WO2025059779A1

WO2025059779A1 - Plant-based or fungal-based micro-carrier scaffolds optimized for large-scale cell-culture production and other applications

Info

Publication number: WO2025059779A1
Application number: PCT/CA2024/051265
Authority: WO
Inventors: Andrew PELLING; Anna CANTO; Cesar TISCHER; Ryan Hickey; Michelle BAMJI-MIRZA; Natalie CRAWFORD; Paula Cristina DE SOUSA FARIA TISCHER; Colin Russell; Keenan MACLAREN; Daniel MODULEVSKY
Original assignee: Spiderwort Biotechnologies Inc
Current assignee: Spiderwort Biotechnologies Inc
Priority date: 2023-09-22
Filing date: 2024-09-20
Publication date: 2025-03-27
Anticipated expiration: 2026-03-22

Abstract

The present invention relates to plant-based or fungal-based cellulose particles for in-vitro growth/production of cells. These scaffold particles are derived from plant or fruit sources by decellularizing, mercerizing, oxidizing or bleaching a plant tissue. A method of using the scaffold particles is also described for in-vitro production/proliferation of cells in a container/culture vessel. The method involves a step of mixing a culture medium containing cells in a container that is pre-filled with scaffold particles and allowing the cells to interact with the scaffold particles in optimized growth conditions for increased proliferation / production of cells.

Description

PLANT-BASED OR FUNGAL-BASED MICRO-CARRIER SCAFFOLDS OPTIMIZED FOR LARGE-SCALE CELL-CULTURE PRODUCTION AND OTHER APPLICATIONS

Field of the Invention

[1 ] The invention relates to scaffold particles, microcarriers or beads. More specifically, the invention relates to plant-based or fungal-based cellulose scaffold particles, microcarriers or beads for in-vitro proliferation of cells.

Background of the Invention

[2] Most animal cells need to stay adhered to surfaces to grow in vitro. In a smaller scale research, adherent cells are grown on treated cell culture plastic or glass surfaces that facilitate cell adhesion and are enzymatically or chemically removed when they fully cover the surface of the culture dishes. The global consensus for in vitro cell production at a commercial scale is to use bioreactors filled with biodegradable polymer microcarriers, such as gelatin and polysaccharides as conventional 2-dimensional cell culture is not practical on large industrial scales, such as those required for food production. Instead, 3-dimensional spheroids or microcarriers can be used to provide surface attachment sites for cells to grow while at the same time increasing surface area for scalability. Most of these materials are biodegradable polymers, like gelatin, or polysaccharides such as dextran (Chen et al., 2019) and modified cellulose (BioCradle | Asahi Kasei Corporation Fibers & Textiles). However, some of these polymers are not ideal for food-production, biomedical, tissue engineering, or therapeutic applications due to the requirement for costly pre and post-processing steps.

Summary of the Invention

[3] The invention describes plant-based or fungal-based cellulose scaffold particles that can be employed in- vitro production, proliferation, or growth of cells. The scaffold particles, microcarriers or beads may be derived from any plant source by decellularizing, mercerizing, bleaching or oxidizing the plant tissue. A plant tissue or a fruit tissue may be pre-treated to obtain scaffold particles by any known decellularization, mercerization, oxidization or bleaching techniques. In certain embodiments, fungal-based cellulose microcarriers are also envisioned.

[4] The invention further describes a method of in-vitro proliferation, production or manufacturing of cells by means of the plant-based or fungal-based cellulose scaffold particles. The method involves a step of adding a batch of culture medium comprising cells in a container that is pre-filled with plant-based or fungal-based cellulose scaffold particles followed by which the cells are allowed to grow on the surface of the scaffold particles for a period of at least 1 day. The method may involve certain pre-processing steps where the scaffold particles/microcarriers/beads are pre-treated and/or modified before the in-vitro process begins. The method may also involve modulating or optimizing the growth conditions or agitation parameters to increase cellular proliferation. The growth of cells can be monitored by any known cell viability or metabolic assay.

[5] The plant-based or fungal-based cellulose scaffold particles could be employed in various application. For instance, the particles may prove useful in cellular in-vitro meat production, cultured meat production, agriculture, tissue engineering, 3D tissue culturing, proliferation of cell-banks, stem cells, in-vitro drug testing, protein harvesting, biomolecule amplification, virus and vaccine production, plasmid amplification, production of metabolites, production of cosmetic actives, production of biopharmaceuticals, production of recombinant proteins, biological filtration, waste-water treatment, industrial cleaning operations, or food production. The particles/microcarriers/beads could also be used as edible scaffolds for the production of dairy products.

Brief Description of Drawings

[6] Figure 1 is an illustrative embodiment of cells that were seeded in a proportion of 4x10⁷ cells for 0.4 to 50 g (wet weight) plant-based beads and left overnight without agitation in 30 mL complete media.

[7] Figure 2 is an illustrative embodiment showing the effect of the number of intermittent agitations during seeding procedures on cell attachment on the beads; A) After 4 hours of attachment (2 min swirling, 30 min rest, 8 times); B) After 20 hours of growth (30 min mixing, 1 hour off).

[8] Figure 3 is an illustrative embodiment with microscopy data showing the influence of the initial concentration of beads and the ratio of cell/bead on attachments.

[9] Figure 4 is an illustrative embodiment showing 0.3 g of beads with roughly 40 million cells, after 4h of attachment.

[10] Figure 5 is an illustrative embodiment showing 0.3 g of beads with roughly 40 million cells, after 20h of attachment.

[1 1] Figure 6 is an illustrative embodiment showing the correlation between the cell number and the protein content on the beads from the 0.3 to 2.5 g beads contained in the spinner flask, determined from the suspension aliquot at days 4 and 8.

[12] Figure 7 is an illustrative embodiment showing beads (Homogenized, Non-Homogenized, and Chemically treated) with L-15 media (during the wash step).

[13] Figure 8 is an illustrative embodiment showing samples (250 uL) that were taken for microscopy and protein analysis. For chemical (middle) and non-homogenized beads (right), it was observed that the beads settled within 10-15 minutes. For homogenized beads (left), the beads didn’t settle at all, even if left O/N. [14] Figure 9 is an illustrative embodiment showing the samples after the first wash after staining with Congo Red and centrifuging for 5 minutes at 1000g (left = non-homogenized/ right=chemical) and it was observed that beads were still floating.

[15] Figure 10 is an illustrative embodiment showing the samples after 3 PBS washes, beads were resuspended in 500uL of PBS and left in the fridge over the weekend.

[16] Figure 11 is an illustrative embodiment showing beads that were left in the fridge over the weekend.

[17] Figure 12 [A, B & C] is an illustrative embodiment showing day 5 microscopy data from the beads/cells of chemically treated and non-homogenized cellulose-based material.

[18] Figure 13 is an illustrative embodiment showing microscopy data of the homogenized beads (sole beads without cells).

[19] Figure 14 is an illustrative embodiment showing images taken at 10X magnification, scale bar = 200 urn. Figure 14 A) & B) show 4 day samples of dried and wet bead samples respectively. Figure 14 C), D), & E) show the wet bead samples, where C) & D) show 1 1 day samples, and E) shows 15 day sample.

[20] Figure 15 is an illustrative embodiment showing CHSE-214 cell spheroids sampled on day 18 from 125 mL spinner flask cultured with wet and dry plant microcarriers, stained with 0.4% trypan blue viability dye, scale bar = 200 urn. Figure 15 A) & B) shows spheroids from spinner flasks with dry plant microcarriers and Figure 15 C) & D) shows spheroids from spinner flasks with wet plant microcarriers.

[21] Figure 16 is an illustrative embodiment showing inverted microscope images taken at 10X magnification of CHSE-214 cells (as shown in Figure 16 A, B, & C) and 3T3 GFP cells (as shown in Figure 16 D, E, & F) seeded with various beads/microcarriers, taken at 24 hours post seeding. Figure 16 A) & D) shows cells seeded with wet beads, Figure 16 B) & E) shows dry beads, and Figure 16 C) & F) show Cytodex-1 beads.

[22] Figure 17 is an illustrative embodiment showing 3T3 GFP cells stained with 0.4% trypan blue viability dye, imaged at 8X magnification. Figure 17 A) & D) show 3T3 cells with Cytodex-1 beads and Figure 17 B) shows 3T3 cells with dried beads, Figure 17 C) & E) show 3T3 cells with wet beads.

[23] Figure 18 is an illustrative embodiment showing interpolated total protein content of 3T3 cell microcarrier samples from BCA assay comparing the total protein concentration among dry beads, wet beads, and commercial Cytodex-1 at days 0, 1 , and 2 of cell culture.

[24] Figure 19 [A, B] is an illustrative embodiment showing 3T3 GFP cells that were seeded with microcarriers/beads then stained with Trypan Blue viability dye, then the number of live vs dead cells were counted. [25] Figure 20 [A-H] is an illustrative embodiments showing images of 3T3 GFP cells incubated with plant microcarriers after 24 hours (in Figure 20 A-D) and 48 hours (in Figure 20 E-H) post attachment phase taken at 10X magnification. Agitation conditions during attachment phase were 30 minute static (in Figure 20 A & E), 1 hour static (in Figure 20 B & F), no agitation (in Figure 20 C & G), and centrifuged (in Figure 20 D & H).

[26] Figure 21 is an illustrative embodiment showing images taken at 10X (i.e. Figure 21 A-D) and 20X (i.e. Figure 21 E-F) magnification of 3T3 GFP cells 48 hours post attachment phase. Images of 3T3 cells seeded with plant microcarriers are shown in Figure 21 A, C & E, and with succinylated cellulose beads are shown in Figure 21 B, D, & F taken from 30 minutes agitation plate in Figure 21 A & B and from no agitation plate in Figure 21 C, D, E, & F.

[27] Figure 22 is an illustrative embodiment showing BOA assay interpolated total protein content of 3T3 cell bead samples, 48 hours post attachment phase after various agitation conditions.

[28] Figure 23 shows HEK 293 cells seeded with succinylated and mercerized cellulose microcarriers in a 6- well plate. Scale bar = 250 pm.

[29] Figure 24 shows Vero cells seeded with succinylated and mercerized cellulose microcarriers in a 6-well plate. Scale bar = 250 pm.

[30] Figure 25 shows Vero cells seeded with succinylated and mercerized cellulose microcarriers in a 6-well plate. Single cellulosic particles circled in black, and cells displaying late phase adhesion morphology highlighted with red arrow. Scale bar = 250 pm.

[31] Figure 26 shows Microscopy of 3T3-GFP cells on plant derived cellulose based microcarriers. Scale bar = 250 pm.

[32] Figure 27 shows Microscopy of C2C12 cells on plant derived cellulose based microcarriers. Scale bar = 250 pm.

[33] Figure 28 shows microscopy of 3T3-GFP cells on plant derived cellulose based microcarriers. Scale bar = 250 pm.

[34] Figure 29 shows microscopy of C2C12 cells on plant derived cellulose based microcarriers. Scale bar = 250 pm.

[35] Figure 30 shows 3T3-GFP cells seeded with mercerized cellulose microcarriers in a 6-well plate after 48 hours (left column) and 15 days (right column). Scale bar = 250 pm.

[36] Figure 31 shows C2C12 cells seeded with mercerized cellulose microcarriers in a 6-well plate after 48 hours (left column) and 15 days (right column). Scale bar = 250 pm. [37] Figure 32 shows Fluorescence microscopy of 3T3-GFP cells after 21 days in culture. Cell nuclei stained with Hoechst (blue) and cellulose particles stained with Congo Red (red). Scale bars = 250 pm.

[38] Figure 33 shows Fluorescence microscopy of C2C12 cells after 21 days in culture. Cell nuclei stained with Hoechst (blue) on cellulose particles stained with Congo Red (red). Scale bars = 250 pm.

[39] Figure 34 shows plate layout for 3T3-GFP cell seeding and post attachment phase conditions on different sizes of lyophilized pellets of mercerized cellulose microcarriers. Wells crossed out with an X represent unused wells.

[40] Figure 35 shows microscopy of 3T3-GFP cells (green) 24 hours post seeding on lyophilized cellulose microcarrier pellets. Scale bar = 250 pm.

[41] Figure 36 shows microscopy of 3T3-GFP cells (green) 24 hours post seeding on lyophilized cellulose microcarrier pellets pre-soaked in 1X DMEM, 10% FBS, 1 % antibiotic-antimycotic. Scale bar = 250 pm.

[42] Figure 37 shows microscopy of 3T3-GFP cells (green) 9 days post seeding on broken apart lyophilized cellulose microcarrier pellets. Scale bar = 250 pm.

[43] Figure 38 shows overlay of brightfield and fluorescence microscopy images of 3T3-GFP cells (green) 14 days post seeding on broken up 0.15 mL lyophilized cellulose microcarrier pellets with 0.30 mL (high volume) media added post attachment phase. Scale bar = 50 pm.

[44] Figure 39 shows fluorescence microscopy of 3T3-GFP cells (green) seeded on cross-linked cellulose microcarriers at 2.5 and 10 X magnifications. Scale bar for 2.5X magnification is 750 pm, and for 10X magnification is 200 pm.

[45] Figure 40 shows microscopy of 3T3-GFP and C2C12 cells seeded on cross-linked cellulose microcarriers. Scale bar = 200 pm.

[46] Figure 41 shows fluorescence microscopy of 3T3-GFP and C2C12 cells after 7 days in culture. Cell nuclei stained with Hoechst (cyan) and cellulose particles stained with Congo Red (red). Scale bar for 4X magnification is 500 pm, and for 10X magnification is 200 pm.

Detailed Description of the Invention

[47] The following description is of preferred embodiments by way of example only and without limitation to the combination of features necessary for carrying the invention into effect.

[48] All terms are intended to be understood as they would be understood by a person skilled in the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure pertains. The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. [49] Although various features of the present disclosure can be described in the context of a single embodiment, the features can also be provided separately or in any suitable combination. Conversely, although the present disclosure can be described herein in the context of separate embodiments for clarity, the present disclosure can also be implemented in a single embodiment.

[50] The following definitions supplement those in the art and are directed to the current application. Accordingly, the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

Definitions

[51] In this application, the use of the singular includes the plural unless specifically stated otherwise. It must be noted that, as used in the specification, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise.

[52] In this application, the use of "or" means "and/or" unless stated otherwise. The terms "and/or" and "any combination thereof and their grammatical equivalents as used herein, can be used interchangeably. These terms can convey that any and all combinations are specifically contemplated. The term "or" can be used conjunctively or disjunctively, unless the context specifically refers to a disjunctive use.

[53] Furthermore, use of the term "including" as well as other forms, such as "include", "includes," and "included," is not limiting.

[54] Reference in the specification to "some embodiments," "an embodiment," "one embodiment" “alternate embodiment”, or "other embodiments" means that a particular feature, structure, or characteristic described in connection with the embodiments is included in at least some embodiments, but not necessarily all embodiments, of the present disclosures.

[55] As used in this specification and claim(s), the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include") or "containing" (and any form of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method or composition of the present disclosure, and vice versa. Furthermore, compositions of the present disclosure can be used to achieve methods of the present disclosure.

[56] The term "about" in relation to a reference numerical value and its grammatical equivalents as used herein can include the numerical value itself and a range of values plus or minus 10% from that numerical value. The term "about" or "approximately" means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, "about" can mean a range of up to 20%, up to 10%, up to 5%, or up to 1 % of a given value. In another example, the amount "about 10" includes 10 and any amounts from 9 to 11 .

[57] Embodiments

[58] The invention will now be described by means of the following exemplary embodiments. A person skilled in the art would understand that the embodiments and examples provided merely describe the nature of the invention and any embodiment or example not specifically recited still falls within the scope of this invention.

[59] In an embodiment of the invention, plant-based or fungal-based cellulose scaffold particles are provided for in-vitro proliferation of cells. The scaffold particles are derived from plant sources which are employable as stable scaffolds for in-vitro growth, culture and proliferation of cells. The particles may also be referred to as microcarriers, mercerized cellulosic material or beads.

[60] In another embodiment, the scaffold particles may be obtained from any plant tissue by decellularizing, mercerizing, bleaching or oxidizing the plant tissue. These plant tissues may be pre-treated by any known decellularization, mercerization, oxidization or bleaching techniques known in the art.

[61] In certain embodiments, the plant or fungal tissue may be cellulose-based, chitin- based, chitosan-based, lignin-based, lignan-based, hemicellulose-based, or pectin-based, or any combination thereof. In certain embodiments, the plant or fungal tissue may comprise a tissue from apple hypanthium (Malus pumila) tissue, a fern (Monilophytes) tissue, a turnip (Brassica rapa) root tissue, a gingko branch tissue, a horsetail (equisetum) tissue, a hermocallis hybrid leaf tissue, a kale (Brassica oleracea) stem tissue, a conifers Douglas Fir (Pseudotsuga menziesii) tissue, a cactus fruit (pitaya) flesh tissue, a Maculata Vinca tissue, an Aquatic Lotus (Nelumbo nucifera) tissue, a Tulip (Tulipa gesneriana) petal tissue, a Plantain (Musa paradisiaca) tissue, a broccoli (Brassica oleracea) stem tissue, a maple leaf (Acer psuedoplatanus) stem tissue, a beet (Beta vulgaris) primary root tissue, a green onion (Allium cepa) tissue, a orchid (Orchidaceae) tissue, turnip (Brassica rapa) stem tissue, a leek (Allium ampeloprasum) tissue, a maple (Acer) tree branch tissue, a celery (Apium graveolens) tissue, a green onion (Allium cepa) stem tissue, a pine tissue, an aloe vera tissue, a watermelon (Citrullus lanatus var. lanatus) tissue, a Creeping Jenny (Lysimachia nummularia) tissue, a cactae tissue, a Lychnis Alpina tissue, a rhubarb (Rheum rhabarbarum) tissue, a pumpkin flesh (Cucurbita pepo) tissue, a Dracena (Asparagaceae) stem tissue, a Spiderwort (Tradescantia virginiana) stem tissue, an Asparagus (Asparagus officinalis) stem tissue, a mushroom (Fungi) tissue, a fennel (Foeniculum vulgare) tissue, a rose (Rosa) tissue, a carrot (Daucus carota) tissue, or a pear (Pomaceous) tissue, or a genetically altered tissue produced via direct genome modification or through selective breeding, or any combinations thereof.

[62] In certain embodiments, the scaffold particles may be derived from any plant or fruit sources. A few nonlimiting examples of plant tissues could be apple, pear, banana, mango, lotus root, wood, bamboo, cotton linters, cotton stalks, cotton fabric waste, cotton wool, soybean husk, corn cob, water hyacinth, coconut shells, oil palm fronds, oil palm biomass residue, rice husk, sugar cane bagasse, jute, ramie, flax fibers, flax straw, wheat straw, sorghum stalks, sisal fibers, potato or mangosteen. [63] In certain other embodiments, the scaffold particles have exposed polar groups with an augmented charge on their surface. The derived scaffold particles, microcarriers or beads are biocompatible and optionally biodegradable. The scaffold particles are capable of facilitating cell growth in suspension and contribute to the textural properties, cellular organization or cellular differentiation in a final product.

[64] In certain embodiments, the particles, microcarriers, or beads, are substantially circular, spherical, diskshaped or oval in shape. Optionally, the particles are structured as folded sheets of cellulose arranged in the form of irregular microspheres.

[65] In certain embodiments, the surface of scaffold particles is modified by a chemical treatment or by a physical treatment. The modification is capable of improving and enhancing the proliferation of cells. The modification allows increasing cellular attachment to the surface of the scaffold particles, by increasing cellular entrapment to the surface of the scaffold particles or by increasing cell proliferation capacity on the particles.

[66] In certain embodiments, the surface of the scaffold particles is physically treated by various physical modifications known in the art. In an exemplary embodiment, the physical treatment is by freeze-drying or lyophilization, re-hydration, heating, freezing with or without liquid nitrogen. The scaffold particles of claim 11 , wherein the surface is chemically treated by functionalizing the particle surface, cross-linking or by treating the particle surface with a pharmaceutically acceptable compound or a combination thereof.

[67] In certain embodiments, the surface of the scaffold particles is functionalized by providing a functional group that creates a charge on the particle surface or by succinylating the particle surface. Functionalizing the surface allows for increased attachment of cells to the surface of the particles or increases proliferation of cells. In an exemplary embodiment, the functional group may be a primary amine, a tertiary amine, a quaternary compound, a denatured collagen, gelatin or a combination thereof. In certain embodiments, the scaffold particles are pretreated with hydrogen peroxide, sodium bicarbonate, sodium hydroxide, gelatin, fetal bovine serum, glycine, citric acid or ascorbic acid.

[68] A person skilled in the art would understand that the scaffold particles may be treated with any pharmaceutically acceptable compound. In an exemplary embodiment, the pharmaceutically acceptable compound may be hydrogen peroxide, sodium bicarbonate, RGD motif, fibronectin, cadherins, integrins, nectins, afadin, p-catenin/aE-catenin, E-cadherin, vitronectin, cell attachment factors, vitamins, ascorbic acid, gelatin, serum, fetal bovine serum, charged amino acids, glycine or any and food-grade acceptable compounds. In another exemplary embodiment, the scaffold particles may be pre-treated with hydrogen peroxide.

[69] In some embodiments, the scaffold particles are subjected to cross-linking with a cross-linker agent to create cross-linked cellulose microcarriers. The cross-linker agent may be citric acid or paraformaldehyde.

[70] The size of the scaffold particles may vary greatly depending on the plant tissue used to derive the particles. Their shapes may vary greatly too. In an exemplary embodiment, the size of the scaffold particles may range from 1 pm to 1000 pm. In another exemplary embodiment, the size of the particles may range from 20 pm to 700 pm. In another exemplary embodiments, the size of the particles may range from 40 pm to 150 pm.

[71] In another embodiment of the invention, wherein the attachment of cells to the scaffold particles is dependent on one or more particle characteristics, wherein the one or more particle characteristics are particle size, particle surface tension, particle topography, or particle roundness. The attachment of cells also depends on the growth conditions and culture vessel parameters. The factors that may affect the attachment of cells may include the culture medium, agitation, temperature, pH and other growth conditions/parameters. Moreover, the size, surface modifications (chemically treated, physically modified, succinylated, mercerized, lyophilized etc), number, and the surface charge of the scaffold particles can also impact the attachment and proliferation of cells on the particles. In certain embodiments, the proliferation of cells is directly correlated to the number of scaffold particles in the culture vessel or culture medium. The scaffold particles could be employed for in-vitro proliferation, production or growth of a variety of cells including animal cells, plant cells, or human cells.

[72] In another embodiment of the invention, the plant-based or fungal-based cellulose scaffold particles, microcarriers or beads could be employed for cellular in-vitro meat cell-production, cultured meat cell-production, agriculture, tissue engineering, 3D tissue culturing, proliferation of cell-banks, stem cells, in-vitro drug testing, protein harvesting, biomolecule amplification, virus and vaccine production, plasmid amplification, production of metabolites, production of cosmetic actives, production of biopharmaceuticals, production of recombinant proteins, biological filtration, waste-water treatment, industrial cleaning operations, or food production. In certain embodiments, the cellulose scaffold particles could be employed as edible scaffolds for the production of dairy products.

[73] In another embodiment of the invention, a method of in-vitro proliferation, production or manufacture of cells is provided. The method involves the step of adding a batch of culture medium comprising cells in a container filled with plant-based or fungal-based cellulose scaffold particles as discussed and defined hereinbefore. The method further involves allowing the cells to grow on the surface of the scaffold particles for a period of at least 1 day. The growth phase i.e. the phase where the cells are allowed to grow on the particles, could vary greatly depending on the purpose, for instance, it could range from 1 day to a month or even more. The growth phase could be interrupted to collect a batch of proliferated cells and then resumed by adding fresh culture medium, fresh batch of starter cells, fresh scaffold particles etc., thereby allowing the growth phase to continue indefinitely. The growth of cells can be monitored by any known cell viability or metabolic assays in the art. These assays could prove as indicators of depleting nutrients, or depleting surface area for growth, based on which the growth conditions can be altered. The growth of cells on the surface of the particles is generally in the form of a monolayer, spheroids or cellular aggregates, or both. The cellular aggregates could have a variety of shapes including spheroidal.

[74] In certain embodiments, the method further comprises agitation of the container or culture vessel at regular or irregular intervals after the step of adding the cells to the container-filled with scaffold particles/beads, or after the growth phase. This agitation, shaking or mixing step could help increase the attachment and proliferation of cells. In certain embodiments, the agitation step after the step of adding the cells increases the attachment of cells to the surface of the scaffold particles and contributes to movement, motion or diffusion of of gasses and the culture medium within the container. In certain embodiments, the agitation step after the growth phase begins i.e. when the cells are allowed to grow on the particles directs the growth of cells as monolayer, spheroids or cellular aggregates, improves the viability of cells, and increases the proliferation of cells on the surface of the scaffold particles.

[75] The agitation parameters can vary greatly. For instance, the agitation step may be carried out continuously or intermittently. It could be carried out by manual means i.e. manual means or by automated means e.g. by means of spectrophotometer plate reader. The agitation step can be carried out for a few minutes or for a few hours or for a few days. For instance, the agitation step can be carried out for a duration of 0.5, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, or 60 minutes.

[76] In certain embodiments, the agitation step may be carried out with a static of a few minutes, a few hours or a few days. For instance, the static between agitation sessions could be 300 minutes, 270 minutes, 240 minutes, 210 minutes, 180 minutes, 150 minutes, 120 minutes, 90 minutes, 60 minutes or 30 minutes.

[77] In certain embodiments, the method further comprises a step of spinning an impeller within the container at a speed of 20 rpm - 90 rpm at regular or irregular intervals. The spinning speeds can vary greatly and it can be as low as 10 rpm and as high as 100 rpm. The spinning step is introduced as it may increase the proliferation of cells by allowing more time for the cells to interact with the surface of the scaffold particles and it may contribute to the movement, motion or diffusion of the culture medium within the container. The spinning step may be carried out continuously or intermittently. It may be carried out for a few minutes, a few hours or for a few days. For instance, the spinning step may be carried out at 30-minute intervals or at one-hour intervals. In certain embodiments, the spinning step is carried out once every hour after step b) followed by every four hours. In an exemplary embodiment, the spinning step may be carried out for a period of 10- 60 minutes. In certain other embodiment, the spinning step may be carried out for 20 to 40 minutes. In certain embodiments, the spinning step is carried out at 30 rpm to 80 rpm.

[78] In certain embodiments, the method i.e. the cell culture and proliferation technique can be carried out at a temperature ranging from 15°C to 45°C. For instance, the method may be carried out at ambient temperature. In certain exemplary embodiments, the method may be carried out in the presence or absence of light. The method may also be carried out in the presence or absence of carbon dioxide and any known buffer solution e.g. HEPES or any other buffer solution for cells.

[79] In certain embodiments, multiple batches of culture medium containing cells are added to the container at regular intervals. In certain other embodiments, extra batches of scaffold particles may be added to the container at regular intervals. [80] In certain embodiments, the method may comprise a pre-processing step prior to the initial step of adding the culture medium containing cells to the container. For instance, the scaffold particles may be pre-processed by washing the scaffold particles using a salt solution, or pre-treating the scaffold particles chemically with a pharmaceutically acceptable compound, pre-treating the scaffold particles physically, or modifying the surface of the scaffold particles. In certain embodiments, the washing step may comprise an incubation step in which the scaffold particles are incubated in the salt solution overnight. The salt solution could be any salt solution that is generally known in the art. For instance, PBS. In certain specific embodiments, the salt solution may be Ca²⁺/Mg²⁺ free. In certain embodiments, any culture medium known or used for promoting cell growth on microcarriers or beads could be employed in this invention. For instance, the culture medium could be with or without FBS, with or without FBS substitute, with or without antibiotics, with or without glucose, with or without indicator, or with or without CO2 buffering solution. In some exemplary embodiments, the culture medium may be DMEM, MEM, L- 15, Opti-MEM, MEM Alpha, McCoy’s 5A, RPMI-1640, F-12 or Hanks' Balanced Salt Solution.

[81] In certain embodiments, the pharmaceutically acceptable compound for pre-treating the scaffold particles may be a peptide, a drug, an antibody, a cofactor, or an enzyme. In certain exemplary embodiments, the pharmaceutically acceptable compound is hydrogen peroxide, bicarbonate, RGD motif, fibronectin, cadherins, integrins, gelatin, serum, fetal bovine serum, vitamins, ascorbic acid, glycine, nectins, afadin, p-catenin/aE- catenin, E-cadherin, cell attachment factor, or charged amino acids or any other food-grade acceptable compound. For instance, the scaffold particles are pre-treated with hydrogen peroxide at a concentration ranging from 0.1 % to 10%. In certain exemplary embodiments, the scaffold particles are pre-treated with bicarbonate salt at a concentration ranging from 1 % to 10%. In certain other exemplary embodiments, the scaffold particles are pretreated with sodium hydroxide at a concentration ranging from 1 % to 10%. In some embodiments, the scaffold particles are subjected to cross-linking with a cross-linker agent to create cross-linked cellulose microcarriers. The cross-linker agent may be citric acid or paraformaldehyde.

[82] In certain embodiments, the scaffold particles are pre-treated physically by homogenization, dispersion, low-shear mixing, high-shear mixing, ultrasonic processor, emulsifying equipment, lyophilization, mercerization, micron ization, re-hydration, heating, freezing with orwithout liquid nitrogen, or sonication. In certain embodiments, the pre-treatment step could be employed to modify the surface of the scaffold particles or to enhance the proliferation of cells by increasing cellular attachment to the surface of the scaffold particles, or by increasing cellular entrapment to the surface of the scaffold particles or by increasing cell proliferation capacity on the particles.

[83] In certain embodiments, the surface modification of the scaffold particles may be carried out by functionalizing the surface of the scaffold particles. For instance, the functionalization may be carried out by providing a functional group that creates a charge on the surface of the scaffold particles. Any known functional groups can be employed to charge the scaffold particle surface. For instance, the functional group could be a primary amine, a tertiary amine, a quaternary compound, denatured collagen, gelatin, fetal bovine serum, vitamins, amino acids, gelatin or a combination thereof. The surface modification may also be carried out by succinylating the surface of the scaffold particles or by lyophilizing the scaffold particles, or cross-linking the scaffold particles. In some embodiments, the method may include subjecting the scaffold particles to cross-linking with a cross-linker agent to create cross-linked cellulose microcarriers. The cross-linker agent may be citric acid or paraformaldehyde.

[84] In certain embodiments, the method may also comprise a sterilization step prior to the initial step of mixing the culture medium with the scaffold particles, where the scaffold particles are sterilized for a pre-determined period of time. Any known sterilization technique may be employed. For instance, the sterilization step may be carried out by autoclaving, water steam treatment, chemical steam treatment, ethylene oxide treatment, alcohol treatment, UV irradiation, Gamma irradiation or pasteurization. The sterilization step may be carried out for a few seconds, or a few minutes, or a few hours. In an exemplary embodiment, the sterilization step may be carried out for 10 to 60 minutes, or from 1 to 85 minutes, or from 0.5 minutes to 120 minutes, or for a greater time span if needed.

[85] In certain embodiments, the method may additionally comprise a step of screening the scaffold particles prior to the initial step of mixing the culture medium containing cells with the scaffold particles. For instance, the scaffold particles may be screened prior to filling in the container. In certain embodiments, the screening step may involve selecting specific sized scaffold particles. For instance, the scaffold particles may be screened to select smaller-sized particles, or particles that are within a particular size range, or of a particular shape. In certain embodiments, the screening step may involve selection of scaffold particles with varying sizes to obtain an effective particle size distribution. In certain other embodiments, the screening step may involve quantification of the number of scaffold particles. In some embodiments, the quantification step may be followed by increasing the number of scaffold particles if the number of particles are found below a threshold amount.

[86] In certain specific embodiments, the culture vessel or container may be filled with scaffold particles sized in the range of 1 pm to 1000 pm. In certain exemplary embodiments, the size of the scaffold particles may range from 20 pm to 700 pm. In certain other embodiments, the size of the scaffold particles may range from 40 pm to 150 pm.

[87] In certain embodiments, the method additionally comprises a purification step where the scaffold particles are purified after the pre-processing step.

[88] In certain embodiments, the method may comprise a quantification step to quantify the proliferation or growth of cells. There are various quantification and viability assays known in the art which can be employed in the present invention. For instance, the quantification step may be carried out by a biomolecule quantification assay, cell-quantification assay, DNA quantification assay, genome quantification assay, cell-adherence quantification analysis, cell metabolism assay, metabolite assay, microscopy, protein assay, protein quantification assay or quantification of cell-specific antigens.

[89] In certain other embodiments, the quantification step is followed by an optimization step to increase the proliferation of cells, or to increase cellular attachment to scaffold particles. For instance, the optimization step may be carried out to optimize the growth conditions in the container, to optimize the cell culture conditions, to optimize the number of scaffold particles, to optimize the size of the scaffold particles, to optimize the agitation parameters in the container, to optimize the cell to scaffold particle ratio or a combination thereof. In some cases, the optimization step may be modulated to direct or modulate the growth of cells. For instance, the growth conditions may be optimized to allow the cells to form a monolayer on the scaffold particles. In some other embodiments, the optimization step may be carried out to allow the cells to grow in the form of cellular aggregates or spheroids on the surface of the scaffold particles.

[90] In certain embodiments, the in-vitro proliferation of cells may be increased by increasing the number of scaffold particles in the container. In certain other embodiments, the in-vitro proliferation may be increased by changing, altering or selecting the size of the scaffold particles in the container. In certain embodiments, the container may be filled with a solution comprising the scaffold particles. Any known containers, flasks, culture vessels or plates known for in-vitro cell-culturing may be employed in this invention. For instance, the container may be a T-175 flask, a 6-well plate, well plates, a siliconized plate or flask, a Hyperflask, a Spinner flask, a Cell stack culture chamber, a vessel, a bioreactor, a cell culture microreactor, a cell-culture chip, a microfluidic cell culture device, a lab-on-a-chip device or the combination of two or more of this recipients or assembled laminates.

[91] In certain embodiments, the method further involves a step of separating the growth of cells from the scaffold particles by mechanical or enzymatic action. The proliferated cells can be separated or harvested from the surface of the scaffold particles by any known mechanical means or via enzymatic action, for instance, cellulase, trypsin, TrypLE etc.

[92] Experimental Data

[93] Preliminary studies conducted by the inventors demonstrated edible cellulose-based scaffold obtained from apple were capable of holding a population of fish cells competently, being surrounded by them. It was observed that the scaffold increased the area for cell growth and also retained the mechanical strength as needed.

[94] The inventors envisioned utilizing scaffold particles from a variety of plant sources and plant parts that could prove useful for in-vitro production or proliferation of cells. In an exemplary embodiment, the inventors utilized cellulose-based apple particles as a starting material to act as a commercial bead (similar to Cytodex-1 , commercial dextran beads) and to create an environment for cell attachment and proliferation. This technology was investigated in the context of expanding cell populations for various applications e.g. alternative meat production.

[95] The inventors conducted experiments that helped in increasing cellular attachment and proliferation of cells in a controlled environment (e.g. bench scale). The experiments also demonstrated beads/microcarrier effectiveness for cell attachment and proliferation e.g. fish cells.

[96] I. Assessing scaffold particles or beads as an appropriate edible scaffold for CHSE-214 cells in an exemplary embodiment. [97] Objective: The objective of this experiment was to determine whether the plant based cellulose scaffold beads I particles (e.g. mercerized apple particles) provide a good scaffold for cells to adhere and proliferate in a spinner flask system.

[98] Protocol: In this exemplary embodiment, the cells utilized were adherent CHSE-214 from Chinook salmon embryo cells, and the protocol was based on a commercial beads protocol: Cytiva’s Cytodex-1 Corning dissolvable microcarriers. However, a person skilled in the art would understand that plant, animal or human cells could be proliferated on the scaffold particles I microcarrier beads.

[99] Processing of Beads: The plant based cellulose beads/particles were processed using a similar methodology to Cytodex-1 beads (Cytiva).The plant based cellulose beads I particles were washed in Ca²⁺, Mg²⁺- free PBS. The centrifugation and autoclaving parameters could vary. The inventors centrifuged the beads/particles at least 5,000 rpm using JLA 16.250 rotor for more than 10 minutes, and transferred to a glass bottle then sterilized by autoclaving at 121 °C, 15 psi for 20 minutes. The number of beads in grams was determined and transferred to the growing flask.

[100] a. Cell for seeding: In this exemplary embodiment, the inventors used CHSE-214 cells, growing in T-175 flasks, were washed with 1xPBS, detached with trypsin-EDTA and transferred to 50 mL centrifuge tubes for centrifugation at 10OOxg for 5 minutes. The cell pellet was resuspended in a pre-determined volume to transfer to the growing flask to contain 2x10⁷ CHSE-214 cells in complete media: L-15 media, 10% FBS, 1x antibiotic/antimycotic. The growth conditions were closed receptacles, ambient temperature and shielded from light.

[101] b. Plant based bead/cell staining: An aliquot of the bead/cell suspension was transferred to a 1.5 mL microcentrifuge tube. The tubes were centrifuged for 2000xg for 5 minutes, the culture media was removed, and the pellet (a collection of beads and cells) was fixed with 4% paraformaldehyde for at least 1 h to several days. Next, the beads were washed 3x with PBS (each wash 1000 x g for 5 minutes), stained with 1 ug/mL Hoechst (1 :1000 dilution in PBS of 1 mg/mL stock) for 30 minutes in the dark, washed 3x with PBS, stained with 0.1 % Congo red in PBS for 30 minutes, washed 3x with PBS, and then half of the suspension was placed on a slide and covered with a coverslip. In later experiments, the centrifugation steps were increased to 2000 x g for 5 minutes because the material was still in suspension at lower speeds. In later experiments, the beads were allowed to settle in the microfuge tube once the aliquot was collected and the supernatant containing the dead cells was removed and the beads were washed twice with PBS before the fixation step.

[102] c. Plant based bead/cell protein assessment: A 1 mL plant-based bead/cell suspension aliquot was transferred to a microfuge tube. The material was centrifuged at 2000 x g for 5 minutes and washed in ice-cold PBS 2x. Next, the pellet was resuspended in ice-cold Tris-EDTA SDS lysis buffer (10 mM Tris HCI, 1 mM EDTA, 0.5% SDS, 1 mM PMSF/DTT, 1 ug/mL DNase), incubated for 30 minutes on ice, sonicated for 30 seconds and then the protein lysate was clarified by centrifugation at 12,000 rpm, 10 min at 4°C. In parallel, CHSE-214 cells were harvested from a static dish, counted with a hemocytometer, and serially diluted to correlate, on a pre- prepared graph, the cell number and the protein content. Next, the protein content in the samples and the cell dilutions were determined using the Pierce BCA Protein Assay Kit - Reducing Agent Compatible using the manufacturer’s instructions.

[103] II. Protocol of attachment and proliferation experiments with the plant based beads

[104] a. Test to analyze CHSE-214 cells attachment to plant-based beads (0.4 g): 0.4 g of 1 % wet plant based beads were weighed, washed, and incubated overnight with PBS as described in processing beads. In the glass bottle containing the 0.4 g of beads, the PBS was replaced with L-15 media to condition the beads and then replaced with complete culture media. The 0.4 g of plant based beads were seeded with 4x10⁷ cells (from 2x T- 175 dishes) and the attachment step was performed using intermittent shaking: 2-min swirling by hand, 30-min stand, repeated 1x. After this attachment phase, the suspension was left overnight without agitation. The next day, an aliquot of the material was removed, stained as outlined above, and visualized by microscopy. A similar procedure was followed with Cytodex-1 beads (Cytiva).

[105] b. Test to analyze CHSE-214 cell proliferation on plant based beads (50 g): 50g of beads were washed and sterilized as described in the steps above and were conditioned in serum-free media, followed by conditioning in complete growth media before being transferred to a 125 mL spinner flask. The beads were seeded and submitted to an attachment phase including 50 rpm for 2 min, 30-minute rest, and repeat 2 times. The growing experiment was repeated for 7 and 9 days with the increased quantity of the plant based cellulose (50 g), and aliquots were taken (washed before fixation thereby removing the unattached cells) for staining and microscopy.

[106] c. Test to analyze the optimal ratio between beads and cells: Spinner flasks containing the 0.3 and 2.5 g beads were washed with L-15 media by allowing the beads to settle for 30 min to 1 h, removing the existing media and replacing it with L-15 media before adding complete fresh media once again. The beads were seeded with 4x10⁷ cells and the attachment phase included a 2 min swirl, 30 min off, and a repeat of 8x (4 hours). After the attachment procedure, the cells were left to grow in intermittent mixing at 50 rpm, 30-min on, 1-h off, and then changed to 30 minutes on, with 4 h off. Aliquots were taken after attachment and then after 1 , 4, 5, 8, and 12 days. Microscopy was performed and described below. Aliquots were also processed for protein level calculation. The protein level was calculated from the aliquoted beads, it was determined that the cells are proliferating on plant based beads between Day 4 and Day 8.

[107] d. Test three different preparations of Cellulose-based starting material to analyze particle size and cell attachment: Three different types of Cellulose-based starting material were tested: Standard (Nonhomogenized), Chemically treated and Physically treated. The chemical and physically treated were analyzed with the objective of investigating if (1) they had smaller size particles, and (2) observe if they had higher cellular attachment. The standard beads acted as a control in this experiment. The number of cells to beads ration was 1 x T-175 dish for 0.2 g beads

[108] i) General preparation of the beads: Plant based scaffold beads/microcarriers were transferred into 250mL centrifuge bottles and washed with 75 mL of PBS for 2 minutes continuously. Next, the particles were centrifuged for 10 minutes, followed by a second washing step using 50 mL fresh Ca2+ and Mg2+ free PBS. The plant based particles were transferred to a 500 mL glass bottle, completed with 100 mL fresh Ca2+ and Mg2+ free PBS, and sterilized (121 °C, 15 psi for 20 minutes). A 0.2 g of the material was then transferred into their respective spinner flask, and washed with L-15 medium (without FBS)

[109] ii) Culture procedure: 3 x T-175 flasks containing CHSE-214 cells were washed, trypsinized, and the cell concentration was measured using a hemocytometer. The L-15 medium was replaced by 25 mL of complete culture medium (with 10% FBS). A quantity of 2x10⁷ cells was added in each spinner flask, swirled for 2 minutes, and then left for 30 minutes. This procedure was repeated at least 10 times (5 hours), 100 mL of media was added, and intermittent agitation (50 rpm) overnight in the room occurred. The cell attachment and growth were monitored every 2 days for the next 7-10 days, by removing 0.25 mL samples and visualizing them under a microscope.

[1 10] iii) Bead/Cell staining: An aliquot of the bead/cell suspension was transferred to a 1 .5 mL microcentrifuge tube. The tubes were centrifuged for 2000xg for 5 minutes, the culture media was removed, and the pellet (i.e. a collection of beads and cells) was fixed with 4% paraformaldehyde for at least 1 h to several days. Next, the beads were washed 3x with PBS, each wash was at 1000 g for 5 minutes, and stained with 1 uL of the Green Biotracker cell labelling. These were further incubated for 20 min at room temperature in the dark. Next, the scaffold particles/beads were washed 3x with PBS (each wash 1000 g for 5 minutes), stained with 0.1 % Congo red in PBS for 30 minutes, and washed again 3x with PBS. A 100 uL of suspension was placed on a microscope slide. A drop of Fluoroshield with DAPI mounting medium was added on a glass slide, the coverslip was placed on the drop of mounting media, and the sample was examined using the BX53 microscope with the following specifications: a) Used DAPI filter to identify the presence of cells; b) without shifting the image or the focus, switched to TXRED or TRITC, to identify the scaffold; c) thereafter the two images were merged.

[1 11] III Results

[1 12] Indication: In the above exemplary embodiment, the first indication that CHSE-214 cells seem to attach to plant based beads (0.4 g) was observed by the inventors when cells were seeded in a proportion of 4x10⁷ cells for 0.4 to 50 g (wet weight) plant based beads and left overnight without agitation in 30 mL complete media (as shown in Figure 1). Figure 1 is an illustrative embodiment showing cells that were seeded in a proportion of 4x10⁷ cells for 0.4 to 50 g (wet weight) plant based beads and left overnight without agitation in 30 mL complete media. 40 million cells (CHSE-214) were seeded with 0.4 g beads (wet weight). The data show cells within the crevices of the large beads (roughly 400 pm) and surrounding beads (Figure 2) .

[1 13] Figure 2 shows the effect of the number of intermittent seeding procedures on cell attachment on the beads; Figure 2A) shows the effect after 4 hours of attachment (2 min swirling, 30 min rest, 8 times); Figure 2B) shows the effect after 20 hours of growth (30 min mixing, 1 hour off).

[1 14] Further evidence: Inventors further observed that CHSE-214 cells seem to proliferate on plant based beads (50 g) when plant based beads (50 g) were seeded with 4x10⁷ cells in a spinner flask with continuous agitation at 50 rpm for up to 9 days. It was observed that the cells were still attached to the beads after 9 days, demonstrating that the cells were not only attached but also proliferating, on the beads. The cells were found again within the crevices of large beads. The beads were washed before fixation suggesting that this attachment is likely real. The results of the experiment adding 50 g of wet cellulose which corresponds to 0.5 g of dry cellulose can be seen in Figure 3. Figure 3 provides microscopy data showing the influence of the initial concentration of beads and the ratio of cell/bead on attachments.

[1 15] Optimal ratio of beads:cells: The spinner flask with 0.3 g of beads at 4h and 20 h exhibited cellular attachment (as shown in Figure 4 and Figure 5). Figure 4 shows 0.3 g of beads with roughly 40 million cells, after 4h of attachment. In, Figure 5, 0.3 g of beads with roughly 40 million cells, after 20h of attachment are shown.

[1 16] A correlation between the cell number and the protein content on the beads from the 0.3 to 2.5 g beads was performed from the suspension aliquot at days 4 and 8. The spinner flask containing 2.5 g beads exhibited higher concentration of protein when compared to the one containing 0.3 g for both days analyzed. Thus can be suggested that the spinner flask with 2.5 g of beads demonstrated greater number of cells than the counterpart (as shown in Figure 6). Figure 6 shows correlation between the cell number and the protein content on the beads from the 0.3 to 2.5 g beads containing the spinner flask was determined from the suspension aliquot at days 4 and 8. The beads were seeded with 4x10⁷ cells and only increased to 5x10⁷ cells after 8 days in culture.

[1 17] Preparations: The inventors tested three different preparations of cellulose-based starting material to further analyze particle size and cellular attachment. The rationale for performing this experiment was performed in order to increase the small bead fraction and consequently cell attachment, by analyzing physically (homogenization) and chemically (using a more strong base in the process) treated cellulose-based beads.

[1 18] The homogenized beads demonstrated a constant dispersion in the media with no precipitation. To avoid complexity, those beads were not utilized in this experiment. However, physically modified beads can be utilized using various preparation techniques known in the art. During the washing step in the Spinner flask with L-15 media, although the homogenized beads did not settle, chemical beads settled within 10 minutes, and nonhomogenized beads settled within 15 minutes.

[1 19] The chemically treated beads demonstrated homogeneous particle sizes (ranging from 150-400 urn) and exhibited pockets of cell clusters. They behaved similarly to a population of beads present in the non-homogenized preparation. The salmon cells were visualized by microscopy on non-homogenized and chemically treated beads on day 5 and day 14. This was also a collection of cells indicative of cell growth.

[120] Details of these experiments can be found in the figures. Figure 7 shows beads (Homogenized, Non- Homogenized, and Chemically treated) with L-15 media (during the wash step). Figure 8 shows samples (250uL) that were taken for microscopy and protein analysis. For chemical (middle) and non-homogenized beads (right), it was observed that the beads settled within 10-15 minutes. For homogenized beads (left), the beads didn’t settle at all, even if left overnight. Figure 9 shows the samples after first wash after staining with Congo Red and centrifuging for 5 minutes at 1000 xg (left = non-homogenized/ right=chemical) and it was observed that beads were still floating. Figure 10 shows the samples after 3 PBS washes, beads were resuspended in 500uL of PBS and left in the fridge over the weekend. Figure 11 shows beads that were left in the fridge over the weekend. Figure 12 [A, B, & C] shows day 5 microscopy data from the beads/cells of chemically treated and nonhomogenized cellulose-based material. Figure 13 shows microscopy data of the homogenized beads (sole beads without cells).

[121] Enzymes: The proliferated cells can be separated from the cellulose particles/beads by degrading away the particles/beads with enzymes e.g. cellulase, trypsin, TrypLE, and thus, easily free the cells from the micro carrier particles. Various other separation mechanisms can be explored apart from degrading the particles via enzymatic action or hydrolyzation.

[122] Protein concentration as a tool for cell quantification: For cell quantification, that usually is performed after microcarriers hydrolyzation- i.e. dextranase to hydrolyze commercial dextran beads - detaching them to the medium to be stained. Cellulose is insoluble and therefore harder to hydrolyze, and thus the cell quantity can optionally be measured by the protein content using the Pierce BCA Protein Assay. For that, a standard curve is prepared with CHSE-214 cells harvested from a static dish, counted with a hemocytometer and serially diluted to correlate, results were graphed to correlate the cell number and the protein content. In this way, the protein content in the samples and the cell dilutions were determined using the Pierce BCA Protein Assay Kit - Reducing Agent.

[123] Intermittent spinning: Usually the cell growth is obtained in bioreactors by gently stirring the cells and rotating the blades at a rate that keeps them in suspension without exceeding the physical resistance of the cellular membrane. At the same time, cells need time to interact with the bead surface and adhere. To test if intermittent spinning improves the procedure, intermittent spinning with established conditions was utilized after some trials.

[124] Intermittent attachment procedure: The beads were seeded with cells and the attachment phase included: 2 min swirl, 30 min off, this was repeated 8x (4 hours). Intermittent growth: After the attachment procedure, the cells were let to grow in the following loop: swirl at 50 rpm for 30-min, 1 h static, 30 minutes swirl, 50 rpm, followed by 4 h static.

[125] IV Additional Preparation and Quantification Methods

[126] a. Cell Culture

[127] CHSE-214 cells were cultured in T175 flasks with L-15 media supplemented with 10% FBS, 1 % antibiotic/antimycotic. Cells were subcultured at 1 :2 to 1 :4 cell suspension to new media ratios, incubated at 21 °C, without CO2. The Cells were subcultured every 5-7 days and media was changed every 3 days.

[128] 3T3 GFP cells were cultured in T75 vented flasks at 37 °C, 5% CO2, in complete growth medium: DMEM, 10% FBS, 1x antibiotic/antimycotic. Cells were subcultured at 1 :25 ratio, and were split every 3-4 days. When subcultured all cells were washed once with 1X PBS then detached with trypsin-EDTA and transferred to 50 mL centrifuge tubes for centrifugation at 1500 x rpm for 5 minutes. The cell pellet was re-suspended in complete growth medium to transfer to a fresh culture flask at the appropriate sub-culturing ratio. [129] b. 6-well plate Siliconization with Dow SYLGARD TM 184 Silicone Elastomer kit

[130] Anti-adherence 6-well plates were prepared by siliconization using Dow SYLGARD TM 184 Silicone Elastomer kit. 50 mL of silicone elastomer mix was prepared for 5X 6-well plates.

[131] In a fume hood, 5 mL of reagent A was added to 45 mL reagent B in a 50 mL falcon tube (1 :10 ratio of reagent A to B), then mixed gently by inversion for 5-10 minutes, while avoiding bubble formation. In a BSC, approximately 1-2 mL of the silicone mixture was added to each well of a 6 well plate, which were then rocked to ensure the silicone mix fully covered the bottom of the wells. The plates were then left to cure overnight in the BSC at room temperature. Plates were sterilized the following day by fully submerging in 70% ethanol, and left to dry in BSC during a 1 hour UV cycle with the lids off.

[132] c. Microcarrier Bead preparation for spinner flasks

[133] 0.2 g of dried plant microcarriers was measured and resuspended in 20 mL of Ca/Mg²⁺ free PBS to make a 1 % cellulose solution. The final solution of dried resuspended cellulose was then weighed and recorded. Equal weight of 1 % dried resuspended material (approximately 20 g) was measured of the 1 % mercerized (wet) cellulose resulting in approximately 20 g of 1 % cellulose of both dried and wet mercerized material.

[134] Cellulose beads were then washed twice with 75 mL of PBS, mixed well after each wash for 2 minutes then centrifuged at 8000 rpm for 10 minutes to pellet beads. After washing the total volume was brought up to 100 mL of PBS, and then the beads were sterilized in the autoclave at 121 °C, 15 psi for 30 minutes. Once beads had cooled, beads were transferred to 2X 50 mL falcon tubes and washed with 30 mL of complete growth medium. Using trypan blue stain and a hemocytometer, the concentration of beads/mL was calculated.

[135] d. Scaffold/Microcarrier/Bead & Cytodex-1 microcarrier preparation for 6-well plates

[136] 0.1 g of Cytiva Cytodex-1 beads and 1.0 g of microcarrier wet material was measured and resuspended in 25 mL Ca/Mg²⁺ free PBS then left to rehydrate at room temperature for at least 1 hour. Beads were then sterilized by autoclaving at 121 °C, 15 psi for 30 minutes. After autoclaving, beads were transferred to sterile 50 mL falcon tubes then washed 2X with 20 mL of sterile Ca/Mg²⁺ free PBS and stored overnight at 4°C. The next day the beads were resuspended, then transferred to 2X 50 mL falcon tubes and washed with 15 mL of complete growth medium. Once the beads settled, the total volume was adjusted to get a final concentration of beads in suspension of 0.012 g/mL.

[137] e. Spinner flask (SF) cell & microcarrier bead seeding

[138] Once cell and bead concentrations were calculated, the amount of cells and beads required to get a 16:1 cell to bead ratio was determined. Spinner flasks were seeded with 4.8x107 cells. Therefore, the number of beads required for a 16:1 cell to bead ratio was 3x106 beads per spinner flask. [139] Beads were diluted appropriately to get 3x106 beads in 30 mL total volume. Cells were centrifuged and resuspended in 10 mL of complete growth medium. Spinner flasks were then seeded with 30 mL of the prepared bead suspension and 5 mL of cell suspension, for a total volume of 35 mL per spinner flask, then covered in aluminum foil and put on stir plates kept at room temperature.

[140] After spinner flask seeding with beads and cells, continuous agitation cycle was programmed for the attachment phase (4 hours post seeding). Specifically, continuous agitation was carried out for 60 mins stir at 50 rpm, 10 mins static and left on a continuous agitation cycle for 4 hours.

[141] After 4 hour attachment phase, volume was topped off to a total volume of 100 mL with complete growth medium, and stir plates were programed for intermittent agitation of the Spinner flasks where intermittent agitation was carried out for 30 mins stir at 50 rpm, 4 hours static and the spinner flasks were left on intermittent agitation program for 15 days.

[142] f. Cell & Bead seeding in 6-well plates

[143] Once cell and bead concentrations were calculated, the appropriate volume of cell suspension was prepared at a concentration of 4x105 cell/mL. Of the cell suspension, 0.5 mL was added to each well of the 6-well plates.

[144] 0.5 mL of the appropriate bead preparations were then added to the corresponding wells of all 6-well plates, then T-rocked for 10-20 seconds and placed in the incubator for the 4 hour attachment phase (3T3 at 37°C and 5% CO2, CHSE-214 at 22 °C & no CO2). During the 4 hour attachment phase, plates were agitated every 30 minutes by T-rocking for 2 minutes. Any deviations from these agitation conditions are listed below. After 4 hour attachment phase, wells were topped up with an additional 1 mL of complete growth medium (final volume 2 mL/well).

[145] q. Cell viability stain and counting:

[146] Bead samples were transferred to 2 mL tubes and allowed to settle, then the supernatant was removed from all samples (volume sampled 1-2 mL) leaving 50 pL of the remaining sample. Beads were then washed 1X with PBS, then trypsinized with 200 uL Trypsin-EDTA and left on a shaker (moderate agitation) for 10-20 minutes (cell line dependant) at room temperature.

[147] After trypsinization, 300 pL of the appropriate complete growth medium was added to each sample to quench the trypsinization. Samples were then centrifuged at 2000 rpm for 5 minutes at room temperature. After centrifugation, the supernatant was removed to get a final volume of 50 pL.

[148] 50 uL of the bead suspension was mixed with 50 pL of trypan blue viability dye (1 :1 ratio cell susp. to dye), then 10 pL added to each side of the hemocytometer. Number of live and dead cells were counted, and the level of aggregation remaining after trypsinizing cells was observed. [149] h. Cell & scaffolding staining and microscopy

[150] An aliquot of the bead/cell suspension was transferred to a 1.5 mL microcentrifuge tube. The samples were left for 10-15 minutes for the beads to settle after which the culture media was removed, and the beads were washed 3X with 1 mL of 1 X PBS. After allowing the beads to settle the pellet (a collection of beads and cells) was then fixed with 4% paraformaldehyde for at least 1 h to several days.

[151] Next, the beads were washed 3x with PBS (each wash had a settling time of 10-15 minutes), stained with 1 pg/mL Hoechst (1 :1000 dilution in PBS of 1 mg/mL stock) for 30 minutes in the dark, washed 3x with PBS, stained with 0.1 % Congo red in PBS for 30 minutes, washed 3x with PBS, then after the last wash 900 pL of the supernatant was removed leaving the beads/cell sample in a volume of 100 pL. 50 pL of suspension was added to a slide and covered with a coverslip. Images were taken using the Olympus SZX16 microscope.

[152] L Microscopy with Inverted Microscope

[153] Images were taken on inverted microscope with Fisherbrand C-mount camera connected by HDMI cable to camera and at the other end connected to HDMI-USB video capture adapter. All images were taken with phase contrast. Images of each well at 4X and 10X were taken and at least one video of the plate being agitated to show the spheroid attachment.

[154] j. Protein extraction & protein quantification

[155] A 0.25-1 mL sample was transferred to a 2 mL tube, all samples were then washed 2X with 1X PBS. After the second wash, the supernatant was removed and beads/cells were resuspended in 150-200 pL of ice-cold Tris- EDTA SDS lysis buffer (10 mM Tris HCI, 1 mM EDTA, 0.5% SDS, 1 mM PMSF/DTT, 1 pg/mL DNase), then incubated on ice for 30 minutes.

[156] After incubations, the samples were clarified by centrifugation at 14,000 rpm for 15 minutes at 4°C, then the supernatants transferred to new tubes and stored either on ice for immediate use or at -20°C. Protein quantification was done using Pierce™ BCA Protein Assay Kit, with all standards run in triplicate and samples run in duplicate. BCA working reagent was prepared by mixing 50 parts of Pierce BCA reagent A with 1 part Reagent B (i.e. 50:1 , Reagent A:B). 25 pL of prepared BSA standards and samples were transferred to each of their corresponding wells of the 96 well plate. i. BSA Standards: 2000, 1500, 1000, 750, 500, 250, 125, and 25 pg/mL ii. Cell lysate reference standards at concentrations 1x106, 3.16x105, 1x105, 3.16x104, and 1x104 cell/mL.

Hi. Experimental bead/cell samples [157] After all standards and samples were added to the 96 well plate, 200 pL of prepared working reagent was added to each well, then the plate was covered with a plate sealer, wrapped in aluminum foil then incubated at 37°C for 30 minutes.

[158] After the 30 minute incubation, the absorbance was measured at 562 nm. The resulting absorbances were blank subtracted and were used to generate a standard curve with the BSA standards which was in turn used to determine the protein concentration of all samples.

[159] V Performance Comparison of dry versus wet plant microcarriers in Spinner flasks

[160] a. Lyophilization of Cellulose

[161] 1. Objective

[162] The objective of this experiment was to determine if lyophilization of cellulose has an effect on cell attachment and proliferation in comparison to wet/non-lyophilized cellulose material.

[163] 2. Treatments

[164] In this experiment, 2 x 125 mL Spinner flasks (SF) were seeded with 4.8x107 of CHSE-214 cells and 3x106 beads per SF, with a cell to bead ratio of 16:1. Lyophilized cellulose was resuspended in PBS to make a 1 % solution in one SF and 1 % wet mercerized cellulose in a second SF. The spinner flasks were sampled at days 0, 1 , 4, 8, 11 , and 15, at each time point samples taken for the following analytical methods: i. Staining and microscopy ii. Total protein quantification via BCA assay.

[165] b. Miniaturized model for Cell attachment to plant microcarriers

[166] 1 . Objective

[167] The objective was to determine if a 6-well plate format is an appropriate scale down model for a short term cell attachment and proliferation on the scaffold beads I microcarriers for increasing experimental throughput.

[168] 2. Treatments

[169] Anti-adherence 6-well plates, one seeded with 3T3 GFP cells, the other with CHSE-214 cells, both seeded at 2x105 cells/well and 0.006 g/well of each of the following microcarrier/bead types: i. Lyophilized cellulose ii. Cytiva Cytodex-1 microcarriers iii. 1% mercerized cellulose.

[170] Plates were sampled at 4 hours and 24 hours post seeding, at each time point samples taken for the following analytical methods: i. Cell count and viability staining ii. Staining and microscopy iii. Protein quantification, BCA assay.

[171] c. Testing automated agitation with plate reader for cell Attachment phase

[172] 1. Objective

[173] The objective was to test if automated agitation using a spectrophotometer plate reader, under no CO2 conditions affects cell attachment to plant microcarriers.

[174] 2. Treatments

[175] 2X siliconized 6-well plates were each seeded at 2x105 cells/well of 3T3 GFP cells with 0.006 g/well of each of the following microcarrier/bead types: i. Cytiva Cytodex-1 microcarriers ii. 1% mercerized cellulose

[176] One of the 6-well plates was agitated using an automated program on a spectrophotometer plate reader, where the plate was incubated at 37°C, with no CO2 and the complete growth media supplemented with 25 mM HEPES buffer. The second 6-well plate was agitated manually as described in the previous section, under regular growth conditions for 3T3 cells.

[177] Microscopy was done at 4, 24 & 48 hours for all plates, and samples were taken at 24 hours and 48 hours post seeding, and samples taken for the following analytical methods: i. Cell count/viability staining at 24 hours ii. Protein quantification, BCA assay at 24 and 48 hours.

[178] d. Cell attachment phase testing of agitation conditions

[179] 1. Objective [180] The objective was to test various agitation conditions during the attachment phase to maximize cell adherence to plant microcarriers and do preliminary testing on modified (succinylated) cellulose beads.

[181] 2. Treatments

[182] For this experiment, 4X siliconized 6-well plates each seeded at 2x105 cells/well of 3T3 GFP cells with 0.006 g/well of each of the following microcarrier/bead types: i. Cytiva Cytodex-1 microcarrier ii. 1 % mercerized cellulose

Hi. Succinylated cellulose beads

[183] During the 4 hour attachment phase, one of each of the 6-well plates underwent one of each of the following agitation conditions: i. 30 minutes static, 2 minutes manual agitation for 4 hours ii. 1 hour static, 2 minutes manual agitation for 4 hours

Hi. no agitation. iv. plate centrifuged at 200 x g for 5 mins after seeding and before attachment phase. After centrifugation the plate was left untouched for the attachment phase (no agitation).

[184] Microscopy was done at 4, 24 & 48 hours for all plates, and samples were taken at 48 hours post seeding for the following analytical methods: i. Protein quantification, BCA assay.

[185] e. Discussion

[186] 1. Performance Comparison of Dry versus Wet plant microcarriers in Spinner flasks

[187] The objective of this experiment was to determine if lyophilization (dried) of plant-based cellulose has an effect on cell attachment and proliferation in comparison to mercerized (wet) cellulose material.

[188] In the samples obtained on days 11 and 15, cell aggregates were observed in both the wet and dried bead samples. Spheroid cell aggregates ranging in size were first observed in both wet and dry samples that were taken at day 4. These aggregates were also observed at day 11 and 15 only in the wet beads samples.

[189] As seen in Figure 14, some of these aggregates are free floating (see Figure 14 A), but some appear to be attached or closely associated to the beads (see Figure 14 B-E). Figure 14 shows images taken at 10X magnification, scale bar = 200 um. Images A) & B) show 4 day samples of dried and wet beads respectively. Images C), D), & E) show the wet bead samples, where C) & D) show 11 day samples, and E) shows 15 day sample.

[190] Figure 15 shows CHSE-214 cell aggregates/spheroids sampled on day 18 from 125 mL SF cultured with wet and dry plant microcarriers, stained with 0.4% trypan blue viability dye, imaged at 10X magnification, scale bar = 200 um. Figure 15 A) & B) shows cell aggregates/spheroids from SF with dry plant microcarriers. Figure 15 C) & D) shows cell aggregates/spheroids from SF with wet plant microcarriers.

[191] Additional samples were taken at day 18 and stained with trypan blue showed many spheroid/cell aggregates of viable cells in both the wet and dry bead samples. As seen in Figure 15, some of the spheroid structures are again free floating but appear to have formed a complex with fragments of cellulose (Figure 15, A & B), while other aggregates appear to be attached to the beads (Figure 15 C & D). In conclusion, the presence of spheroids/cell aggregates found in samples taken at later time points, suggest that rather than forming a monolayer along the surface of the beads they prefer to aggregate and eventually form spheroid structures/large cell aggregates.

[192] Furthermore, samples taken on day 18, showed not only more of the cell aggregates/spheroids in both the dry and wet bead samples, but also some of the cell aggregates/spheroids attached to full beads or complexed with bead fragments. This may suggest that there is some level of attachment between the cell aggregates/spheroids and the plant microcarriers at some point during their formation, and some of the cell aggregates/spheroids breaking away from the beads once they reach a certain diameter or mass, taking fragments of the beads with them as they break off.

[193] 2. Miniaturized model for observing cellular attachment to plant microcarriers

[194] In order to test more conditions for optimizing cell attachment and proliferation, scaling down to a higher throughput model was needed. Therefore a 6-well plate format was tested as a possible model for looking at short term cell attachment and proliferation on plant microcarriers.

[195] At 24 hours, with both 3T3 and CHSE-214 cell types (as can be seen in Figure 16) cell aggregate/spheroid formation was seen in both dry and wet bead conditions. However no aggregates/spheroids were observed in wells where cells were cultured with cytodex-1 beads. Figure 16 show inverted microscope images taken at 10X magnification of CHSE-214 cells (see Figure 16 A, B, & C) and 3T3 GFP cells (Figure 16 D, E, & F) seeded with various beads/microcarriers, taken at 24 hours post seeding. Figure 16 A) & D) shows cells seeded with wet beads, Figure 16 B) & E) shows dry beads, and Figure 16 C) & F) show Cytodex-1 beads.

[196] Interestingly, strong adhesion of some spheroids to wet beads was seen in Figure 17 C & D. Approximately 20% of the cell aggregates/spheroids observed in wet beads conditions appeared attached to the beads, therefore indicating some level of cell attachment to the plant microcarriers that can be further optimized. Additionally, microscopy showed that after 48 hours cells attached to Cytodex-1 beads appeared to have poor viability as can be seen in Figure 17 D, where the cells stained blue from the trypan blue viability dye.

[197] Figure 17 shows 3T3 GFP cell aggregates/spheroids stained with 0.4% trypan blue viability dye, imaged at 8X magnification. Figure 17 A) & D) show 3T3 cells with Cytodex-1 beads. Figure 17 B) show 3T3 cells with dried beads, Figure 17 C) & E) show 3T3 cells with wet beads. It is pertinent to note that samples A), B), and C) were taken at 24 hours post attachment phase, and samples D) and E) were taken at 48 hours post attachment. The dry and wet beads also stained blue.

[198] Interpolated protein concentrations show that wet bead conditions have a higher level of proliferation over 48 hours than the dry bead conditions, and additionally appears to have the greatest protein content at day 0, 4 hours post seeding (as can be seen in Figure 18). This suggests that cells seeded in wet bead conditions have a higher level of early proliferation than cells seeded with dry and Cytodex-1 beads.

[199] In cytodex-1 samples, over 2.5-fold increase in total protein content was seen indicating proliferation after 24 hours. However after 48 hours the total protein content begins to drop indicating that cell death begins to occur. Wet beads additionally appear to have the greatest protein content at 48 hours and observe consistent cell growth over the 48 hour time course which is not seen on Cytodex-1 beads.

[200] Figure 18 provides interpolated total protein content of 3T3 cell microcarrier samples from BCA assay comparing the total protein concentration among dry beads, wet beads, and commercial Cytodex-1 at days 0, 1 , and 2 of cell culture. There were average of 2 replicates per sample, blanks were subtracted and interpolated using kit BSA standards 4PL curve fitted.

[201 ] Figure 19 [A, B] shows 3T3 GFP cells that were seeded with microcarriers/beads then stained with T rypan Blue viability dye, then the number of live vs dead cells were counted.

[202] At 24 hours, wet bead samples were found to have live cells and higher % viability indicating overall cell growth and health on plant microcarriers (Figure 19)

[203] In conclusion, microscopy shows evidence of early cell attachment to the wet beads after 4 hour attachment phase, and at later time points cell aggregation/spheroid formation can be seen with aggregates both free floating and attached to the wet beads. This aggregation/spheroid formation is specific to plant microcarriers, as no spheroids or cell aggregates can be seen in the cytodex-1 samples

[204] From microscopy observations, estimated attachment of cell aggregates/spheroids to wet beads was approximately 20%. Further evidence of early 3T3 cell attachment and proliferation to wet beads at 4 hour time points was found through protein analysis of cell/bead samples.

[205] In cytodex-1 samples, good initial cell attachment and proliferation was observed with over 2.5-fold increase in total protein content after 24 hours. However after 48 hours any additional proliferation was not observed and in contrast a significant decrease in cell viability was seen, indicating that the cells on the cytodex- 1 beads began to die after 48 hours under these conditions. In comparison, wet bead samples showed a steady increase in total protein content overthe 48 hours, starting with a higher amount of initial protein content, indicating better initial cell viability and a steady amount of cell proliferation. Additionally, increase in cell viability and number of live cells counted at 24 and 48 hours was also observed.

[206] 3. Cell attachment phase testing of agitation conditions

[207] After optimizing a high throughput scale down model, various agitation conditions during the attachment phase were tested to maximize cell adherence to plant microcarriers. In addition to testing agitation conditions, the inventors also began preliminary testing on modified (succinylated) cellulose beads.

[208] At 24 hours post seeding, similar amount of cell aggregation/spheroids in suspension and attached to plant microcarriers under all agitation conditions were observed (as seen in Figure 20[A-H]). Interestingly, in all other conditions but the 30 minute agitation condition, a small population of beads was seen that had single cell adherence and monolayer formation to plant microcarriers (Figure 20). Figure 20 shows images of 3T3 GFP cells incubated with plant microcarriers after 24 hours (in Figure 20 A-D) and 48 hours (in Figure 20 E-H) post attachment phase taken at 10X magnification. Agitation conditions during attachment phase were 30 minute static (in figure 20 A & E), 1 hour static (in Figure 20 B & F), no agitation (in Figure 20 C & G), and centrifuged (in Figure 20 D & H).

[209] Between the 1 hour, centrifuged and no agitation conditions, no significant difference was seen in the amount of plant microcarriers that had this type of cell morphology. Some beads were seen to be densely populated with cells in a monolayer surrounding the bead (in Figure 20 H). This type of cell morphology is the same as is seen on commercial microcarriers such as Cytodex-1 .

[210] Figure 21 shows images taken at 10X (i.e. Figure 21 A-D) and 20X (i.e. Figure 21 E-F) magnification of 3T3 GFP cells 48 hours post attachment phase. Images of 3T3 cells seeded with plant microcarriers are shown in Figure 21 A, C & E, and with succinylated cellulose beads are shown in Figure 21 B, D, & F taken from 30 minutes agitation plate in Figure 21 A & B and from no agitation plate in Figure 21 C, D, E, & F.

[211] As seen in Figure 21 , succinylated beads appear to have a better affinity for 3T3 cell attachment under all agitation conditions tested during the attachment phase in comparison to the plant microcarriers at both 24 and 48 hours, where 40-50% of the cell aggregates/spheroids appeared to be attached to the succinylated beads for all the conditions. The succinylated beads were observed to have cells fully adhered to the beads, indicated by the cell morphology shown in Figure 21 E & F. Cells with this morphology indicate the presence of focal adhesion points attaching to the extracellular surfaces.

[212] While the agitation condition appeared to have no effect on the cell attachment efficiency to the succinylated beads, for plant microcarriers the conditions with longer static periods appeared to increase the amount of cell monolayer formation on the beads (as can be seen in Figure 21). [213] Figure 22 provide BCA assay interpolated total protein content of 3T3 cell bead samples, 48 hours post attachment phase after various agitation conditions. Average of 2 replicates per sample were taken with blank subtracted and interpolated using kit BSA standards 4PL curve fitted.

[214] All samples and standards %CV were <10%. The standard curve R² was >98%. The 1x106 cell/mL 3T3 cell reference standard total protein was 250 ug/mL. Interpolated protein concentrations show that wet beads samples have the greatest protein content in the no agitation condition, followed closely by the 1 hour condition. Interestingly this was a large increase in comparison to the 30 minute agitation condition that has been used in all previous experiments in 6-well format. In both the 1 hour and no agitation conditions the total protein content for the plant microcarriers is comparable to the commercial Cytodex-1 beads. Interestingly, the total protein content observed in the succinylated beads remained fairly constant across the agitation conditions. Based on the microscopy observations, it appeared that the increased cell attachment efficiency seen in the succinylated beads does not directly correlate to the total protein content.

[215] Protein analysis and microscopy showed that with 1 hour static period and no agitation conditions during the attachment phase, an increase in overall cell attachment and cell proliferation was observed in comparison to the 30 minute static condition. Microscopic images showed that conditions with less agitation showed more plant microcarriers with cell monolayer formation.

[216] Furthermore, succinylated beads were tested and microscopy showed they had a greater affinity for cell attachment than the plant microcarriers. This was seen for both cell aggregate/spheroid attachment and monolayer formation along the beads. However this was not seen in the total protein results, as the plant microcarriers had higher protein levels than the succinylated beads for all agitation conditions.

[217] These experiments and data obtained by the inventors prove that plant-based cellulose scaffold beads/particles e.g. beads constituted from apple cellulose are able to retain attached cells in bioreactor conditions as surface area for cell proliferation. Plant based cellulose beads are a promising tool for cellular growth for cellular in-vitro meat cell-production, cultured meat cell-production, agriculture, tissue engineering, 3D tissue culturing, proliferation of cell-banks, stem cells, in-vitro drug testing, protein harvesting, virus and vaccine production, biomolecule amplification, plasmid amplification, production of metabolites, production of cosmetic actives, production of biopharmaceuticals, production of recombinant proteins, biological filtration, waste-water treatment, industrial cleaning operations, or food production.

[218] Plant based cellulose beads are effective to hold adherent cells and have the potential to increase the surface area of cells grown in bioreactors Cells grown with plant microcarriers have comparable levels of cell proliferation to commercial microcarriers and have also shown evidence of improved cell viability, thus increasing the total yield of viable healthy cells produced with plant microcarriers. In fact, plant microcarriers act as a scaffold for cell attachment and proliferation and further mediate formation of 3D cell aggregates defined as spheroids, which can give an advantage in scale up cell production. [219] The inventors also observed that cellular growth and attachment could vary depending on the size of the particles, and therefore, in an embodiment of the invention, microparticles or beads of a variety of sizes, e.g. a combination of small and large sized particles could be used. The surface of area of the particles could be varied, as this is an important parameter as the microcarriers increase the adherent area in bioreactors. Various other parameters could be optimized, for instance the times and rotation of intermittent spinning to increase cellular production. Optimization of cell culture and agitation conditions can also modify cell morphology from 3D spheroid structures to formation of cell monolayers along the surface of plant microcarriers.

[220] The inventors also observed that there were clusters of cells on the beads during proliferation, which suggested that the cells were proliferating on the beads. The protein assay also demonstrated that the cells were proliferating on the beads.

[221] Accordingly, the microcarrier beads/particles are capable of mimicking the function of the beads for cell attachment and proliferation in bioreactors. In fact, the inventive beads are better especially for commercial tissue engineering and food applications as commercially available beads need to be dissolved or washed out, while the plant-based beads would be utilized not only as a scaffold for cell attachment and proliferation but also to improve the textural properties of the final product.

[222] VI. Assessing HEK 293 Cell Attachment to Microcarriers

[223] This experiment was aimed at testing the attachment efficiency of HEK 293 cells to plant-derived cellulose- based microcarriers both with and without succinylation modification. Microscopy images taken after 24 hours and up to 5 days post seeding showed low levels of cell adhesion to the microcarriers, with better cell attachment efficiency observed with the succinylated cellulose material in comparison to the mercerized cellulose material. Additionally, less cell aggregation and spheroid formation was observed in both conditions compared to previously tested cell lines. However, no changes in cell morphology indicating late stage cell to extracellular matrix (ECM) adhesion were observed with either of the microcarrier types.

[224] a. Context

[225] Due to the biocompatible nature of the mercerized cellulosic material, additional studies looking into its application and use as a microcarrier for cell culturing purposes were conducted. To test the attachment and proliferative capacity of additional cell lines to the plant-derived cellulose-based microcarriers, HEK 293 cells were selected as a commonly used cell line that has many applications within research and the biotechnology and pharmaceutical industry. HEK 293 cells were of particular interest as this cell line is frequently used for production of vaccines, viral vectors, and recombinant proteins, and therefore is very commonly used with commercial microcarriers in bioreactors.

[226] b. Methods

[227] Materials & Equipment Table I. List of Materials

.. . . . e i- Catalog .. .

Material Supplier .. . Notes

Number

Sterile, non cleanroom, Microcarriers lot

Mercerized Apples Prepared in-house N/AMER-15 prepared on 2023-09-

15 (8.91% mass density)

Non-sterile lot prepared

Succinylated Mercerized Apples Prepared in-house N/ASUC-24 on 2024-06-21 (4.2% mass density)

Phosphate Buffered Saline (PBS) 1X Intermountain (Cytiva _pR„ Lot: 21802221 , without Ca & Mg Hyclone) bbb-Hbb-ixo exp: 2024-02-18

DMEM with L-Glutamine, 4.5g/L Corning MT-10-013-CV Maintained sterile

Glucose and Sodium Pyruvate

Fetal Bovine Serum Thermo Scientific J66993.03 Lot: X251071

Containing penicillin,

Antibiotic-Antimycotic (100X) Gibco 15240062 streptomycin and amphotericin B

Table II. List of Equipment

Equipment Supplier Catalog Number Notes

Biosafety Cabinet Scilogex HB120-S N/A

Inverted microscope BioTek Synergy Mx N/A

Autoclave Steris AMSCO 250LS SN: 032271914

[228] c. Protocol i. 6-well Plate Siliconization:

Dow SYLGARD™ 184 Silicone Elastomer kit was used to siliconize TC-treated 6-well plates. As per the kit instructions, Reagent A was mixed with Reagent B in a 1 :10 ratio (A:B), then mixed slowly by inversion for a minimum of 30 minutes. 2 mL of the mixture was added per well of a 6-well plate, then left to cure for 24 hours at room temperature. The plates were then sterilized in a BSC by submerging in 70% isopropyl alcohol, followed by a 60-minute UV sterilization cycle. ii. Preparation of Mercerized Material

A stock solution of mercerized cellulose material was diluted to 1 % mass density using 1X PBS, from which 1 g of material was transferred to a new tube and further diluted with 25 mL of 1X PBS. Succinylated cellulose material, at a mass density of 4.2%, was diluted with 42 mL of 1X PBS. Then both preparations were autoclaved with a sterilization cycle at 121 °C for 25 minutes. After autoclaving, both preparations were sterilly washed 2X with 1X PBS by first allowing the material to settle for 15 minutes and then carefully removing the supernatant. Then 25 mL of fresh PBS was added. This was repeated an additional time for 2 washes. After the second wash, the supernatant was replaced with 30 mL of complete growth medium (DMEM, 1 % antimycotic & antibiotic) then left to incubate at 4°C overnight. After the 24 hour incubation, each microcarrier preparation was further diluted with complete growth medium to a final concentration of 0.03% mass density.

Hi. Cell and Microcarrier Seeding

HEK 293 cells were trypsinized and counted using a Countess automated cell counter. A working stock of cell suspension was prepared and diluted to a final concentration of 1.0 x10⁵ cells/mL using complete growth media. The microcarrier preparations were then seeded in the wells of a 6-well plate in duplicate from the stock solution prepared at 0.03% mass density, at an inoculation volume of 1 .0 mL per well. The cells from the working stock prepared at 1.0 x10⁵ cells/mL were then inoculated at a volume of 0.5 mL in each well of the 6-well plate dropwise, therefore resulting in 5.0 x10⁴ cells added per well. After seeding the microcarriers and the cells, the 6-well plate was placed in a 37°C, 5% CO2 incubator for a 4 hour attachment phase. After the 4 hour attachment phase, all wells were topped up to a total volume of 2 mL using fresh complete growth media, and the samples were then placed back at 37°C, 5% CO2 incubator for up to 5 days. iv. Microscopy Imaging and Staining

An inverted Fisherbrand microscope was used for phase contrast imaging at all timepoints using a OMAX A35180U3 18 MP USB3.0 camera (part number TP11800A) to record images with ToupView software at 4912x3684 resolution, format RGB24, using auto white balance, at 10X magnification. The plates were imaged with the inverted microscope at 24, 48, and 5 days post seeding. [229] d. Results

[230] Figure 23 shows HEK 293 cells seeded with succinylated and mercerized material in a 6-well plate. Scale bar = 250 pm.

[231] Microscopy images taken at 24 hours post seeding showed HEK 293 cells attached to both the regular mercerized material and the succinylated material. While the attachment efficiency for both microcarrier types remained low, a slight increase in percent cell attachment efficiency was observed with the succinylated material (approximately 30-50%) in comparison to the mercerized control (approximately 20-40%). Additionally, after 24 hours and up to 5 days, some cell aggregation was observed in the presence of both microcarriers. However, the level of aggregation appeared to be relatively low in comparison to other cell lines observed in previous experiments, namely 3T3-GFP and C2C12 cell lines. No notable difference in the level of cell aggregation was observed between the succinylated and mercerized material, with the majority of the cells forming small/loosely attached and irregularly shaped aggregates instead of the compact and large cell aggregates/spheroids seen in other cell lines, for example as seen with 3T3-GFP cells. Furthermore, no increase in cell aggregate size was observed after 5 days in culture with either of the microcarrier types.

[232] After 5 days in culture, little to no high quality cell adhesion was observed in either microcarrier condition. High quality adhesion refers to the cells beginning late stage or Phase 3 cell adhesion by creating focal adhesions to the extracellular matrix and reorganization of the cytoskeleton. This late phase cell adhesion is indicated by observations of flattened and spread/branching cell morphology along the surface of the culture vessel/scaffold, creating a typical monolayer. Cells both adhered and un-attached to the both types of microcarrier were observed as either aggregated clusters or rounded single cells, with no observations indicating late phase cell adhesion.

[233] Notably, after 5 days post seeding, early signs of cell death were observed, indicating poor cell health under these experimental parameters. Observations included typical hallmarks of cell death and apoptosis such as membrane blebbing, formation of apoptotic bodies and nuclear condensation. This may be due to the extended time course of the experiment without additional media supplementation and splitting of the cells.

[234] e. Discussion

[235] It is important to note that the results found in this study are based solely on microscopy observations; thus, they are only qualitative and not quantitative. In future studies, using a quantitative method for cell attachment efficiency will give more insight on how various cell lines adhere to the plant-derived cellulose-based microcarriers and the effect that additional modifications to the microcarriers has on cell adhesion.

[236] There are two main types of cell adhesion; cell-cell adhesion mediated by cadherin receptor domains, and cell-ECM adhesion largely mediated by binding of cell integrin receptors to various motifs in the ECM. The interplay between these two types of adhesion can greatly affect the cell’s localization, structure and functionality in vivo and in vitro, and therefore the internal “cross-talk” between these two types of cell adhesion is highly regulated. Cell adhesion to the ECM can be defined by three phases. Briefly, Phase I involves cell sedimentation and initial cell attachment mediated by electrostatic interactions. Phase II is mediated by the interactions between integrin receptors and ECM-associated ligands, resulting in flattening and the beginning of the cell body spreading. Lastly, the cell spreading and flattening continues in Phase III, as structural reorganization of the cytoskeleton begins and focal adhesions are formed with the ECM resulting in stronger, stable cell to ECM adhesion. The cell morphology observed in this experiment is indicative of early phase cell adhesion, indicating that additional treatments or modifications to the plant derived cellulose based microcarriers may be required for stable cell adhesion to occur. Additionally, it is notable that HEK 293 cells are known to take longer to adhere to culture vessels, and once adhered do not create very strong anchorage to the surface of vessels, thus can detach quite easily. The late observations of poor cell health and early signs of cell death may be in part attributed to this.

[237] VII. Assessing Vero Cell Attachment to Microcarriers

[238] This experiment was designed to test the attachment efficiency of Vero cells to plant-derived cellulose- based microcarriers both with and without succinylation modification. Microscopy after 24 hours and up to 96 hours showed an observable increase in cell attachment to succinylated microcarriers, with some evidence of late stage cell adhesion, all of which was not observed in the mercerized cellulose material. While some cell attachment was observed in the mercerized cellulose material, the overall percentage of cell adhesion was considerably less than was seen in the succinylated material.

[239] a. Context

[240] Due to the biocompatible nature of the mercerized cellulosic material, additional studies looking into its application and use as a microcarrier for cell culturing purposes were conducted. To test the attachment and proliferative capacity of more cell lines to the plant-derived cellulose-based microcarriers, Vero cells were selected as a commonly used cell line that has many applications within research and the biotechnology industry. Vero cells were of particular interest as this cell line is considered standard for vaccine/viral vector production, and therefore are very commonly used with commercial microcarriers in bioreactors.

[241] b. Methods

[242] Materials & Equipment

Table III. List of Materials

. M.at .er .ia .l S ©upplier . Catalog .. .

N.um .ber Notes

Sterile, non clean-

Mercerized Apples Prepared in-house N/AMER-15 room, Microcarriers lot prepared on 2023-09- 15 (8.91% mass density)

Non-sterile lot prepared

DMEM with L-Glutamine, 4.5g/L Corning MT-10-013-CV Maintained sterile

Glucose and Sodium Pyruvate

Fetal Bovine Serum Thermo Scientific J66993.03 Lot: X251071

Containing penicillin,

Antibiotic-Antimycotic (100X) Gibco 15240062 streptomycin and amphotericin B

Table IV. List of Equipment

Equipment Supplier Catalog Number Notes

Biosafety Cabinet Scilogex HB120-S N/A

Inverted microscope BioTek Synergy Mx N/A

Autoclave Steris AMSCO 250LS SN: 032271914

[243] c. Protocol i. 6-well plate Siliconization:

Dow SYLGARD™ 184 Silicone Elastomer kit was used to siliconize TC-treated 6-well plates. As per the kit instructions, Reagent A was mixed with Reagent B in a 1 :10 ratio (A:B), then mixed slowly by inversion for a minimum of 30 minutes. 2 mL of the mixture was added per well of a 6-well plate, then left to cure for 24 hours at room temperature. The plates were then sterilized in a BSC by submerging them in 70% isopropyl alcohol, followed by a 60-minute UV sterilization cycle. ii. Preparation of Mercerized Material

A stock solution of mercerized cellulose material was diluted to 1 % mass density using 1X PBS, from which 1 g of material was transferred to a new tube and further diluted with 25 mL of 1X PBS. Succinylated cellulose material, at a mass density of 4.2%, was diluted with 42 mL of 1X PBS. Both preparations were autoclaved with a sterilization cycle at 121 °C for 25 minutes. After autoclaving, both preparations were sterilly washed twice with 1X PBS by first allowing the material to settle for 15 minutes, carefully removing the supernatant, and then adding 25 mL of fresh PBS. This was repeated an additional time for a total of two washes. After the second wash, the supernatant was replaced with 30 mL of complete growth medium (DMEM, 10% FBS, 1 % antimycotic & antibiotic) and was then left to incubate at 4°C overnight. After the 24 hour incubation, each microcarrier preparation was further diluted with complete growth medium to a final concentration of 0.03 % mass density.

Hi. Cell and microcarrier seeding and media change

Vero cells were trypsinized and counted using a Countess automated cell counter. A working stock of cell suspension was prepared and diluted to a final concentration of 7.70 x10⁴ cells/mL using complete growth media. The microcarrier preparations were then seeded in the wells of a 6-well plate from the stock solution prepared at 0.03% mass density, at an inoculation volume of 0.75 mL per well. The cells from the working stock prepared at 7.70 x10⁴ cells/mL were then inoculated at a volume of 0.5 mL and were added to each well of the 6-well plate in a drop-wise fashion, therefore resulting in 3.85 x10⁴ cells added per well. After seeding the microcarriers and the cells, the 6-well plate was placed in a 37°C, 5% CO2 incubator for a 4 hour attachment phase. After the 4 hour attachment phase, all wells were topped up to a total volume of 2 mL using fresh complete growth media, and then the samples were placed back in the 37°C, 5% CO2 incubator for up to 5 days. iv. Microscopy Imaging and Staining

An inverted Fisherbrand microscope was used for phase contrast imaging at all timepoints using a OMAX A35180U3 18 MP USB3.0 camera (part number TP11800A) to record images with ToupView software at 4912x3684 resolution, format RGB24, using auto white balance, at 10X magnification. Plates were imaged with the inverted microscope at 4, 24, 48, 72, 96 hours and 7 days post seeding.

[244] d. Results

[245] Figure 24 shows Vero cells seeded with succinylated and mercerized cellulose material in a 6-well plate. Scale bar = 250 pm. [246] Figure 25 shows Vero cells seeded with succinylated and mercerized cellulose material in a 6-well plate. Single cellulosic particles circled in black, and cells displaying late phase adhesion morphology highlighted with red arrow. Scale bar = 250 pm.

[247] Microscopy images taken after 24 hours post seeding showed Vero cells attached to both the regular mercerized cellulose material and the succinylated cellulose material. While the attachment efficiency for the mercerized cellulose material remained quite low, an observable increase in percent cell attachment efficiency was observed with the succinylated material (approximately 50-70%) in comparison to the mercerized control (approximately 10-30%). Additionally, after 24 hours and up to 96 hours post seeding, very low levels of cell aggregation were observed in the presence of both microcarriers types with no large cell aggregates/spheroid formation observed. No notable difference in level of cell aggregation was observed between the succinylated and mercerized cellulose material, with the majority of the cells forming small/loosely attached aggregates instead of the compact and large aggregates/spheroids seen in other cell lines, for example with 3T3-GFP cells. Furthermore, no increase in aggregate size or the level of aggregation was observed after 4 days in culture with either of the microcarrier types.

[248] After 4 days in culture, Vero cells seeded with the succinylated microcarriers appeared to have more cells fully adhered to individual particles, beginning to show late phase cell adhesion as indicated by the cell morphology. Microscopy observations after 24 hours and up to 96 hours post seeding showed this change in the quality of cell adhesion as a flattening and spreading of the cell body to the microcarrier surface, indicative of high quality stable cell adhesion. This was observably more apparent in succinylated microcarriers in comparison to the mercerized microcarriers which showed little to no signs of late phase cell adhesion or monolayer formation along the surface of the particles, indicated by the rounded single cell morphology and lack of cell flattening and spreading. High quality adhesion refers to the cells in late stage or Phase 3 adhesion which involves creating focal adhesions to the extracellular matrix (ECM) and reorganization of the cytoskeleton. This late phase cell adhesion is indicated by observations of flattened and spread/branching cell morphology along the surface of the culture vessel, creating a typical monolayer. Both early and late phase cell morphology was observed with the succinylated cellulose microcarriers, while with the mercerized microcarriers only observed cell morphology associated with initial cell attachment.

[249] e. Discussion

[250] It is important to note that the results found in this study are based solely on microscopy observations; thus, they are only qualitative and not quantitative. In future studies, using a quantitative method for cell attachment efficiency will give more insight on how different cell lines adhere to and proliferate on the cellulose-based microcarriers.

[251] As explained earlier, cell adhesion to the ECM can be defined by three phases. While the cell morphology observed in with the mercerized microcarriers is indicative of early phase cell adhesion, some late phase cell adhesion was observed with the succinylated cellulose material. This indicates that additional modification, such as increasing the amount of surface charge of the substrate as is the case in the succinylated microcarriers, moderately increases the quality of Vero cell adhesion to the microcarrier surface.

[252] Additionally, it should be noted that the cells used for this experiment were seeded very shortly after thawing, thus may have still been in the lag phase of their growth cycle after thawing. Lag phase refers to the initial phase in a cell growth curve, directly after seeding when initial cell proliferation is stunted as the cells get used to their new environment, after which they will enter into the exponential growth phase. This is often seen in cells after thawing from cryopreservation, as they grow slower than normal while they acclimate and recover from the stress of a freeze-thaw cycle. This post-thawing lag phase can be extended depending on how much the cells were stressed during the freezing and thawing process. Depending on the freezing down process of the cells, this may have had an impact on the cell behavior and proliferative capacity of the cells throughout the duration of this experiment.

[253] VIII. Effect of Glycine on Cell Attachment to Mercerized Material

[254] This experiment was aimed at studying the effect that glycine treatment of plant-derived cellulose-based microcarriers has on cell attachment efficiency. It was found that mercerized material pre-treated with a 1 M glycine solution had no observable effect after 72 hours on both 3T3-GFP and C2C12 cell attachment efficiency and quality in comparison to control cellulose microcarriers treated with media.

[255] a. Context

[256] Due to the biocompatible nature of the mercerized cellulosic material, additional studies looking into its application and use as a microcarrier for cell culturing purposes were conducted. As seen in previous studies, some cell attachment to the cellulose particles is observed; however, the efficiency of this is quite low for what would be expected with commercial microcarriers. The aim of this study was to utilize the slow release properties of the mercerized material with glycine to allow for its sustained release after seeding cells, which was hypothesised to aid in cell attachment. Glycine is one of three amino acids that make up the RGD motif, a well documented adhesion peptide, and one of the main domains found in the extracellular matrix (ECM) responsible for cell attachment. Therefore, glycine is a commonly used coating material for culturing adherent cells.

[257] b. Methods

[258] Materials & Equipment

Table V. List of Materials

. M.at .er .ia . S ©upp her . Catalog .. .

N.umber Notes

Sterile, non clean-

. M.ercer .ized . A . pp i es r P,rep . ■ . M/A nm- d r room, Microcarriers lot

^Kared in-house N/AMER-15 prepared . on 2023- _n0_n9-

15 (8.91 % mass density)

DMEM with L-Glutamine, 4.5g/L Corning MT-10-013-CV Maintained sterile

Glucose and Sodium Pyruvate

Fetal Bovine Serum Thermo Scientific J66993.03 Lot: X251071

Containing penicillin,

Antibiotic-Antimycotic (100X) Gibco 15240062 streptomycin and

Amphotericin B

Glycine, Electrophoresis Grade, MP „ . .... .^Monoonn rr .■ , TM / i \ Fisher Scientific CN808822 _K N_l /_/ A_A

Biomedica s™ (1 kg)

Table VI. List of Equipment

Equipment Supplier Catalog Number Notes

Inverted microscope BioTek Synergy Mx N/A

Benchtop Centrifuge Thermo Scientific Sorvall ST 16R SN: 42530122

37°C Water Bath Fisher Scientific Isotemp GPD 10 Model: FSGPD10

SN: 300266216

[259] c. Protocol i. 6-well plate Siliconization:

Dow SYLGARD™ 184 Silicone Elastomer kit was used to siliconize TC-treated 6-well plates. As per the kit instructions, Reagent A was mixed with Reagent B in a 1 :10 ratio (A:B), then mixed slowly by inversion for a minimum of 30 minutes. 2 mL of the mixture was added per well of a 6-well plate, then left to cure for 24 hours at room temperature. Plates were then sterilized in a BSC by submerging in 70% isopropyl alcohol, followed by a 60-minute UV sterilization cycle. ii. Glycine Coating of Mercerized Material: A stock solution of mercerized cellulose material was diluted to 1 % mass density using 1X PBS, from which 2X 2 g of material was measured and further diluted with 25 mL of 1X PBS. The material was then autoclaved with a sterilization cycle at 121 °C for 25 minutes. After autoclaving, the material was then washed 3X with 25 mL of either sterile distilled water or with 1X PBS for the glycine treatment and control treatment respectively. Between washes, the material was left for 15 minutes undisturbed to settle, after which the supernatant was removed and replaced with fresh solution. Afterthe washes and removal of the supernatant, 35 mL of complete growth media was added to the control material, and it was stored at 4°C for up to 2 weeks. The supernatant of the glycine treatment material was replaced with 35 mL of 0.9% NaCI and was added to the mercerized material and inverted several times to mix then incubated for 2 hours. The saline supernatant was then removed and replaced with 1 M glycine in distilled, deionized water, 0.22 pm filter sterilized, then left at 4°C for 24 hours. After a 24 hour incubation period, the microcarriers were washed 3X with 1X PBS in the same manner as previously described. The material was then stored in fresh 1X PBS at 4°C for up to 96 hours. Prior to seeding glycine treated Microcarriers , the supernatant was removed and replaced with complete growth medium (DMEM, 10% FBS, 1 % antimycotic & antibiotic solution containing penicillin, streptomycin and amphotericin B) and incubated at room temperature for a minimum of 30 minutes.

Hi. Cell and microcarrier seeding

3T3-GFP and C2C12 cells were trypsinized and counted using a Countess automated cell counter. A working stock of cell suspension was prepared at final concentration of 1.0 x10⁵ cells/mL using complete growth media. The microcarriers were then seeded in the wells of a 6-well plate from the stock solution prepared at 0.03% mass density, at an inoculation volume of 0.7 mL per well. Of the 1 .0 x10⁵ cells/mL cells working stock, 0.5 mL was then added to each well of the corresponding 6-well plate drop-wise, therefore seeding 5.0 x10⁴ cells and diluting microcarriers to 0.015% mass density per well. After seeding the microcarriers and cells, the 6-well plates were gently rocked to ensure complete coverage of the bottom of the well, then placed in a 37°C, 5% CO2 incubator for 4 hours. Following the 4 hour attachment phase, the total volume of each well was topped up to 2 mL, and then the samples were placed back in the incubator for up to 72 hours. iv. Microscopy Imaging

An Inverted Fisherbrand microscope was used for phase contrast imaging at all timepoints using a OMAX A35180U3 18 MP USB3.0 camera (part number TP11800A) to record images with ToupView software at 4912x3684 resolution, format RGB24, using auto white balance, with images taken at 10X magnification. Plates were imaged with the inverted microscope at 4, 24, and 72 hours post seeding for both cell lines.

[260] d. Results

[261] Figure 26 shows Microscopy of 3T3-GFP cells on plant derived cellulose based microcarriers. Scale bar = 250 pm. [262] Figure 27 shows Microscopy of C2C12 cells on plant derived cellulose based microcarriers. Scale bar = 250 pm.

[263] Microscopy images taken at 4, 24 and 72 hours post seeding showed no observable change in cell adherence to the cellulose particles after glycine treatment in comparison to the control mercerized cellulose material treated with media. Similar levels of cell adhesion was observed in both the glycine treated and control conditions after the 4 hour attachment phase, that being relatively low (<50%). This was observed in both the 3T3- GFP and C2C12 cell lines. As seen in previous experiments, cell aggregation and spheroid formation was observed in both cell lines and conditions after 24 hours and up to 72 hours post seeding. Greater levels of cell aggregation/spheroid formation with larger average diameters were observed in the 3T3-GFP cells in relation to the C2C12 cells; however, no difference was observed between each treatment condition within each cell line. After 24 hours, a population of cell aggregates and spheroids were seen attached to the cellulosic particles, with similar levels of this observed in both conditions for each cell line.

[264] Additionally, no change in the quality of cell adhesion was seen in either the glycine treated or control wells, as indicated by the rounded single cell morphology and aggregation/spheroid formation. High quality adhesion refers to the cells beginning late stage or Phase 3 cell adhesion by creating focal adhesions to the extracellular matrix and reorganization of the cytoskeleton. This late phase cell adhesion is indicated by observations of flattened and spread/branching cell morphology along the surface of the culture, creating a typical monolayer. Cells both adhered and un-attached to the glycine treated and control microcarriers were observed as either aggregated clusters/spheroids or rounded single cells, with no observations indicating late phase cell adhesion.

[265] e Discussion

[266] It is important to note that the results found in this study are based solely on microscopy observations; thus, they are only qualitative and not quantitative. In future studies, using a quantitative method for cell attachment efficiency will give more insight on the effect glycine pre-treatments and other coatings have on cell adhesion.

[267] As discussed earlier, cell adhesion to the ECM can be defined by three phases. The cell morphology observed in this experiment is indicative of early phase cell adhesion, indicating that additional treatments or modifications to the plant derived cellulose based microcarriers may be required for stable cell adhesion to occur.

[268] IX. Effect of FBS Treated Mercerized Material on Cell Attachment

[269] This experiment presents data on the effect of FBS treatment on cell attachment to plant-derived cellulose- based microcarriers. It was found that mercerized material pre-treated with either 10% or 20% FBS had no observable effect, after 72 hours, on 3T3-GFP and C2C12 cell attachment efficiency in comparison to the PBS treated control microcarriers.

[270] a. Context [271] Due to the biocompatible nature of the mercerized cellulosic material, additional studies looking into its application and use as a microcarrier for cell culturing purposes were conducted. As seen in previous studies, some cell attachment to the cellulose particles is observed; however, the efficiency of this is quite low for what would be expected with commercial microcarriers. The aim of this study was to increase the total protein and growth factors found in FBS, adsorbed to the Microcarriers by leveraging the slow release properties of the cellulose particles documented in previous studies. It was hypothesised that this would increase the protein content within the mercerized cellulose material and aid with cell attachment as well as create a more suitable microenvironment for optimal cell proliferation.

[272] b. Methods

Materials & Equipment

Table VII. List of Materials

.. . . . e i- Catalog .. .

Material Supplier .. . Notes

Number

Sterile, non cleanroom, Microcarriers lot

Mercerized Apples Prepared in-house N/AMER-9 prepared on 2023-04-

28 (8.23% mass density)

Phosphate Buffered Saline (PBS) 1X Intermountain (Cytiva _R„„ _pR„ _{i yfi} Lot: 21802221 , without Ca & Mg Hyclone) bbb-Hbb-lXb exp: 2024-02-18

DMEM with L-Glutamine, 4.5g/L Corning MT-10-013-CV Maintained sterile

Glucose and Sodium Pyruvate

Fetal Bovine Serum Thermo Scientific J66993.03 Lot: X251071

Containing Penicillin,

Antibiotic-Antimycotic (100X) Gibco 15240062 Streptomycin and

Amphotericin B

Table VIII. List of Equipment

Equipment Supplier Catalog Number Notes

Inverted microscope BioTek Synergy Mx N/A CO₂ Incubator (37°C, 5% CO₂) Thermo Scientific HERAcell vios 160i SN: 42457396

Benchtop Centrifuge Thermo Scientific Sorvall ST 16R SN: 42530122

37°C Water Bath Fisher Scientific Isotemp GPD 10 Model: FSGPD10

SN: 300266216

[273] c. Protocol i. 6-well plate Siliconization:

Dow SYLGARD™ 184 Silicone Elastomer kit was used to siliconize TC-treated 6-well plates. As per the kit instructions, Reagent A was mixed with Reagent B in a 1 :10 ratio (A:B), then mixed slowly by inversion for a minimum of 30 minutes. 2 mL of the mixture was added per well of a 6-well plate, then left to cure for 24 hours at room temperature. Plates were then sterilized in a BSC by submerging in 70% Isopropyl alcohol, followed by a 60 minute UV sterilization cycle. ii. FBS Treatment of Mercerized Material:

A stock solution of mercerized cellulose material was diluted to 1 % mass density using 1X PBS, from which 1 g of material was further diluted with 25 mL of 1X PBS. The material was then autoclaved with a sterilization cycle at 121 °C for 25 minutes. After autoclaving, the material was washed twice with 1X PBS by first allowing the material to settle for 15 minutes and then carefully removing the supernatant. 25 mL of fresh PBS was then added, and the washing process was repeated an additional time for a total of two washes. After the second wash, the material was divided equally between two tubes (0.5 g of 1 % mass density). Then, after settling, the supernatant was removed leaving the microcarrier fraction in a 5 mL volume. 15 mL of complete growth medium (DMEM, 1 % antimycotic & antibiotic solution containing penicillin, streptomycin and amphotericin B) containing either 10% or 20% FBS was added to the corresponding tubes for a total volume of 20 mL then left to incubate at 4°C overnight (Note: the concentration of FBS is calculated prior to addition to the microcarrier fraction). After the 24 hour incubation, the microcarriers was further diluted with the appropriate media and FBS concentration to a final concentration of 0.012% (after dividing the material in two tubes and then resuspending in 20 mL total volume, the mass density was diluted to 0.025% mass density). iii. Cell and microcarrier seeding:

3T3-GFP and C2C12 cells were trypsinized and counted using a Countess 3 automated cell counter. A working stock of cell suspension was prepared and diluted to a final concentration of 1 .0 x10⁵ cells/mL using complete growth media. The Microcarriers was then seeded in the wells of a 6-well plate from the stock solution prepared at 0.012%, at an inoculation volume of 0.7 mL per well. 0.5 mL of the 1.0 x10⁵ cells/mL prepared cell suspension was then added to each well of the corresponding 6-well plate drop-wise, therefore resulting in a final cell concentration of 5.0 x10⁴ cells/well, and a dilution of Microcarriers to 0.008% mass density per well. After seeding the Microcarriers and the cells, the 6-well plates were gently rocked to ensure complete coverage of the bottom of the well, then placed in a 37C, 5% CO2 incubator for 4 hours. Following the 4 hour attachment phase, the total volume of each well was topped up to 2 mL, and then the samples were placed back in the incubator for up to 72 hours.

[274] iv. Microscopy Imaging:

[275] An Inverted Fisherbrand microscope was used for phase contrast imaging at all timepoints using a OMAX A35180U3 18 MP USB3.0 camera (part number TP11800A) to record images with ToupView software at 4912x3684 resolution, format RGB24, using auto white balance, with images taken at 10X magnification. Plates were imaged at 24 and 48 hours post seeding for both cell lines, and additionally at 72 hours for 3T3-GFP cells.

[276] d. Results

[277] Figure 28 shows microscopy of 3T3-GFP cells on plant derived cellulose based microcarriers. Scale bar = 250 pm.

[278] Figure 29 shows microscopy of C2C12 cells on plant derived cellulose based microcarriers. Scale bar = 250 pm.

[279] Microscopy images taken up to 72 hours post 3T3-GFP cell seeding showed no observable qualitative difference between either concentrations of the FBS treated mercerized cellulose material and the PBS control in terms of cell attachment efficiency, that being the approximate percentage of cells attached to the microcarriers. Relatively low levels of cell attachment were observed in both the 10% and 20% FBS treatment conditions as well as the PBS control, that being the number of cells adhered to each particle and the number of particles with cells attached. In addition, similar levels of cell aggregation and spheroid formation were observed in all conditions, with comparable amounts of cell aggregate/spheroid attachment to cellulosic particles of each condition.

[280] The microscopy results showed that the C2C12 cells behaved similarly to the 3T3-GFP cells, with all treatments displaying no observable difference between conditions. Images of C2C12 cells were taken up to 48 hours post seeding and showed a similar degree of cell attachment between both FBS treated conditions and the PBS control. Notably after 48 hours, a large population of cells adhered to the silicon well rather than the cellulosic particles was observed, as seen in all C2C12 conditions. Interestingly, while the C2C12 cells did appear to aggregate and form some large aggregate/spheroid structures after 24 hours, it was observed that fewer aggregates/spheroids were present with these cells after 48 hours in comparison to the 3T3-GFP cells. Additionally, the aggregates formed in the C2C12 cells were more often seen as irregularly shaped aggregates and most often smaller than observed in the 3T3-GFP cells.

[281] e. Discussion [282] It is important to note that the results found in this study are based solely on microscopy observations, thus are only qualitative and not quantitative. In future studies, using a quantitative method for cell attachment efficiency will give more insight on the effect FBS pre-treatment with plant-derived cellulose-based microcarriers has on cell adhesion. This was also the first study to look at C2C12 cell attachment and behavior in low-adhesion conditions with plant-derived cellulose-based microcarriers, and unsurprisingly C2C12 began forming cell aggregates/spheroid structures after 24 hours post-seeding. However, the C2C12 cells appeared to aggregate less than has been observed with the 3T3-GFP cells, and they appeared to form looser and more irregularly shaped aggregates. This suggests that the C2C12 cells form cell to cell attachments less readily than the 3T3- GFP cells, and may be an interesting cell line to explore cell adhesion to the plant-derived cellulose-based microcarriers. Another important consideration is the difference in cell types used in this experiment. 3T3-GFP cells are a fibroblast cell type derived from a mouse embryo, whereas C2C12 cells are mouse muscle myoblast cells. These two cell types display very different phenotypes in vitro and in vivo, and as neither cell type in contact is inhibited, they also display different phenotypes in high density environments and 3D culture. For example, myoblasts are precursor cells that upon differentiation form multinucleated myotubes, a process that involves cytoskeletal rearrangement and cell body alignment followed by cell to cell fusion. Myoblast differentiation is regulated by a number of different signalling pathways, some of which can be onset by a reduction of various growth factors in the culture medium which can occur in high density cell population. Therefore, during prolonged experimental conditions where media nutrients and growth factors may become depleted over time, C2C12 cells may undergo physiological changes that alter various phenotypes such as cell morphology and adhesion preferences.

[283] X. Effect of Ascorbic Acid and Gelatin on Cell Attachment to Mercerized Material

[284] This experiment presents data on the effect that gelatin or ascorbic acid treatment of plant-derived cellulose-based microcarriers has on cell attachment efficiency and quality. It was found that mercerized material pre-treated with 10% gelatin or 100 pM ascorbic acid had no observable effect after 48 hours, but they appeared to improve cell attachment efficiency and adhesion quality after 15 days post seeding in both 3T3-GFP and C2C12 cell lines in comparison to control cellulose microcarriers treated with media. With the increased level of late phase cell adhesion observed, additionally fewer cell aggregates/spheroids were observed with the treated microcarriers in both cell lines.

[285] a. Context

[286] Due to the biocompatible nature of the mercerized cellulosic material, additional studies looking into its application and use as a microcarrier for cell culturing purposes were conducted. As seen in previous studies, some cell attachment is observed to the cellulose particles; however, the efficiency of this is quite low for what would be expected with commercial microcarriers. In this study, lab grade gelatin was used as a pretreatment of the mercerized cellulose material, to provide additional extracellular matrix molecules for the cells to interact with. Early in vitro biocompatibility studies using 10% gelatin combined with decellularized apples have shown to help increase cell attachment. In parallel, ascorbic acid was used as a media supplement with the aim to increase cell collagen deposition in the ECM and increase overall cell attachment to the cellulose particles.

[287] b. Methods

[288] Materials & Equipment

Table IX. List of Materials

.. . . . e i- Catalog .. .

Material Supplier .. . Notes

Number

Sterile, non cleanroom, Microcarriers lot

Mercerized Apples Prepared in-house N/AMER-15 prepared on 2023-09-

15 (8.91% mass density)

DMEM with L-Glutamine, 4.5g/L Corning MT-10-013-CV Maintained sterile

Glucose and Sodium Pyruvate

Fetal Bovine Serum Thermo Scientific J66993.03 Lot: X251071

Containing Penicillin,

Antibiotic-Antimycotic (100X) Gibco 15240062 Streptomycin and

Gibco Amphotericin B

Gelatin from Porcine skin Sigma-Aldrich G1890-100G N/A

Ascorbic Acid LabChem LC115309 Lot: J224-15

Table X. List of Equipment

Equipment Supplier Catalog Number Notes

Biosafety Cabinet Scilogex HB120-S N/A

Benchtop Centrifuge Thermo Scientific Sorvall ST 16R SN: 42530122 37°C Water Bath Fisher Scientific Isotemp GPD 10 Model: FSGPD10

SN: 300266216

Autoclave Steris AMSCO 250LS SN: 032271914

[289] c. Protocol i. 6-well Plate Siliconization:

Dow SYLGARD™ 184 Silicone Elastomer kit was used to siliconize TC-treated 6-well plates. As per the kit instructions, Reagent A was mixed with Reagent B in a 1 :10 ratio (A:B), then mixed slowly by inversion for a minimum of 30 minutes. 2 mL of the mixture was added per well of a 6-well plate, then left to cure for 24 hours at room temperature. The plates were then sterilized in a BSC by submerging in 70% isopropyl alcohol, followed by a 60-minute UV sterilization cycle. ii. Preparation of 10% Gelatin

Gelatin from porcine skin (Sigma Aldrich) was prepared at 10% (mass/volume) in complete growth medium (DMEM, 1 % antimycotic & antibiotic solution containing Penicillin, Streptomycin and Gibco Amphotericin B). Solution was then autoclaved using a 30 minute liquid cycle, and it was stored for up to 1 month at 4°C. Prior to use, the solidified gelatin mixture was put in a water bath set to 37°C for a minimum of 30 minutes for it to return to a liquid phase.

Hi. Preparation of Ascorbic Acid

A stock solution of ascorbic acid was prepared at a concentration of 50 mg/mL in 1X PBS. It was then filter sterilized with a 0.22 pm syringe filter and then stored at 4°C away from light for up to 48 hours. Cells were supplemented with 100 pg/mL of ascorbic acid at time of seeding with Microcarriers. iv. Preparation of Mercerized Material

A stock solution of mercerized cellulose material was diluted to 1 % mass density using 1 X PBS, from which 1.5 g of material was further diluted with 25 mL of 1X PBS. The material was then autoclaved with a sterilization cycle at 121 °C for 25 minutes. After autoclaving, the cellulose material was washed twice with 1X PBS by first allowing the material to settle for 15 minutes and then carefully removing the supernatant. 25 mL of fresh PBS was then added, and the washing process was repeated an additional time for a total of two washes. After the second wash, the material was divided equally between three tubes (0.5 g of 1 % mass density). Then, after settling, the supernatant was removed leaving the microcarrier fraction in a 5 mL volume. 15 mL of complete growth medium with 10% gelatin was added to one tube and 15 mL complete growth medium was added to the remaining 2 tubes, for a total volume of 20 mL per tube then left to incubate at 4°C overnight (Note: the concentration of gelatin is calculated prior to addition to the microcarrier fraction). After the 24 hour incubation, the Microcarriers was further diluted with the appropriate media condition to a final concentration of 0.03%. v. Cell and Microcarrier Seeding and Media Change

3T3-GFP and C2C12 cells were trypsinized and counted using a Countess 3 automated cell counter. A working stock of cell suspension was prepared and diluted to a final concentration of 1 .0 x10⁵ cells/mL using complete growth media. Microcarriers were then seeded in the wells of a 6-well plate from the stock solutions prepared at 0.03%, at an inoculation volume of 0.75 mL per well. Each treatment condition was plated in duplicate per cell type. 0.5 mL of the 1 .0 x10⁵ cells/mL cells working stock was then added to each well of the corresponding 6-well plate drop-wise, therefore resulting in a final cell concentration of 5.0 x10⁴cells/mL. After seeding the Microcarriers and the cells, the plates were incubated in the BSC for 30 minutes at room temperature for the cells to settle on the solidified gelatin. Then they were placed in a 37°C, 5% CO2 incubator for a 4 hour attachment phase. After the 4 hour attachment phase, all wells were topped up with 0.75 mL of fresh complete growth media, and then they were placed back at 37°C, 5% CO2 incubator for 7 days. After 7 days, the media was replaced by first combining the replicates of each condition, then centrifuging at 500 xg for 5 minutes to pellet all cells, and finally leaving for 15 minutes undisturbed to allow for the cellulose particles to settle. After all material had settled, the supernatant was removed and replaced with fresh complete growth media, and the samples were replated on new siliconized 6-well plates. The cells were incubated for up to 21 days, after which they were fixed and stained. vi. Microscopy Imaging and Staining

An inverted Fisherbrand microscope was used for phase contrast imaging at all timepoints using a OMAX A35180U3 18 MP USB3.0 camera (part number TP11800A) to record images with ToupView software at 4912x3684 resolution, format RGB24, using auto white balance, at 10X magnification. The plates were imaged with the inverted microscope at 24, 48, and 72 hours and 15 days post seeding for both cell lines. After 21 days post seeding, both replicates from each condition were combined then washed 2X with 1X PBS, fixed using 4% paraformaldehyde, and stored at 4°C for up to 3 months. After fixing, the samples were stained using Hoechst dye diluted 1 :1000 with 1X PBS, followed by staining with Congo Red diluted with 1X PBS to 0.02%. All samples were then imaged at 2X, 4X, and 10X magnification using an Olympus SZX16 microscope.

[290] d. Results

[291] Figure 30 shows 3T3-GFP cells seeded with mercerized cellulose material in a 6-well plate after 48 hours (left column) and 15 days (right column). Scale bar = 250 pm. [292] Figure 31 shows C2C12 cells seeded with mercerized cellulose material in a 6-well plate after 48 hours (left column) and 15 days (right column). Scale bar = 250 pm.

[293] Figure 32 shows Fluorescence microscopy of 3T3-GFP cells after 21 days in culture. Cell nuclei stained with Hoechst (blue) and cellulose particles stained with Congo Red (red). Scale bars = 250 pm.

[294] Figure 33 shows Fluorescence microscopy of C2C12 cells after 21 days in culture. Cell nuclei stained with Hoechst (blue) on Cellulose particles stained with Congo Red (red). Scale bars = 250 pm.

[295] Microscopy imaging at early time points (48 hours post seeding) showed that cellulose microcarriers treated with either ascorbic acid or 10% gelatin did not result in an observable difference in early cell adhesion to the cellulose particles in comparison to the control microcarriers treated with only media. In all tested conditions, low cell attachment efficiency (<50%) was observed 48 hours post seeding in both cell lines. Notably, in the ascorbic acid treated material, the cells appeared to form fewer aggregates after 48 hours of incubation, with more rounded single cells observed still in suspension indicating a lack of attachment. This was observed in both 3T3- GFP and C2C12 cell lines; however, it was more apparent in the C2C12 cells.

[296] At later time points, 15 and 21 days post seeding, improved cell attachment was observed with an increase in both cell attachment efficiency and quality of adhesion with both the ascorbic acid and gelatin treated microcarriers when compared to the control conditions. Microscopy showed improved 3T3-GFP cell attachment efficiency with ascorbic acid and gelatin treated microcarriers, with approximately 50-70% cell attachment, in comparison to the control treatment, which had a cell attachment efficiency of approximately 25-50%. Similarly, the C2C12 cells also saw an improvement in attachment efficiency with the treated groups compared to the control, with an approximate efficiency of 60-80% and 25-50% respectively. This improvement was observed to be slightly more pronounced in the C2C12 cells in comparison to the 3T3-GFP cells.

[297] Additionally, a decrease in the number of cell aggregates/spheroids formed with the treatment conditions than was seen in the controls was observed at later time points. Microscopy of the 3T3-GFP cells at 15 and 21 days showed quite large cell aggregates/spheroids in the media control condition, both attached to the microcarriers and free floating, the diameter of which was comparable to the size of the cellulosic particles. In contrast, fewer cell aggregates/spheroids with a smaller average diameter were observed with both the ascorbic acid and gelatin treated microcarriers, with no observable difference between the two conditions. In comparison, the C2C12 cells with the media control microcarriers formed less compact cell/aggregate/spheroid structures but still appeared highly aggregated, both attached to the microcarriers and free floating. Similar to the 3T3-GFP cells, fewer cell aggregates/spheroids were observed with the C2C12 cells in the presence of the ascorbic acid and gelatin treated material. While some cell aggregates/spheroids were still present in both treatment conditions, this was considerably less than was observed in the media control.

[298] Observations at day 15 and 21 also showed that both the gelatin and ascorbic acid treated material appeared to have more cells fully adhered to individual particles, beginning to create a cell monolayer as indicated by the cell morphology. This was seen in both the 3T3-GFP and C2C12 cell lines and was interestingly not observed in either of the media treated controls. This change in the quality of cell adhesion was observed as a flattening and spreading of the cell body to the microcarrier surface, indicative of high quality stable cell adhesion. This was observably more apparent in both the treated conditions in comparison to the media control group, which showed little to no signs of monolayer formation along the surface of the particles, indicated by the rounded single cell morphology and cell aggregate/spheroid formation seen at early time points and in the media controls. High quality adhesion refers to the cells in late stage or Phase 3 adhesion which involves creating focal adhesions to the extracellular matrix (ECM) and reorganization of the cytoskeleton. This late phase cell adhesion is indicated by observations of flattened and spread/branching cell morphology along the surface of the culture vessel, creating a typical monolayer. Both early phase and late phase cell morphology was observed in the treatment conditions for both cell lines, while cell morphology only associated with initial adhesion phases was seen in the media controls.

[299] e. Discussion

[300] It is important to note that the results found in this study are based solely on microscopy observations; thus, they are only qualitative and not quantitative. In future studies, using a quantitative method for cell attachment efficiency will give more insight on the effect gelatin and ascorbic acid pre-treatments and other coatings have on cell adhesion.

[301] As discussed earlier, there are two main types of cell adhesion; cell-cell adhesion mediated by cadherin receptor domains, and cell-ECM adhesion largely mediated by binding of cell integrin receptors to various motifs in the ECM, which can be defined by three phases. This type of late phase cell-ECM adhesion was observed at the later time points in the treatment conditions. Both of these treatment types were chosen as conditions to increase ECM proteins associated with the microcarriers to promote cell adhesion. Ascorbic acid supplementation is well documented to promote ECM deposition in both myoblast and fibroblast cells lines and gelatin is derived from collagen which is a main component of the ECM in vivo.

[302] XI. Cell Seeding onto Lyophilized Mercerized Material

[303] This experiment presents data on the effect of seeding cells onto lyophilized plant-derived cellulose-based microcarriers has on cell attachment efficiency and quality over time. Additionally, a variety of lyophilized cellulose pellet sizes and media treatment conditions were tested to determine the optimal seeding parameters for lyophilized plant-derived cellulose-based microcarriers. It was found that 3T3-GFP cells seeded onto lyophilized pellets of mercerized cellulose material had no observable impact on cell attachment efficiency or quality after 24 hours, but appeared to result in some high quality adhesion at later time points. It was also shown that seeding directly onto lyophilized pellets, prepared from 0.1 and 0.15 mL of mercerized cellulose material resulted in better cell viability after 24 hours and up to 9 days post seeding compared to cells seeded on 0.2 mL pellets which resulted in low cell viability after 24 hours.

[304] a. Context [305] Due to the biocompatible nature of the mercerized cellulosic material, additional studies looking into its application and use as a microcarrier for cell culturing purposes were conducted. The use of lyophilized mercerized cellulose material was thought may help maximize cell to cellulose surface contact for a longer period of time rather than relying on the kinetic cell to particle interactions in suspension. It was hypothesised this would give the cells additional time to fully adhere to the surface of the cellulose microcarriers, increasing the cell to cellulose microcarrier attachment efficiency. After cell attachment, the material could then be broken apart into the individual microcarrier particles, thus increasing the total surface area for the cells to proliferate upon.

[306] b. Methods

[307] Materials & Equipment

Table XI. List of Materials

. M.at .er .ia .l S eupp il-ier . Catalog .. .

N.um .ber Notes

Sterile, non cleanroom, Microcarriers lot

Mercerized Apples Prepared in-house N/AMER-9 prepared on 2023-04-

28 (8.23% mass density)

Non-sterile lot prepared

Phosphate Buffered Saline (PBS) 1X Intermountain (Cytiva R Q PR I YA Lot: 21802221 , without Ca & Mg Hyclone) bbb-Hbb-ixo exp: 2024-02-18

DMEM with L-Glutamine, 4.5g/L Corning MT-10-013-CV Maintained sterile

Glucose and Sodium Pyruvate

Fetal Bovine Serum Thermo Scientific J66993.03 Lot: X251071

Containing penicillin,

Antibiotic-Antimycotic (100X) Gibco 15240062 streptomycin and

Gibco amphotericin B

Table XII. List of Equipment

Equipment Supplier Catalog Number Notes

Biosafety Cabinet Scilogex HB120-S N/A

Inverted microscope BioTek Synergy Mx N/A

CountessTM 3 Invitrogen AMQAX2000 SN: 2187A23046709 Microscope Olympus SZX16 SN: 5L11 120

Lyophilizer Buchi Lyovapor™ L-200 SN: 1500032648

[308] c. Protocol i. Lyophilization of Mercerized Material

Mercerized cellulose material was diluted to 4.5% mass density with 1X PBS then autoclaved at 121 °C for a 30 minute sterilization cycle. After autoclaving, the material was stored at 4°C overnight. The next day, the material was further diluted with 1X PBS to 3% mass density and was mixed 30 times between two 3 mL syringes. After mixing, the material was then dispensed into the wells of a 96-well plate in volumes of 0.10, 0.15 and 0.20 mL per well, filling a minimum of 5 wells per volume. Once all of the material was added, the plate was then tapped several times then centrifuged at 500x g for 15 minutes to collect all of the cellulose material to the bottom of the well, and get a relatively flat layer of material in each well. The plate was placed in the -20°C freezer overnight, and then it was lyophilized. After lyophilization, the resulting mercerized cellulose pellets were transferred to a new sterile falcon tube and stored at room temperature until use (24 hours). ii. Cell and microcarrier seeding and media change

3T3-GFP cells were trypsinized and counted using a Countess 3 automated cell counter. A working stock of cell suspension was prepared and diluted to a final concentration of 3.33 x10⁵ cells/mL using complete growth media. The pellets of mercerized cellulose material were transferred to the wells of a 24-well plate as shown in Figure 34. Prior to cell seeding, 0.15 mL of complete growth medium was added to four of the prepared wells, fully saturating the cellulose pellets within each of the wells before the addition of cells. This was then referred to as the pre-soak condition. Media was added dropwise around the sides of the well, careful not to disturb the cellulose pellets. Cells were then carefully inoculated dropwise directly onto the pellets in a volume of 0.15 mL, therefore adding 5.00 x10⁴ cells per well. After cells were seeded, the plate was transferred to a 37°C, 5% CO2 incubator for a 4 hour attachment phase. After the 4 hour attachment phase, the wells were topped up with either a low volume (0.15 mL) or a high volume (0.3 mL) of complete growth media as shown in Figure 35. No top-up media was added to the pre-soak conditions. The plate was then placed back in the 37°C, 5% CO2 incubator for up to 14 days. After 6 days, the pellets of the 24-well plate were then transferred using sterile tweezers to the wells of a siliconized 6- well plate. 2 mL of fresh complete growth media was added to each well, then all pellets were manually broken apart using a pipet tip. The 6-well plates were then transferred back into the 37°C, 5% CO2 incubator for up to 14 days post seeding. iii. Microscopy Imaging

Microscopy imaging of 3T3-GFP cells was performed at 24 hours, 9 days, and 14 days post seeding using an Olympus SZX16 microscope at 24 hours and 9 days at 4X and 10X magnifications, and a BX53 upright microscope was used for images taken at 14 days post seeding at 40X magnification.

[309] d. Results

[310] Figure 34 shows plate layout for 3T3-GFP cell seeding and post attachment phase conditions on different sizes of lyophilized pellets of mercerized cellulose material. Wells crossed out with an X represent unused wells.

[311] Figure 35 shows microscopy of 3T3-GFP cells (green) 24 hours post seeding on lyophilized cellulose pellets. Scale bar = 250 pm.

[312] Figure 36 shows microscopy of 3T3-GFP cells (green) 24 hours post seeding on lyophilized cellulose pellets pre-soaked in 1X DMEM, 10% FBS, 1 % antibiotic-antimycotic. Scale bar = 250 pm.

[313] Figure 37 shows microscopy of 3T3-GFP cells (green) 9 days post seeding on broken apart lyophilized cellulose pellets. Scale bar = 250 pm.

[314] Figure 38 shows overlay of brightfield and fluorescence microscopy images of 3T3-GFP cells (green) 14 days post seeding on broken up 0.15 mL lyophilized cellulose pellet with 0.30 mL (high volume) media added post attachment phase. Scale bar = 50 pm.

[315] Microscopy taken 24 hours post seeding showed 3T3-GFP cells on the surface of the lyophilized cellulose pellets in all conditions except for the 0.20 mL pellet with 0.15 mL of media (low volume) condition, in which no GFP signal was observed. Between the various sizes of lyophilized cellulose pellets, the 0.2 mL pellets appeared to have the fewest viable cells adhered to the surface at 24 hours and additionally at 9 days after the pellet had been broken apart in media. This considerable decrease in GFP signal observed with the 0.2 mL pellets was seen in both the 0.15 mL media (low volume) and the pre-soaked conditions. Observations taken at the time of cell seeding noted that upon addition of the cell suspension to the pre-soak conditioned pellets, the cellulose pellets rapidly began breaking apart in solution. Additionally, it was noted that when seeding cells with the 0.2 mL pellets, all of the cell suspension/media was immediately absorbed by the cellulose pellet, whereas upon seeding the 0.1 and 0.15 mL pellets, they immediately became fully saturated, allowing additional media to pool in the wells. Based on these observations, one may suggest that the lack of cells noted in the 0.2 mL pellets may have been caused by dehydration of the Microcarriers and associated cells during incubation or due to the reduced volume of media directly available to the cells. This would also explain the presence of some cells observed in the pre-soak condition with the 0.2 mL cellulose pellet, as additional media was present during the 4 hour attachment phase that would not have been in the 0.15 mL media (low volume) condition.

[316] While a mild increase in cells was observed in the pre-soak condition with the 0.2 mL cellulose pellets, this was not seen with the 0.15 mL pellets, which when compared to 0.15 mL (low volume) and 0.3 mL (high volume) conditions, a similar amount of GFP signal was visually observed at 24 hours, and a moderate decrease in signal at 9 days post seeding. Microscopy after 24 hours and up to 9 days showed that for both the 0.1 and 0.15 mL cellulose pellets, similar levels of GFP signal intensity was observed in the 0.15 (low volume) and 0.3 mL (high volume) conditions at 24 hours, and in the 0.3 mL media (high volume) condition at 9 days. Furthermore, no observable change in GFP signal intensity was noted between the 0.1 and 0.15 mL cellulose pellets in either condition of media volume at 24 hours and 9 days post seeding. Images taken at 24 hours showed that the cells observed on the surface of the cellulose pellets displayed a more rounded morphology and no observable aggregation or spheroid formation. This indicates that while the cellulose pellet conditions do not cause early cell to cell aggregation, it appears that seeding directly onto the pellets also does not promote late phase cell adhesion after 24 hours. After the pellets were broken apart and transferred to low attachment 6-well plates, microscopy taken at 9 days showed evidence of cell aggregation/spheroid formation in all conditions where GFP signal was observed. Cell aggregates/spheroids were particularly notable in both the 0.1 and 0.15 mL cellulose pellets with 0.3 mL media (high volume) condition, as these were the conditions with the most GFP signal observed at 9 days. An important distinction to note is that all observations of GFP signal intensity and the corresponding cell viability are all based solely on visual observations, thus are only qualitative results and not quantitative.

[317] Microscopy taken at 14 days post seeding showed a moderate amount of 3T3-GFP cell attachment and quality adhesion to individual cellulose particles from the broken up 0.15 mL lyophilized cellulose pellet, 0.30 mL media (high volume) condition. Evidence of both early and late phase cell adhesion was observed on the plant- derived cellulose-based microcarriers, indicating that while the quality of cell adhesion appears to be improved at 14 days compared to 24 hours post seeding, the amount of quality adhesion observed still remains relatively low, with little monolayer formation compared to cells observed in regular culturing vessels. High quality adhesion refers to the cells in late stage or Phase 3 adhesion which involves creating focal adhesions to the extracellular matrix (ECM) and reorganization of the cytoskeleton. This late phase cell adhesion is indicated by observations of flattened and spread/branching cell morphology along the surface of the culture vessel, creating a typical monolayer. This change in the quality of cell adhesion was observed as a flattening and spreading of the cell body to the microcarrier surface, indicative of high quality, stable cell adhesion, while low quality cell adhesion was observed as rounded single cells with moderate level of aggregation, both of which were observed on various cellulosic particles from the 0.15 mL pellet with 0.3 mL media (high volume) condition at 14 days.

[318] e. Discussion

[319] It is important to note that the results found in this study are based solely on microscopy observations; thus, they are only qualitative and not quantitative. In future studies, using a quantitative method for cell attachment efficiency will give more insight on the optimal seeding conditions for this method and the effect that cell seeding directly onto lyophilized Microcarriers has on cell adhesion.

[320] As discussed earlier, there are two main types of cell adhesion: cell-cell adhesion mediated by cadherin receptor domains and cell-ECM adhesion largely mediated by binding of cell integrin receptors to various motifs in the ECM which can be defined by three phases. This type of late phase cell-ECM adhesion was observed at the later time points in some of the microcarriers.

[321] Accordingly, from the above experiments it is evident that the microcarrier beads/scaffold particles are capable of mimicking the function of the commercially available beads for cell attachment and proliferation in bioreactors. In fact, the inventive beads/microcarriers are better especially for commercial tissue engineering and food applications as commercially available beads need to be dissolved or washed out, while the plant-based beads would be utilized not only as a scaffold for cell attachment and proliferation but also to improve the textural properties of the final product.

[322] XII. 3T3 and C2C12 Cell Attachment to Cross-linked Cellulose Microcarriers

[323] This experiment was designed to test the effect of seeding cells onto cross-linked plant-derived cellulose- based microcarriers has on cell attachment efficiency and quality overtime. It was found that 3T3-GFP and C2C12 cells seeded onto lyophilized cross-linked microcarriers had similar levels of attachment efficiency, at approximately 40-60% after 24 hours. Microscopy images taken after 24 hours and up to 7 days post seeding showed low levels of cell attachment in both cell types and additionally exhibited cell aggregation and spheroid formation after 48 hours, observed both attached and unadhered to the cross-linked microcarriers. However, after 7 days no changes in cell morphology indicating late phase cell to extracellular matrix (ECM) adhesion were observed in either cell line.

[324] a. Context

Due to the biocompatible nature of Spiderwort’s mercerized cellulosic material, additional studies looking into its application and use as a microcarrier for cell culturing purposes were conducted. Previous experiments looking at cell adhesion to lyophilized mercerized material showed evidence of cell attachment and at later time points late phase cell adhesion. It was hypothesised that using the cross-linked cellulose scaffold would further increase the cell attachment efficiency at early time points, increase the level of late phase cell adhesion, and increase the rate of monolayer formation on the microcarriers. Supporting evidence has been observed in previous in vivo implantation studies that have shown increased cell infiltration and angiogenesis with the cross-linked cellulose indicating a high level of biocompatibility and scaffolding capabilities of the material.

[325] b. Methods

Materials & Equipment Table XIII. List of Materials

Catalog Material Supplier Notes

Number

Sterilized in-process,

Cross-linked Cellulose Prepared in-house N/A and maintained sterile

MT-10-013-CV Maintained sterile

DMEM with L-Glutamine, 4.5g/L Glucose and Sodium Pyruvate

Fetal Bovine Serum Thermo Scientific J66993.03 Lot: X251071

Trypsin, EDTA Sigma-Aldrich C1794 Lot: SLCL0379 Catalog

Material Supplier Notes

Number

Sterilized in-process,

Cross-linked Cellulose Prepared in-house N/A and maintained sterile

Containing penicillin,

Antibiotic-Antimycotic (100X) Gibco 15240062 streptomycin and Gibco amphotericin B

Table XIV. List of Equipment

Equipment Supplier Catalog Number Notes

Inverted microscope BioTek Synergy Mx N/A

Microscope Olympus SZX16 SN: 5L11120

Model: 31193C

Convection Oven Hamilton Beach N/A

Series: A1122CE

CO2 Incubator (37°C, 5% Thermo Scientific HERAcell vios 160i SN: 42457396

CO₂) Benchtop Centrifuge Thermo Scientific Sorvall ST 16R SN: 42530122

Lyophilizer Buchi Lyovapor™ L-200 SN: 1500032648

[326] c. Protocol

6-Well Plate Siliconization:

Dow SYLGARD™ 184 Silicone Elastomer kit was used to siliconize TC-treated 6-well plates. As per the kit instructions, Reagent A was mixed with Reagent B in a 1 :10 ratio (A:B) and then mixed slowly by inversion for a minimum of 30 minutes. 2 mL of the mixture was added per well of a 6-well plate and then left to cure for 24 hours at room temperature (RT). The plates were then sterilized in a biosafety cabinet (BSC) by submerging in 70% isopropyl alcohol, followed by a 60-minute UV sterilization cycle.

Preparation and Sterilization of Cross-linked Cellulose

Briefly, mercerized cellulose material diluted to 4% mass density (weight/weight) was combined with citric acid diluted to 0.25 g/mL in dFTO and mixed by homogenization. The material was then placed in a vacuum system for to remove any bubbles then transferred to a custom-designed temperature monitoring device and placed in a - 80°C freezer overnight. After overnight freezing, the device was then transferred to the lyophilizer and lyophilized overnight. The following day, the device was then transferred to a convection oven set at a predetermined temperature (in the range of 100°C - 140°C) and left for the cross-linking reaction. The cross-linked cellulose pellets were then removed from the device and hydrated with PBS, and this was followed by several washes with 30 mL dPLO at room temperature (RT). After the last water wash, sodium bicarbonate was added to the aerogels, and it was incubated at RT on a shaker. The cross-linked cellulose pellets were then washed again with dPLO as described above, then submerged in 1X PBS and incubated overnight at 4°C. The next day, the cross-linked cellulose pellets were sterilized by washing in 70% ethanol in a BSC, after which the remainder of the process was performed using aseptic technique in a BSC, with sterile reagents and tools. After sterilization, the crosslinked cellulose pellets were washed with sterile dPLO and incubated at RT on a plate shaker, after which they were transferred to a sterile CPD stage and placed in a -80°C freezer for a minimum of 3 hours. The stage was then placed in the lyophilizer and lyophilized overnight. After lyophilization, the final cross-linked cellulose pellets were stored at 4°C.

Cell and Microcarrier Seeding and Media Change

3T3-GFP cells were trypsinized and counted using a Countess 3 automated cell counter. A working stock of cell suspension was prepared and diluted to a final concentration of 1.0 x10⁵ cells/mL using complete growth media (1X DMEM, 10% FBS, 1 % antibiotic/antimycotic). Each cross-linked cellulose pellet was cut in half using sterile surgery scissors and each half was transferred to the well of a siliconized 6-well plate, then hydrated with 0.5 mL of complete growth medium. Once the cross-linked pellets were fully hydrated, they were manually broken apart using sterile forceps, followed by vigorous pipetting up and down to fully disperse the particles. 0.5 mL of the 1.0 x10⁵ cells/mL working stock of cells was then added to each well of the corresponding 6-well plate drop-wise, therefore resulting in a final cell concentration of 5.0 x10⁴cells/well. After seeding the cross-linked cellulose pellets and the cells, the 6-well plate was placed in a 37°C, 5% CO2 incubator for a 4 hour attachment phase. After the 4 hour attachment phase, all wells were topped up with 1 .0 mL of fresh complete growth media, and then they were placed back at 37°C, 5% CO2 incubator for 7 days.

Microscopy Imaging and Staining

An inverted Fisherbrand microscope was used for phase contrast imaging at 48 hours and 7 days post seeding for both cell lines using a OMAX A35180U3 18 MP USB3.0 camera (part number TP1 1800A) to record images with ToupView software at 4912x3684 resolution, format RGB24, using auto white balance, at 10X magnification. 3T3-GFP cells were imaged at 24 and 48 hours and 7 days post seeding using fluorescence microscopy with an Olympus SZX16 microscope at 2.5X and 10X magnifications. After 7 days post seeding, samples from each well of both cell lines were washed 2X with 1X PBS, fixed using 4% paraformaldehyde, and stored at 4°C for up to 3 months. After fixing, the samples were stained using Hoechst dye diluted 1 :1000 with 1X PBS, followed by staining with Congo Red diluted with 1X PBS to 0.02%. All samples were then imaged at 2.5X, and 10X magnification using an Olympus SZX16 microscope.

[327] d. Results

[328] Figure 39 shows fluorescence microscopy of 3T3-GFP cells (green) seeded on cross-linked cellulose microcarriers at 2.5 and 10 X magnifications. Scale bar for 2.5X magnification is 750 pm, and for 10X magnification is 200 pm.

[329] Figure 40 shows microscopy of 3T3-GFP and C2C12 cells seeded on cross-linked cellulose microcarriers. Scale bar = 200 pm.

[330] Figure 41 shows fluorescence microscopy of 3T3-GFP and C2C12 cells after 7 days in culture. Cell nuclei stained with Hoechst (cyan) and cellulose particles stained with Congo Red (red). Scale bar for 4X magnification is 500 pm, and for 10X magnification is 200 pm. [331] Imaging of 3T3-GFP at 24 hours and C2C12 at 48 hours post seeding showed evidence of early cell attachment to the cross-linked cellulose microcarriers. Microscopy observations showed a moderate level of cell attachment efficiency, at approximately 40-60% percent attachment efficiency for both cell lines. Observations of 3T3-GFP cells at 24 hour post seeding showed primarily single cells; however, microscopy at later time points saw increased levels of cell aggregation and spheroid formation. This was observed at both 48 hours and 7 days post seeding in both 3T3-GFP and C2C12 cell lines, with an observable increase in the level of aggregation seen at 7 days. After 48 hours, cell aggregates/spheroids were seen both attached to the cross-linked cellulose microcarriers and free floating. With the increased level of aggregation at later time points, the percent attachment efficiency remained the same, and no increase in cell aggregate size was observed after 7 days in culture with either of the cell types.

[332] No change in quality of cell adhesion was observed after 7 days in culture for both 3T3-GFP and C2C12 cell lines. Additionally, after 7 days post seeding no monolayer formation was observed on the cross-linked cellulose microcarriers, instead cell morphology remained rounded for both cell lines, with high levels of cell to cell aggregation and spheroid formation, and no indications of high quality cell adhesion observed. High quality adhesion refers to the cells beginning late stage or Phase 3 cell adhesion by creating focal adhesions to the extracellular matrix and reorganization of the cytoskeleton. This late phase cell adhesion is indicated by observations of flattened and spread/branching cell morphology along the surface of the culture, creating a typical monolayer. Cells both adhered and unattached to the aerogel microcarriers were observed as either aggregated clusters or rounded single cells, with no observations indicating late phase cell adhesion.

[333] e. Discussion

It is important to note that the results found in this study are based solely on microscopy observations; thus, they are only qualitative and not quantitative. In future studies, using a quantitative method for cell attachment efficiency will give more insight on the optimal seeding, media, and attachment phase conditions for this method and the effect that seeding cells directly onto cross-linked cellulose microcarriers has on cell adhesion in comparison to lyophilized mercerized cellulose material (non-cross-linked) which has been previously studied. As discussed before, there are two main types of cell adhesion: cell-cell adhesion mediated by cadherin receptor domains and cell-ECM adhesion largely mediated by binding of cell integrin receptors to various motifs in the ECM and that cell adhesion to the ECM can be defined by three phases.

[334] Conclusion

[335] It was found that HEK 293 cells seeded with plant-derived cellulose-based microcarriers both with and without succinylation modification resulted in low amounts of early cell adhesion to the particles after 24 hours post seeding and subsequently little change in cell morphology indicating late phase adhesion after 5 days. While overall low cell attachment efficiency was observed with both microcarriers, a slight but observable increase in attachment efficiency was noted with the succinylated cellulose microcarriers in comparison to the mercerized cellulose material. As microcarriers are typically used for cell production in large quantities, getting high attachment efficiency is crucial as this greatly impacts the final yield of cells. While some HEK 293 cell attachment is observed in the presence of the plant-derived cellulose based microcarriers, the level of adhesion to the cellulosic particles is a bit lower than expected, however, the study does provide promising results which could be optimized for early cell adhesion.

[336] It was found that Vero cells seeded with plant-derived cellulose-based microcarriers with succinylation modification resulted in higher amounts of early cell adhesion to the particles after 24 hours post seeding and subsequently changes in cell morphology indicating late phase adhesion after 96 hours. While overall low cell attachment efficiency was observed with the mercerized cellulose material, an observable increase in attachment efficiency was noted with the succinylated microcarriers in comparison to the mercerized cellulose material. As microcarriers are typically used for cell production in large quantities, getting high attachment efficiency is crucial as this greatly impacts the final yield of cells. While some Vero cell attachment is observed in the presence of the plant-derived cellulose-based microcarriers, with increased attachment seen with succinylated modification, the level of adhesion and time course duration to see late phase cell-ECM adhesion to the cellulosic particles is a bit lower than expected, however, the study does provide promising results which could be optimized for early cell adhesion.

[337] It was found that treatment of plant derived cellulose based microcarriers with glycine had no observable effect on 3T3-GFP and C2C12 cell attachment efficiency or morphology after incubation up to 72 hours. As microcarriers are typically used for cell production in large quantities, getting high attachment efficiency is crucial as this greatly impacts the final yield of cells. While some cell attachment is observed in the presence of the plant- derived cellulose based microcarriers, the level of adhesion to the cellulosic particles is a bit lower than expected, however, the study does provide promising results which could be optimized for early cell adhesion.

[338] It was found that overnight treatment of mercerized cellulose with varying concentrations of FBS had no observable effect on 3T3-GFP and C2C12 cell attachment efficiency after incubation up to 72 and 48 hours respectively. As microcarriers are typically used for cell production in large quantities, getting high attachment efficiency is crucial as this greatly impacts the final yield of cells. While some cell attachment is observed in the presence of the plant-derived cellulose-based microcarriers, the level of adhesion to the cellulosic particles is a bit lower than expected, however, the study does provide promising results which could be optimized for early cell adhesion.

[339] It was found that treatment of the plant-derived cellulose-based microcarriers with 10% gelatin or ascorbic acid pre-seeding had little effect on early attachment at 48 hours, but at later time points was seen to increase attachment efficiency and late stage cell adhesion while decreasing cell aggregation/spheroid formation in both 3T3-GFP and C2C12 cells. As microcarriers are typically used for cell production in large quantities, getting high attachment efficiency is crucial as this greatly impacts the final yield of cells. While some cell attachment is observed in the presence of the plant-derived cellulose-based microcarriers, and has been improved by the addition of gelatin and ascorbic acid, the level of adhesion and time course duration to see late phase cell-ECM adhesion to the cellulosic particles is a bit lower than expected, however, the study does provide promising results which could be optimized for early cell adhesion.

[340] It was found that 3T3-GFP cells seeded onto lyophilized pellets of plant-derived cellulose-based microcarriers had no observable impact on cell attachment efficiency or quality after 24 hours, but after 14 days in culture some high quality cell adhesion was observed. At early time points, no late phase cell adhesion was observed in any of the conditions, indicating that seeding directly onto the dried material has no observable impact on early cell adhesion and attachment efficiency. Additionally, no significant difference in cell viability or attachment efficiency was observed between media conditions tested before and after the attachment phase, with possibly a slight decrease in the number of viable cells observed in the pre-soaked condition at later time points. It was also shown that seeding directly onto lyophilized cellulose pellets, prepared from 0.1 and 0.15 mL of mercerized material resulted in better cell viability after 24 hours and up to 9 days post seeding, in comparison to the 0.2 mL pellets, which had little to no cells observed after 24 hour. These results may be reflective of the apparent balance required between the pre-soak condition, which had the most media present, providing additional protein and growth factors required by the cells to adhere, and the larger pellet sizes with no media during the attachment phase, which had a sponge-like effect of soaking up all the cell-laden, therefore maximizing cell to cellulose interaction. As microcarriers are typically used for cell production in large quantities, getting high attachment efficiency is crucial as this greatly impacts the final yield of cells. While some 3T3-GFP cell attachment is observed on the lyophilized plant-derived cellulose-based microcarriers, the level of adhesion and time course duration to see late phase cell-ECM adhesion to the cellulosic particles is a bit lower than expected, however, the study does provide promising results which could be optimized for early cell adhesion.

[341] It was found that 3T3-GFP and C2C12 cells seeded onto cross-linked plant-derived cellulose-based microcarriers resulted in a moderate level of early cell attachment with no high quality cell adhesion observed after 7 days post seeding. At early time points, cell attachment efficiency was approximately 40-60%, with cell aggregation/spheroid formation observed after 48 hours in both cell lines, indicating that the cross-linked cellulose microcarriers had a mild impact on early cell adhesion and attachment efficiency after 24 hours. Additionally, microscopy showed no signs of late phase cell adhesion, indicating poor adhesion quality and little monolayer formation on the microcarriers. As microcarriers are typically used for cell production in large quantities, getting high attachment efficiency is crucial as this greatly impacts the final yield of cells. While moderate level of 3T3- GFP and C2C12 cell attachment is observed on the cross-linked plant-derived cellulose-based microcarriers, the level of adhesion and time course duration to see late-phase cell-ECM adhesion to the cellulosic particles is a bit lower than expected, however, the study does provide promising results which could be optimized for early cell adhesion.

[342] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. It is not intended that the invention be limited by the specific examples provided within the specification. While the invention has been described with reference to the aforementioned specification, the descriptions and illustrations of the embodiments herein are not meant to be construed in a limiting sense. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. Furthermore, it shall be understood that all aspects of the invention are not limited to the specific depictions, configurations or relative proportions set forth herein which depend upon a variety of conditions and variables. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is therefore contemplated that the invention shall also cover any such alternatives, modifications, variations or equivalents. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

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Claims

1 . Plant-based or fungal-based cellulose scaffold particles for in-vitro proliferation of cells.

2. The scaffold particles of claim 1 , wherein the scaffold particles are obtained by decellularizing, mercerizing, bleaching or oxidizing a plant or a fungal tissue.

3 The scaffold particles of claim 2, wherein the plant or fungal tissue is cellulose-based, chitin- based, chitosan-based, lignin-based, lignan-based, hemicellulose-based, or pectin-based, or any combination thereof.

4 The scaffold particles of claim 3, wherein the plant or fungal tissue is a tissue from apple hypanthium (Malus pumila) tissue, fern (Monilophytes) tissue, turnip (Brassica rapa) root tissue, gingko branch tissue, horsetail (equisetum) tissue, hermocallis hybrid leaf tissue, kale (Brassica oleracea) stem tissue, conifers Douglas Fir (Pseudotsuga menziesii) tissue, cactus fruit (pitaya) flesh tissue, Maculata Vinca tissue, Aquatic Lotus (Nelumbo nucifera) tissue, Tulip (Tulipa gesneriana) petal tissue, Plantain (Musa paradisiaca) tissue, broccoli (Brassica oleracea) stem tissue, maple leaf (Acer psuedoplatanus) stem tissue, beet (Beta vulgaris) primary root tissue, green onion (Allium cepa) tissue, orchid (Orchidaceae) tissue, turnip (Brassica rapa) stem tissue, leek (Allium ampeloprasum) tissue, maple (Acer) tree branch tissue, celery (Apium graveolens) tissue, green onion (Allium cepa) stem tissue, pine tissue, aloe vera tissue, watermelon (Citrullus lanatus var. lanatus) tissue, Creeping Jenny (Lysimachia nummularia) tissue, cactae tissue, Lychnis Alpina tissue, rhubarb (Rheum rhabarbarum) tissue, pumpkin flesh (Cucurbita pepo) tissue, Dracena (Asparagaceae) stem tissue, Spiderwort (Tradescantia virginiana) stem tissue, Asparagus (Asparagus officinalis) stem tissue, mushroom (Fungi) tissue, fennel (Foeniculum vulgare) tissue, rose (Rosa) tissue, carrot (Daucus carota) tissue, or pear (Pomaceous) tissue, or a genetically altered tissue produced via direct genome modification or through selective breeding, or any combinations thereof

5 The scaffold particles of claim 3, wherein the plant tissue is obtained from apple, pear, banana, mango, lotus root, wood, bamboo, cotton linters, cotton stalks, cotton fabric waste, cotton wool, soybean husk, corn cob, water hyacinth, coconut shells, oil palm fronds, oil palm biomass residue, rice husk, sugar cane bagasse, jute, ramie, flax fibers, flax straw, wheat straw, sorghum stalks, sisal fibers, potato or mangosteen.

6 The scaffold particles of claim 2, wherein the scaffold particles have exposed polar groups with an augmented charge on their surface.

7 The scaffold particles of claim 1 , wherein the particles are biocompatible.

8 The scaffold particles of claim 1 , wherein the particles are optionally biodegradable.

9 The scaffold particles of claim 1 , wherein the scaffold particles facilitate cell growth in suspension and contribute to the textural properties, cellular organization or cellular differentiation in a final product.

10 The scaffold particles of claim 1 , wherein the particles are substantially circular, spherical, disk-shaped, or oval in shape.

11 . The scaffold particles of claim 1 , wherein the particles are structured as folded sheets of cellulose arranged in the form of irregular microspheres.

12. The scaffold particles of claim 1 , wherein the surface of scaffold particles is modified by a chemical treatment or by a physical treatment.

13. The scaffold particles of claim 12, wherein the modification enhances the proliferation of cells by increasing cellular attachment to the surface of the scaffold particles, by increasing cellular entrapment to the surface of the scaffold particles or by increasing cell proliferation capacity on the particles.

14. The scaffold particles of claim 12 wherein the surface of the scaffold particles is physically treated by lyophilization or re-hydration.

15. The scaffold particles of claim 12 wherein the surface is chemically treated by functionalizing the particle surface, cross-linking or by treating the particle surface with a pharmaceutically acceptable compound or a combination thereof.

16. The scaffold particles of claim 15, wherein the surface is functionalized by providing a functional group that creates a charge on the particle surface or by succinylating the particle surface.

17. The scaffold particles of claim 16, wherein the functional group is a primary amine, a tertiary amine, a quaternary compound, a denatured collagen, gelatin or a combination thereof.

18. The scaffold particles of claim 15, wherein the pharmaceutically acceptable compound is hydrogen peroxide, sodium bicarbonate, RGD motif, fibronectin, cadherins, integrins, nectins, afadin, p-catenin/aE-catenin, E-cadherin, vitronectin, cell attachment factors, gelatin, vitamins, ascorbic acid, serum, fetal bovine serum, charged amino acids, glycine or any food-grade acceptable compound.

19. The scaffold particles of claim 15, wherein the scaffold particles are subjected to cross-linking with a crosslinker agent to create cross-linked cellulose microcarriers.

20. The scaffold particles of claim 19, wherein the cross-linker agent is citric acid or paraformaldehyde.

21 . The scaffold particles of claim 1 , wherein the scaffold particles are pre-treated with hydrogen peroxide, fetal bovine serum, glycine, ascorbic acid or gelatin.

20. The scaffold particles of claim 1 , wherein the size of the scaffold particles ranges from 20 pm to 700 pm.

22. The scaffold particles of claim 1 , wherein the size of the particles ranges from 40 pm to 150 pm.

23. The scaffold particles of claim 1 , wherein the attachment of cells is dependent on one or more particle characteristics, wherein the one or more particle characteristics are particle size, particle surface tension, particle topography, or particle roundness.

24. The scaffold particles of claim 1 , wherein the proliferation of cells is directly correlated to the number of scaffold particles.

25. The scaffold particles of claim 1 , wherein the scaffold particles are for in-vitro proliferation of animal cells, plant cells, or human cells.

26. Plant-based or fungal-based cellulose scaffold particles according to any one of claims 1-25 for cellular in-vitro meat cell-production, cultured meat cell-production, agriculture, tissue engineering, 3D tissue culturing, proliferation of cell-banks, stem cells, in-vitro drug testing, protein harvesting, virus and vaccine production, biomolecule amplification, plasmid amplification, production of metabolites, production of cosmetic actives, production of biopharmaceuticals, production of recombinant proteins, biological filtration, waste-water treatment, industrial cleaning operations, or food production.

27. Plant-based or fungal-based cellulose scaffold particles according to any one of claims 1-25 as edible scaffolds for the production of dairy products.

28. A method of in-vitro proliferation of cells comprising: a) adding a batch of culture medium comprising cells in a container filled with plant-based or fungal-based cellulose scaffold particles according to any one of claims 1-25; and b) allowing the cells to grow on the surface of the scaffold particles for at least 1 day.

29. The method of claim 28, wherein the growth of cells on the surface of the particles is in the form of a monolayer, cellular aggregates, spheroids or both.

30. The method of claim 28, wherein the method further comprises: agitation of the container at regular or irregular intervals after step a) or after step b).

31 . The method of claim 28, wherein the agitation step after step a) increases the attachment of cells to the surface of the scaffold particles and contributes to diffusion or movement of the culture medium and gasses within the container.

32. The method of claim 28, wherein the agitation step after step b) directs the growth of cells as monolayer or cellular aggregates or spheroids, improves the viability of cells, and increases the proliferation of cells on the surface of the scaffold particles.

33. The method of claim 28, wherein the agitation step is carried out continuously or intermittently.

34. The method of claim 28, wherein the agitation step is carried out manually or by automated means.

35. The method of claim 28, wherein the agitation step is carried out for a duration of 0.5 minute to 60 minutes.

36. The method of claim 28, wherein the agitation step is carried out with a static of 300 minutes, 270 minutes, 240 minutes, 210 minutes, 180 minutes, 150 minutes, 120 minutes, 90 minutes, 60 minutes or 30 minutes.

37. The method of claim 28, wherein the method further comprises: spinning an impeller within the container at 20 rpm - 90 rpm at regular or irregular intervals after step b).

38. The method of claim 37, wherein the spinning step increases the proliferation of cells by allowing more time for the cells to interact with the surface of the scaffold particles and contributes to diffusion or movement of gasses and the culture medium within the container.

39. The method of claim 37, wherein the spinning step is carried out at 30-minute intervals.

40. The method of claim 37, wherein the spinning step is carried out at one-hour intervals.

41 . The method of claim 37, wherein the spinning step is carried out once an hour after step b) followed by every four hours.

42. The method of claims 37-41 , wherein the spinning step is carried out for a period of 20 to 40 minutes.

43. The method of claims 37-41 , wherein the spinning step is carried out at 30 rpm to 80 rpm.

44. The method of claim 27, wherein the method is carried out at a temperature ranging from 15°C to 45°C.

45. The method of claim 28, wherein the method is carried out in the presence or absence of carbon dioxide and any known buffer solution.

46. The method of claim 28, wherein the method is carried out in the absence of light.

47. The method of claim 28, wherein multiple batches of culture medium containing cells are added to the container at regular intervals.

48. The method of claim 28, further comprising a pre-processing step prior to step a), wherein the scaffold particles are pre-processed by: washing the scaffold particles using a salt solution, pre-treating the scaffold particles chemically with a pharmaceutically acceptable compound, pre-treating the scaffold particles physically, or modifying the surface of the scaffold particles.

49. The method of claim 48, wherein the washing step comprises an incubation step in which the scaffold particles are incubated in the salt solution overnight.

50. The method of claim 48, wherein the salt solution is PBS.

51. The method of claim 48, wherein the culture medium is with or without FBS, with or without FBS substitute, with or without antibiotics, with or without glucose, with or without indicator, or with or without CO2 buffering solution.

52. The method of claim 28, wherein the culture medium is DMEM, MEM, Opti-MEM, MEM Alpha, L-15, McCoy’s 5A, RPMI-1640, F-12 or Hanks' Balanced Salt Solution.

53. The method of claim 48, wherein the pharmaceutically acceptable compound is a peptide, a drug, an antibody, a cofactor, a vitamin or an enzyme.

54. The method of claim 48, wherein the pharmaceutically acceptable compound is hydrogen peroxide, bicarbonate, fetal bovine serum, glycine, ascorbic acid, gelatin, RGD motif, fibronectin, cadherins, integrins, nectins, afadin, p-catenin/aE-catenin, E-cadherin, cell attachment factor, or charged amino acids.

55. The method of claim 48, wherein the scaffold particles are pre-treated with hydrogen peroxide, glycine, ascorbic acid, fetal bovine serum, or gelatin, preferable wherein the hydrogen peroxide is used at a concentration ranging from 0.1% to 10%.

56. The method of claim 48, wherein the scaffold particles are pre-treated with bicarbonate salt at a concentration ranging from 1% to 10%.

57. The method of claim 48, wherein the scaffold particles are pre-treated with sodium hydroxide at a concentration ranging from 1% to 10%.

58. The method of claim 48, wherein the scaffold particles are pre-treated physically by homogenization, dispersion, low-shear mixing, high-shear mixing, ultrasonic processor, emulsifying equipment, lyophilization, rehydration, heating, freezing with or without liquid nitrogen, mercerization, micronization or sonication.

59. The method of claim 48, wherein the pre-treatment modifies the surface of the scaffold particles and enhances the proliferation of cells by increasing cellular attachment to the surface of the scaffold particles, by increasing cellular entrapment to the surface of the scaffold particles or by increasing cell proliferation capacity on the particles.

60. The method of claim 48, wherein the modification is carried out by functionalizing the surface of the scaffold particles.

61 . The method of claim 60, wherein the functionalization is carried out by providing a functional group that creates a charge on the surface of the scaffold particles.

62. The scaffold particles of claim 61 , wherein the functional group is a primary amine, a tertiary amine, a quaternary compound, denatured collagen, gelatin or a combination thereof.

63. The method of claim 48, wherein the modification is carried out by succinylating the surface of the scaffold particles.

64. The method of claim 48, wherein the modification is carried out by cross-linking the surface or the scaffold particles using a cross-linker agent to create cross-linked cellulose microcarriers.

65. The method of claim 64, wherein the cross-linker agent is citric acid or paraformaldehyde.

66. The method of claim 28, further comprising a sterilization step prior to step a), wherein the scaffold particles are sterilized for a pre-determined period of time.

67. The method of claim 66, wherein the sterilization step is carried out by autoclaving, water steam treatment, chemical steam treatment, ethylene oxide treatment, alcohol treatment, UV irradiation, Gamma irradiation or pasteurization.

68. The method of claim 67, wherein the sterilization step is carried out for 10 to 60 minutes.

69. The method of claim 28, further comprises a screening step prior to step a), wherein the scaffold particles are screened prior to filling in the container.

70. The method of claim 69, wherein the screening step involves selection of specific sized scaffold particles.

71 . The method of claim 69, wherein the screening step involves selection of scaffold particles with varying sizes to obtain an effective particle size distribution.

72. The method of claim 69, wherein the screening step involves quantification of the number of scaffold particles.

73. The method of claim 72, wherein the quantification step is followed by increasing the number of scaffold particles if found below a threshold amount.

74. The method of claim 28, wherein the container is filled with the scaffold particles sized in the range of 20 pm to 700 pm.

75. The method of claim 28, wherein the container is filled with the scaffold particles sized in the range of 40 pm to 150 pm.

76. The method of claim 48, further comprising a purification step wherein the scaffold particles are purified after the pre-processing step.

77. The method of claim 28, further comprising a quantification step to quantify the proliferation of cells.

78. The method of claim 77, wherein the quantification step is carried out by a biomolecule quantification assay, cell-quantification assay, DNA quantification assay, genome quantification assay, cell-adherence quantification analysis, cell metabolism assay, metabolite assay, microscopy, protein assay or quantification of cell-specific antigens.

79. The method of claim 77, wherein the quantification step is followed by an optimization step to optimize the growth conditions in the container, to optimize the cell culture conditions, to optimize the number of scaffold particles, to optimize the size of the scaffold particles, to optimize the agitation parameters in the container, to optimize the cell to scaffold particle ratio or a combination thereof.

80. The method of claim 79, wherein the optimization step increases the proliferation of cells on the scaffold particles.

81 . The method of claim 79, wherein the optimization step is modulated to direct the growth of cells to either form monolayer, spheroids or cellular aggregates on the surface of the scaffold particles.

82. The method of claim 28, wherein the in-vitro proliferation is increased by increasing the number of scaffold particles in the container.

83. The method of claim 28, wherein the in-vitro proliferation is increased by changing, altering or selecting the size of the scaffold particles in the container.

84. The method of claim 28, wherein the container is filled with a solution comprising the scaffold particles.

85. The method of claim 28, wherein the container is a T-175 flask, a 6-well plate, well plates, a siliconized plate or flask, a Hyperflask, a Spinner flask, a Cell stack culture chamber, a vessel, a bioreactor, a cell culture microreactor, a cell-culture chip, a microfluidic cell culture device, a lab-on-a-chip device orthe combination of two or more of this recipients or assembled laminates.

86. The method of claim 28, wherein the method further comprises a step of separating the growth of cells from the scaffold particles by mechanical, chemical, or enzymatic action.

87. The method of claim 86, wherein the growth of cells is separated by enzymatic action using cellulase, trypsin, or TrypLE.