WO2020234170A1 - Cell-spheroid production in 2d cell culture system - Google Patents

Abstract

A method of producing three-dimensional spheroids of cells, using a two- dimensional cell culture device comprising at least one chamber having a bottom surface comprising separated compartments, comprising the following steps: i. introducing cell culture media comprising cells into the cell culture device, ii. allowing said cells to settle within the compartments, and iii. culturing the cells for a time sufficient for the formation of cell spheroids, wherein in at least 80% of the compartments at least one cell spheroid is formed.

Description

CELL-SPHEROID PRODUCTION IN 2D CELL CULTURE SYSTEM

Description

Field of the Invention

[0001] The present invention relates to the field of cell culture systems.

Specifically, it relates to methods of producing three-dimensional spheroids of cells using a two-dimensional cell culture system comprising a compartmentalized bottom surface and use of cell spheroids produced in such a system.

Background Art

[0002] Scaffold free high cell density cultures have been extensively used in tissue engineering and many other fields of medical science. Three types of culture are mainly used: (1) hanging drop culture, (2) pellet culture and (3) micro-mass culture. (1) Hanging drop culture has been used in a variety of different set ups from cancer research to differentiation studies however handling of many standard culturing systems can be challenging. Additionally, evaporation and limited possibility of media change often require transfer of the generated pellets to well plates for longer culture times. (2) Pellet cultures are frequently used for

chondrogenic differentiation but are sometimes also used for the generation of other organoids. Historically pellet cultures were done in rather big vessels (e.g. 1.5 - 15 ml centrifuging tubes), which led to high consumption of media and laborious maintenance of cultures. This has by some extent been alleviated by the use of compounds blocking cell adherence such as agarose or poly(2-hydroxyethyl methacrylate) (pHEMA) to coat e.g., 96 well plates, which is often referred to as liquid overlay technique. Nevertheless, time and media consumption still leave much room for optimization. (3) Micro-mass culture contrasts itself from the other two methods as it does not predominantly use gravity to mediate pellet/organoid formation. While previously used for developmental studies it has more recently been used for drug testing and chondrogenic differentiation.

[0003] EP2617807A1 discloses methods of generating spheroids using culture substrates having a plurality of dents forming compartments in which cells are cultured to form spheroids. The culture substrate surface of the dent is a non-flat surface, specifically it is a smooth concave plane. The dents are formed by laser irradiation on the culture substrate surface and are placed very close together, leaving almost none of the original surface. At the peripheries of the dents, the synthetic resin material of the culture substrate is melted and piled up to form banks.

[0004] W02017/142410A1 discloses methods of growing spheroids using a round- bottom, optically clear insert plate, which can be reversibly attached to a

standardized microplate. The cells are cultured in the wells of the insert plate, wherein the bottom of each well has a concave arcuate surface (see Fig. 3).

[0005] EP3296018A1 discloses methods of growing arrays of organoids using a cell culture device, which has a surface imprinted with cavities or microwells of various sizes, shapes and depths. Preferably, the wells are round (U) -bottomed, so that the seeded cells are all gathered at the bottom of the cavities.

[0006] US2016/0324991A1 discloses micronized platforms comprising a plurality of testing regions, which may be individual chambers holding tumor spheroids, for in vivo implantation in an animal host. The platform is used to study biosamples in an in vivo setting.

[0007] US2019/0055590A1 discloses methods of generating spheroids, which are useful for screening techniques, using multi-well plates, wherein each of the wells has a lowly absorptive bottom having a U-shaped section.

[0008] EP2759592A1 discloses cell culture devices comprising suspension chambers called culture spaces (see e.g., Fig 3), wherein the surface of each of the culture spaces is processed by glass processing or by forming a functional group thereon by plasma treatment, so that the surface has a water contact angle of 45 degrees or less, i.e. it is a hydrophilic surface.

[0009] Generally, cell culture devices used for the generation of spheroids have a concave bottom, to ensure that cells can aggregate, and/or are anti-adhesive in the hopes of promoting generation of spheroids by preventing cells from sticking to the bottom of the device.

[0010] The unifying downside of these methods is their time and reagent

consumption during longer culture maintenance. One efficient way to alleviate these factors is partial or total automation of pellet generation, maintenance and analysis. While earlier approaches mainly attempted to simplify pellet generation, by e.g. using pipetting automates to distribute cell solutions homogeneously into multi-well plates and letting them aggregate by sedimentation or subsequent manual centrifugation (Welter et al ., 2007), newer systems strife for all in one solutions, often also including automatized analysis. Examples for this are the BioMek Cell Workstation (Beckman Coulter, USA), which was shown to allow fully automated alginate, hanging drop and pellet culture. Another automation setup for pellet culture was presented by BioTek, using NanoShuttle-PL (nano3D

Biosciences, USA) nanoparticles to magnetize cells allowing easier handling with their automatic liquid handling system.

[0011] Most other automated systems for spheroid generation and handling use commercial or in lab produced solutions for hanging drop culture. Tung et al.

presented a custom-made hanging drop plate design, which allowed automated pellet generation and maintenance of molarity for up to two weeks. (Tung et al., 2011) This was achieved by holes in the plate enabling access to the drop from above, therefore allowing for easier media changing and introducing additional reservoirs to minimize evaporation. A similar plate design is the commercially available Perfecta3D hanging drop plate system (3D Biomatrix, USA), which was applied by Beckman Coulter using the BioMek Cell Workstation (Beckman Coulter, USA). Other systems, while employing slightly different plate designs use similar approaches to achieve hanging drop automatization. This includes micro-hole inserts from Eplasia (Kurary, Japan) used by BioTek and the Gravity Plus hanging drop system from InSphero (Switzerland) applied by PerkinElmer, which both used their in-house liquid handling systems for automation. None of the systems showed cultivation times longer than 2 weeks, however easy ways for pellet transfer to standard well plates were presented, which would allow further maintenance of pellets.

[0012] While all of these methods achieve automation and therefore vastly improve on the amount of work-hours and reagents used due to higher precision and no need for constant supervision, these methods remain mostly unused due to the high cost of the equipment involved, which especially for smaller labs makes them often not feasible. To still allow for the generation of high quantities of micro mass pellets with highly repeatable size and morphology, micro-moulding (Babur et al., 2013; Dean et al., 2007; Desroches et al., 2012; Napolitano et al., 2007) and surface patterning (Hardelauf et al., 2011) have been explored as a more affordable option. Some of these methods allow for the design of interestingly shaped scaffold free cell constructs, like honeycombs (Dean et al., 2007), which could have effective applications for regenerative medicine applications. However, most of these methods have a tedious production process, e.g. multistep casting procedures (Babur et al., 2013), and are not easily integrated into standard culturing vessels. To allow optimal usability of such a system easy accessibility to “raw materials”, as well as easy and efficient means of production are necessary. Bruinink and Wintermantel presented an approach, showing that rat bone marrow cells react to groves on standard cell culture plastic by forming pellets (Bruinink and Wintermantel, 2001). This system, however, is rather rudimentary, especially due to a manufacturing process relying on manually scratching the culture surface using a scalpel, and does not produce uniform cell spheroids.

[0013] Therefore, there is an urgent need in the field for an easily mass-producible culturing system for autonomous pellet formation. It is particularly desirable to provide a system using standard cell culturing vessels (e.g. petri dishes) as raw material, for further cost reduction.

Summary of invention

[0014] It is the objective of the present invention to provide a method of producing cell spheroids with uniform size, while at the same time reducing reagent and time consumption.

[0015] The objective is solved by the subject matter of the present invention.

[0016] According to the invention there is provided a method of producing three- dimensional spheroids of cells, using a two-dimensional cell culture device comprising at least one chamber having an adhesive, flat bottom growth surface, which is separated by incisions into compartments, allowing distribution of cell culture media across said at least one chamber, comprising the following steps: i. introducing cell culture media comprising cells into the cell culture device, ii. allowing said cells to settle within the compartments, and

iii. culturing the cells for a time sufficient for the formation of cell spheroids, wherein in at least 80% of the compartments at least one cell spheroid is formed.

[0017] Specifically, cell spheroids are formed in 80, 81, 82, 83, 84, 85, 86, 87, 88,

89, 90, 91, 92, 93, 94, 95, 96, 97, 98, up to 99 or 100% of the compartments.

[0018] According to one embodiment, the compartments on the bottom surface of the two-dimensional cell culture device described herein are separated by incisions or by elevated structures, specifically bulges. Specifically, the incisions or bulges described herein are formed by a laser or a sharp object. Specifically, the bulges described herein are bulges comprising incisions. According to a specific

embodiment, cell spheroids are formed in at least 80% of the compartments and in at least 50, 60, or 70%, preferably at least 80 or 90%, of the incisions or bulges described herein.

[0019] According to a further embodiment, the cells are mammalian cells or invertebrate cells. Specifically, the cells described herein are human cells, preferably human cells derived from cartilage or fat tissue.

[0020] According to yet a further specific embodiment the cells used in the method described herein, are adipose derived stromal/stem cells (ASC) or human articular chondrocytes (HAC).

[0021] According to one embodiment, the method described herein comprises co culturing cells, specifically at least two different kinds of cells. Specifically, the co cultured cells used in the method described herein are ASCs and human articular chondrocytes HACs. Preferably, in a co-culture two different cell types are cultured in a ratio, for example at a ratio of 50:50 or 20:80 in the two-dimensional cell culture device described herein. Specifically, when ASCs and HACs are co-cultured in the cell culture device described herein, they are preferably cultured in a ratio of 80:20, 50:50 or 20:80% ASC:HAC.

[0022] According to a further embodiment, the compartments described herein comprise a diameter of at least 0.1 mm and up to 15 mm. Specifically, the compartments described herein comprise a diameter of at least 0.2, 0.3, 0.4, 0.5,

1.0, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12,

12.5, 13, 13.5, 14, 14.5 or 15 mm. Specifically, the compartments are of a regular shape, such as an angular shape, for example square, rectangular, triangular, regular pentagon, regular hexagon, regular octagon, or a round shape, for example circular or oval. Specifically, the compartments are of an irregular shape, having sides and/or angles of different length and size, for example irregular pentagon, irregular hexagon, or irregular octagon.

[0023] Specifically, the two-dimensional cell culture device described herein comprises at least one chamber. Specifically, the two-dimensional cell culture device described herein comprises at least 2 chambers, specifically at least 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, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75,

76, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100 or more chambers, preferably at least 4, 6, 8, 12, 24, 48 or 96 chambers. [0024] Specifically, the at least one chamber described herein comprises a diameter of about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 130, 140 or 150 mm. The cell culture device described herein may comprise chambers of different sizes and shapes. The chambers described herein may comprise compartments of different sizes and shapes.

[0025] According to one embodiment of the method described herein, the cells are cultured in the cell culture device described herein for at least a day, specifically 3 days, preferably at least 1 week. Specifically, the cells are cultured according to the method described herein for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 days or longer. Specifically, the cells are cultured for at least 1, 2, 3 or 4 weeks. Specifically, the cells are cultured in cell culture media under conditions allowing cell growth, specifically under appropriate temperature and gas mixture. Specifically, the cells are cultured at a temperature between 35 ° C and 38 C, specifically about 37 C. Specifically, the cells are cultured at about 5%

C0₂, 20% 0₂ and 95% relative humidity. The cells are typically cultured under the appropriate oxygen concentration. Cells may also be cultured under hypoxic conditions wherein 0₂ is comprised at a concentration of less than 20 %.

[0026] Specifically, the cell spheroids described herein are generated after culturing cells in the cell culture device described herein for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 days or longer. Specifically, the cell spheroids are generated after about 1, 2, 3, 4, 5 or 6 weeks.

[0027] According to a further embodiment of the invention, the method described herein comprises the step of harvesting the cell spheroids. Specifically, the cell spheroids are harvested after culturing in the cell culture device for at least 1, 2, 3, 4, 5 or 6 days or 1, 2, 3 or 4 weeks.

[0028] Specifically, the cell spheroids produced by the method described herein are of uniform size which correlates to the size of the compartments within the, at least one, chamber of the cell culture device described herein. Specifically, the size of a cell spheroid corresponds to the surface area of the corresponding

compartment. Specifically, a chamber comprising compartments of the same size yields cell spheroids of almost identical or similar size. Specifically, a chamber comprising compartments of different sizes yields cell spheroids of different sizes, corresponding to the surface areas of the different compartments. [0029] According to one embodiment, cell spheroids of uniform size, corresponding to the size of the respective compartments, are formed in at least 80%, preferably at least 90% of the respective compartments. Specifically, cell spheroids of uniform size are produced according to the method provided herein in at least 80, 81, 82,

83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, up to 99 or 100% of the compartments. Specifically, cell spheroids of uniform size, corresponding to the width and/or depth of the respective incisions or bulges, are formed in at least 50, 60, or 70%, preferably at least 80 or 90%, of the incisions or bulges described herein.

[0030] Further provided herein is the use of the cell spheroids produced according to the method described herein in a three-dimensional cell culture. Specifically, the cell spheroids are cultured in a three-dimensional cell culture comprising a hydrogel and/or a scaffold. Specifically, the three-dimensional cell culture comprises a fibrin hydrogel, specifically high and/or low-density fibrin hydrogel.

[0031] Specifically, the cell spheroids produced according to the method described herein are used for regenerative medicine applications, such as in vitro production of cartilage, bone or muscle tissue.

[0032] Specifically, the cell culture device provided herein is used for high- throughput screening of exogenous influences of physical, chemical or biological nature. For example, the cell culture device can be used to screen the effect of chemical compounds or environmental conditions on cell spheroids.

Brief description of drawings

[0033] Figure 1. Size modulation of ASC pellets using different grid sizes (a)

Pellet size 1 mm vs 3 mm and (b) histograms of size distribution (****= p <

0.0001). Bright-field of pellets generated on (c) 1 mm and (d) 3 mm grid plates (pbar: 500pm)

[0034] Figure 2. Bright-field and fluorescence imaging of pellet formation for ASC and 0.5:0.5 ASC:HAC co-culture on a 3mm grid plate over a period of 29 days.

(pbar: 500 pm)

[0035] Figure 3. SEM imaging of different stages during pellet formation (a) Beginning contact loss and retraction at grid borders (b) Rolling up of cell layer (c) Further aggregation and formation of a knob-like structure (d) Fully formed pellet (pbar: 200 pm) [0036] Figure 4. Pellet formation speed of ASC or co-culture (ratio ASC:HAC) on 3 mm grid plates (a) Pellet count over a period of 25 days (b) Duration of reaching a pellet count threshold of 25. *= p < 0.05; **= p < 0.01

[0037] Figure 5. Effect of ASC:HAC ratio on resulting pellet size for 3 mm grid plates (a). Histogram of pellet size distribution for different ASC:HAC ratios: (b) ASC only, (c) 0.8:0.2, (d) 0.5:0.5 and (e) 0.2:0.8. ****= p < 0.0001

[0038] Figure 6. Histological sections of different ASC:HAC ratio pellets grown on 3 mm grid plates, stained for ECM formation (Azan), collagen 2 and GAGs (Alcian). (pbar: 100 pm; pbar inserts: 20 pm)

[0039] Figure 7. Sprouting behavior of ASC pellets (lmm and 3mm) embedded into low and high-density fibrin, and 0.5:0.5 ASC:HAC pellets (3mm) embedded in low density fibrin.

[0040] Figure 8. Bright-field and corresponding histological sections of lmm and 3mm ASC pellets in low and high-density fibrin and 3mm 0.5:0.5 ASC:HAC co culture pellets in low density fibrin after 14 days of culture (pbar bright-field:

500pm pbar histology 100pm).

[0041] Figure 9. Histological sections of ASC-based, grid generated spheroids and pellets after 3 and 5 weeks of culture, stained with AZAN for general matrix deposition, Alcian blue for glycosaminoglycan deposition and collagen type 2. Bar = 100 pm.

[0042] Figure 10. Histological sections of ASC-chondrocytes based, grid generated spheroids and pellets after 3 and 5 weeks of culture, stained with AZAN for general matrix deposition, Alcian blue for glycosaminoglycan deposition and collagen 2. Bar = 100 pm.

[0043] Figure 11. C2C12 cells spreading over the compartment and forming nodules within a non-confluent cell layer.

[0044] Figure 12. Macroscopic images of the grid plate with 3mm grid size (left) and a standard petri dish with flat surface (right).

[0045] Figure 13. SEM image of C02-laser generated incisions within the plastic surface of the cell culture device. Magnification 150x.

[0046] Figure 14. SEM images of plastic surfaces compartmented with three different instruments (left: top view, length indicator: 400pm; right: cross section, length indicator: 100pm). Upper row: Scalpel incisions, middle row: Stanley knife, lower row: fraise. [0047] Figure 15. Bright field images of a scalpel-incised compartmentation of the growth surface with a cell layer of ASC/TERT1. Left: After seven days the cells align (arrow) and aggregate (arrow head) along the incision border. Right: After fourteen days a detachment of the cell layer from the corners is visible.

Description of embodiments

[0048] To overcome the shortcomings of present spheroid cell culture systems such as high consumption of time and reagents, a new system for spheroid generation alleviating these problems is provided herein. As described herein, the bottom surface of two-dimensional cell culture devices, such as for example conventional cell culture dishes, is compartmentalized and cells cultured thereon spontaneously and autonomously self-assemble into three-dimensional cell spheroids.

[0049] According to a paradigm in the field of cell culture, contact inhibition of cells growing on flat surfaces in two-dimensional cell culture hampers the production of three-dimensional cell formations. It is thus highly surprising, that three-dimensional cell spheroids can be produced in the two-dimensional cell culturing device described herein. It is even more surprising, that the size of the spheroids generated is reproducible and determinable by the size of the device’s compartments.

[0050] Advantageously, the cell culture device described herein is designed to be easily mass-producible and to reduce media consumption since numerous cell spheroids can be produced within one chamber. Accordingly, reagent cost and handling time are significantly reduced.

[0051] Unless indicated or defined otherwise, all terms used herein have their usual meaning in the art, which will be clear to the skilled person.

[0052] The terms“comprise”,“contain”,“have” and“include” as used herein can be used synonymously and shall be understood as an open definition, allowing further members or parts or elements.“Consisting” is considered as a closest definition without further elements of the consisting definition feature. Thus “comprising” is broader and contains the“consisting” definition.

[0053] The term“about” as used herein refers to the same value or a value differing by +/-5 % of the given value.

[0054] The term“three-dimensional spheroids of cells", unanimously used with “cell spheroids”, as used herein refers to aggregates of cells in culture that comprise three-dimensional architecture. Specifically, the cell spheroids produced by the method described herein are of uniform size, wherein the size corresponds to the size of the compartments and/or the size of the incisions or bulges separating the compartments. It thus follows, that the size of the cellular spheroids described herein can be controlled by the size of the compartments and/or the incisions or bulges. Specifically, varying the size of the compartments allows producing cell spheroids of a pre-determined size and/or shape.

[0055] Specifically, the cell spheroids described herein do not comprise any supplementary material such as a synthetic matrix. Cellular spheroids represent excellent models for the in vitro study of biological functions of cells, including stem cells and cancer cells, specifically since they allow closely mimicking in vivo behavior.

[0056] According to a specific example, the term cell spheroid encompasses organoids. Specifically, an organoid is a collection of organ-specific cell types that develops from stem cells or organ progenitors and self-organizes into a cellular spheroid. Typically, organoids self-organize their structure through three- dimensional spatial arrangement and spatially restricted lineage commitment in a manner similar to in vivo.

[0057] Specifically, the cell spheroids described herein are not limited in size. Exemplary, cell spheroids described herein comprise as few as 2 cells or up to lxlO⁶ or more cells. In certain cases, a cell spheroid produced by the method described herein comprises about lxlO³, 5xl0³, lxlO⁴, 5xl0⁴, lxlO⁵, 5x10^s, lxlO⁶, 5xl0⁶ or lxlO⁷ cells.

[0058] Specifically, the cell spheroids described herein may be of different shape. The most common shape of a cell spheroid is the round shape; however different cell types yield differently shaped cell pellets. According to a specific example, muscle cells or nerve cells form cell pellets of an elongated architecture, whereas cartilage cells typically form round cell pellets.

[0059] The term“cell” as used herein, is understood to refer to any cell that can be grown in a cell culture system. Specifically, the cells used herein are eukaryotic cells; preferably they are mammalian or invertebrate cells. Mammalian cells used herein can, for example, be of primate origin, such as e.g. human, ape or monkey; rodent origin, such as e.g. mouse, rat or hamster, carnivore origin, such as e.g. cat, dog, or ungulate origin, such as e.g. cattle, horse, pig or deer. For example, mammalian cells used herein can be derived from epithelial tissue, muscle tissue, nervous tissue and/or connective tissue. Specifically, cells used in the method provided herein are stem cells, bone cells, muscle cells, fat cells, skin cells, nerve cells, endothelial cells, or cancer cells. According to a specific embodiment of the method described herein, a single cell type is cultured or multiple cell types are cultured in the device described herein. Specifically, in a so-called co-culture, more than one, preferably two, different cell types are cultured. The different cell types can be cells of the same or different species and can be cells of the same or different tissues. For example, chondrocytes, myocytes, neurons or adipocytes can be co-cultured with stem cells or cancer cells. Specifically, chondrocytes, myocytes, neurons or adipocytes are co-cultured with stem cells or fibroblasts.

[0060] According to a specific example described herein, the cells used in the method provided herein are stem cells, specifically adipose derived stromal/stem cells (ASCs). ASCs are an excellent source for tissue regeneration, including for example regeneration of damaged cartilage, since they are easily available in high cell numbers from subcutaneous fat, and they can be harvested under reduced burden for the donor, compared to for example isolation of bone marrow derived stem cells. Specifically, using the cell culture device described herein, stem cells, such as ASCs, spontaneously form spheroids within about 3 to 4 weeks.

Specifically, using the cell culture device described herein, cell spheroids form autonomously.

[0061] According to a further specific example, the cells used in the method provided herein are chondrocytes, specifically human articular chondrocytes

(HACs). Chondrocytes are the only cell type present in articular cartilage and regulate tissue homeostasis by a fine balance of metabolism that includes both anabolic and catabolic activities. Surprisingly, using the method provided herein, chondrocytes could successfully be grown in a two-dimensional cell culture device and cell spheroids spontaneously formed by chondrocytes could be produced. The produced cell spheroids were of uniform size and shape, corresponding to the size of the compartment. Typically, chondrocytes grown in the cell culture device described herein formed cell pellets after 3 to 4 weeks.

[0062] According to a further example, the cells used in the method provided herein are chondrocytes and stem cells. Specifically, stem cells and chondrocytes are grown in a co-culture at a ratio of about 90:10, 80:20, 70:30, 60:40, 50:50, 40:60, 30:70, 20:80 or 10:90 stem cells to chondrocytes, specifically ASC:HAC.

Surprisingly, the time required for formation of cell spheroids is significantly reduced when stem cells and chondrocytes are grown in co-culture in the cell culture device provided herein. While culturing adipose derived stem cells and chondrocytes separately in the cell culture device described herein, production of uniform cell spheroids is typically achieved within 3 to 6 weeks, depending on initial cell number and age of donor. Co-culture of stem cells and chondrocytes in the present cell culture device, however, yields uniform cell spheroids of

chondrocytes in less than 3 weeks or 2 weeks or even less than 1 week.

[0063] According to a specific embodiment, the cells cultured in the two- dimensional cell culture device described herein are invertebrate cells. Specifically, the invertebrate cells are cells derived from mollusks, annelids or cnidarians or arthropods, such as for example insects or arachnids. Even more specifically, the invertebrate cells used in the method described herein are derived from insects, such as for example the common fruit fly drosophila melanogaster.

[0064] As used herein, the term“two-dimensional cell culture device” is

understood to refer to a device that allows the culturing of cells in a monolayer. Numerous two-dimensional cell culturing devices that are suitable for the method described herein are known to the person skilled in the art. Specifically, the cell culture device used herein is made of plastic or glass material or a mixture thereof. Specifically, the cell culture device used herein is made of a plastic material such as for example polystyrene, polypropylene or polycarbonate. The most commonly used material is polystyrene, which can be colored white for example by the addition of titanium dioxide or black, for example by the addition of carbon, polypropylene is typically used for the construction of plates subject to wide changes in temperature, polycarbonate is cheap and easy to mold.

[0065] Commonly used two-dimensional cell culturing devices according to the method described herein include petri dishes and well plates.

[0066] Specifically, a petri dish is a glass or plastic dish that is typically cylindrical. When the two-dimensional cell culture device is a petri dish it comprises one chamber. Typically, as mentioned above, said chamber is round, however there is no limitation regarding the shape of the chamber(s) of the two-dimensional cell culture device described herein. Various shapes are envisioned herein, beyond the typical round shape, such as for example square, hexagonal, rectangular or triangular chambers.

[0067] Specifically, a well plate, or also called microplate or multi-well plate, is a flat plate with multiple chambers, also called wells. Preferably, the chambers of multi-well plates are round or square; however various shapes are envisioned herein including for example hexagonal, rectangular or triangular wells. Specifically, a cell culture device as described herein comprising more than one chamber may comprise 6, 12, 24, 48, 96, 384 or 1536 chambers or even up to 3456 or 9600 chambers, preferably arranged in a 2:3 rectangular matrix. Specifically, a chamber of a microplate typically can hold somewhere between tens of nanoliters to several milliliters of liquid.

[0068] The cell culture device described herein comprises one or more chambers comprising a diameter of about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or up to 150 mm or any value in between. According to a specific example, the chambers of a cell culture device as described herein comprising 6 or 12 chambers, may comprise a diameter of 10, 14 or 20 mm. The chambers of a cell culture device comprising 24 chambers may, for example, comprise a diameter of 10 or 13 mm, a device comprising 48 chambers may e.g. comprise chambers at a diameter of 6 mm and a device comprising 96 chambers may comprise chambers at a diameter of 5 mm.

[0069] As used herein, the term“bottom growth surface” refers to the side of the layer at the bottom of the cell culture device which comprises the incisions and/or bulges described herein. In other words, the bottom surface of the one or more chambers of the cell culture device described herein is the surface comprising the compartments that are separated. Typically, the bottom surface is the upward facing side of a cell culture device whereupon the cells adhere.

[0070] The term“compartments” as used herein refers to fields that are separated by physical means, for example separated by incisions or bulges. The

compartmentalization described herein separates the bottom surface into fields by elevated or indented lines that are high enough to facilitate formation of the three- dimensional spheroids of cells described herein and low enough to allow

distribution of the cell culture media across the whole chamber.

[0071] The compartments of the device described herein may be of various sizes and shapes. Specifically, the compartments may be squared, rectangular, round, triangular, star-shaped, hexagonal, or may be of irregular shape. A chamber described herein may also comprise compartments of different sizes and/or shapes. According to a specific embodiment of the method provided herein, the compartments described herein comprise a diameter of at least 0.1 mm and up to 10 mm or even longer, such as for example 20, 30, 40, 50, 60, 70, 80, 90 or 100 mm. In the case, where the compartments are squared, they may comprise length and width of 0.1 to 10 mm and in the case where they are rectangular, they may comprise a length of 0.1 to 10 mm and a width of 0.1 to 10 mm.

[0072] Specifically, the size and shape of the compartments is adjusted depending on the cell type to be cultured. For example, to produce three-dimensional cell pellets of muscle cells or nerve cells, the compartments may be of an elongated shape, such as a rectangle comprising a length of about 1 to about 15 mm, specifically, about 2 to about 6, even more specifically about 2.5, 3, 3.5, 4, 4.5, 5,

5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5 or 15 mm, and a width of about 0.2 to about 10 mm, specifically about 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10 mm. According to a specific example, the compartment comprises a length of about 2 mm and a width of about 1 mm, or a length of about 4 or 6 mm and a width of about 1 or 2 mm. According to a further specific example, the compartment comprises a length of 10 or 15 mm, and a width of 0.2, 0.5 or lmm.

[0073] According to yet another example, to produce three-dimensional cell pellets of chondrocytes and/or stem cells, the compartments may be squared, comprising a length and a width of about 0.5 mm to 5 mm. Specifically, the compartments may comprise a length and a width of about 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10 mm or more.

[0074] By adjusting the size and shape of the compartments, the dimensions of the cells in their natural three-dimensional environments can be mimicked. The ease of production of the cell culture device allows adjustment of the device to any cell type’s needs and thus the optimization of the growth environment is easily achieved.

[0075] The term“incision” as used herein, refers to an indentation or a cut in the bottom surface of the cell culture device described herein leading to

compartmentalization of said bottom surface. The incisions described herein can be of a straight or a curved line, preferably the incisions are in straight lines.

Specifically, the incisions described herein are about 20 to 200 pm in depth, specifically about 50 to 150pm, specifically measured from the surface level of the bottom growth surface, preferably about 150pm, and comprise a width of about 50 to 500 pm, specifically about 50, 100, 200, 300, 400, 500 or 600 pm.

[0076] According to a specific embodiment, the incisions comprise a ridge along each edge of the incision. Such ridge may comprise a height of about 40 to 100 pm or more, preferably about 50, 60, 70 or 80 pm, and a width of about 40 to 100pm pm or more, preferably about 50, 60, 70 or 80 pm. Specifically, the incisions are smooth. Specifically, the incision is below the level of the bottom surface and the ridge is above the level of the bottom surface of the cell culture device. Specifically, the ridge comprises overhangs. Specifically, the overhangs are parts of a ridge which stretch outwards from the incision and do not touch the bottom surface. An overhang may comprise a width of about 10 to 40 pm or more. A specific example of an incision comprising ridges is depicted in Figure 13.

[0077] Numerous methods can be used to engrave the incisions described herein. Specifically, the incisions can be created using a laser, or manually using a sharp object, such as for example a scalpel, Stanley knife or a fraise. According to a specific example, the incisions described herein are formed using a computer- guided laser. Use of a computer-guided laser, such as an argon laser or a C0₂ laser, is particularly useful since such a laser cuts the incisions with precision to create incisions of uniform dimensions comprising for example uniform depth and width.

[0078] Specifically, engraving incisions or adding bulges to the bottom surface of the cell culture device described herein removes any coating, with which cell culture devices are typically treated to improve cell adherence. Accordingly, cells cannot adhere along incisions or bulges, but, form cell spheroids therein or thereon.

[0079] The term“bulge” as described herein, refers to an elevation in the bottom surface of the cell culture device described herein leading to compartmentalization of said bottom surface. Specifically, the bulges are of a plastic, ceramics or glass material, preferably plastic such as polystyrene or polycarbonate. The bulges described herein can be of a straight or a curved line, preferably the bulges are in straight lines. Specifically, the bulges described herein comprise a height of at least about 40 to 200 pm, preferably about 50 to 100 pm, and a width of about 40 to 400 pm, preferably about 100 to 300 pm. [0080] According to a specific embodiment, the bulges described herein comprise incisions and may thus be referred to as elevated incisions. In the embodiment, where the bulges comprise incisions, the bottom surface of the cell culture device described herein thus comprises incisions which are above the level of the bottom surface.

[0081] The cell culture device described herein comprises an adhesive bottom growth surface. Specifically, the entire bottom growth surface is adhesive, except for the incisions, bulges and/or ridges. In specific embodiments, the cell culture device described herein comprises a surface modification, such as a surface coating, to facilitate cell surface adhering. Preferably, the device comprises no further surface modifications, except the compartmentalization described herein and such surface modifications that enable or help cells to adhere to the bottom surface of the device. For example, such modification facilitating cell surface adherence may be a fibronectin coat. Specifically, the surface of the cell culture device, specifically the bottom surface, may comprise modifications to make the surface more hydrophilic, such as for example chemical modifications or

attachment of peptides or synthetic polymers, e.g. poly-lysine, to the surface.

According to a specific embodiment, the growth surface of the cell culture device provided herein comprises microstructures providing a certain roughness to the surface of the cell culture device, to aid in the formation of three-dimensional cell spheroids. Such microstructures may be elevations or indentations of sub-cellular size in an irregular or regular pattern.

[0082] Specifically, the cell culture device provided herein comprises no anti adhesive or non-adherent surface modifications that would prevent cells from adhering to the growth surface.

[0083] Prior art cell culture devices, which are used for the formation of cell spheroids typically comprise non-adherent surfaces, so that spheroids are formed in suspension. Such cell culture devices are often technically difficult to produce, as they comprise multiple elaborate separate compartments, that are intended to form individual spheroids, and comprise surface modifications, to make the surface non-adherent. Typically, cells have to be seeded individually into the compartments at predefined cell numbers, making handling of the devices more difficult.

[0084] In contrast, the cell culture device described herein is easy to produce and easy to handle. The device comprises different compartments for the formation of spheroids, but these compartments are only separated by the incisions or bulges described herein, so that cell culture media is distributed evenly across an entire chamber of the device. This has the advantage that seeding of the cells can be done for the entire chamber at once, as cells distribute evenly across the chamber and settle in the compartments and adhere to the bottom growth surface, and in specific embodiments of the device, also settle in the incisions. Furthermore, any media or other substrates, such as e.g. pharmaceuticals, antibodies, antigens, or chemical compounds, can be added to all compartments of a chamber in a single step, thereby significantly simplifying handling of the cell culture. Also, any solutions present in a chamber can be removed in a single step. Specifically, handling of the cell culture is simplified over the time span of the culture, e.g. from seeding the cells, up to the formation of the spheroids and investigation of the same.

[0085] Advantageously, cells adhere to the bottom surface of the two-dimensional cell culturing device described herein in a monolayer and subsequently assemble into cell spheroids of uniform size spontaneously. Surprisingly, cells spontaneously assemble into spheroids, even without non-adhesive coating of the surface of the cell culture device.

[0086] Specifically, speed of spheroid self-assembly and the completeness of monolayer formation varies depending on the cell type and on co-culture ratios. With some cell lines, and in particular in co-culture spheroid formation by aggregation into pellets can be seen within few days, or even within the first 1, 2 or 3 days. Specifically, despite these differences, the overall process of cell spheroid formation has a similar progression. Slight detachment and concentration of the cell layer starts close to the edge of a compartment, specifically close to the incisions or bulges, which is often followed by rolling up of the cell layer. Rolling up of the cell layer causes detachment from the bottom surface of the cell culture device described herein and upon conversion into pellet form, anchor points to the surface are typically lost. Specifically, conversion from monolayer into pellet form is followed by condensation into a spherical shape.

[0087] Some cell types form spheroids by aggregation and condensation instead of a rolling up from the compartment edges, as observed in the ASCs or co-culture. [0088] According to a specific example and depending on the medium, cell type and cellular activity, some cell types may also form non-detaching nodules, which are typically in the shape of half-spheroids.

[0089] Specifically, cell spheroids are formed by spontaneous condensation in the center of the cell monolayer of a compartment, typically following detachment of the monolayer at the edge of the compartment.

[0090] Surprisingly, the cell spheroids formed by the method described herein are of uniform size and their size correlates to the size of the compartments in a chamber. According to a specific example, cell spheroids of ASCs comprised a diameter of about 130 pm when cultured in a two-dimensional cell culture device as described herein comprising squared compartments comprising a length and width of lmm, and comprised a diameter of about 340 pm when cultured in a two- dimensional cell culture device as described herein comprising squared

compartments comprising a length and width of 3mm. Specifically, cell spheroids produced according to the method provided herein vary in size with a standard deviation of only about 10, 20 or 30 %. Preferably, the variation in size is only approximately 10%, however, under conditions where cell spheroids are formed quickly, i.e. within a few days, the variation may be more than 10% since for example spheroid formation in incisions is quicker than in the compartments.

[0091] According to the method described herein, cells are grown in a cell culture media in the device described herein. As used herein, the term“cell culture medium” refers to any liquid or gel-like liquid capable of supporting the growth of cells in an in vitro environment. Cell culture media generally comprise an

appropriate source of energy and compounds which regulate the cell cycle. The person skilled in the art knows how to select cell culture media, depending on the type of cell to be cultured and the outcome to be achieved.

[0092] Specifically, culture media used herein are composed of a complement of amino acids, vitamins, inorganic salts, glucose, and serum as a source of growth factors, hormones, and attachment factors. Serum may also be substituted with albumin alone or in combination with growth factors. In addition to nutrients, the medium ideally also helps maintain pH and osmolality. The cell culture medium may be a natural or an artificial cell culture medium. Specifically, natural media consist solely of naturally occurring biological fluids and are very useful and convenient for a wide range of animal cell culture. Specific examples of natural cell culture media are for example plasma and serum. Artificial or synthetic media are prepared for example by adding nutrients (both organic and inorganic), vitamins, salts, 0₂ and C0₂ gas phases, serum proteins, carbohydrates, cofactors.

Specifically, artificial cell culture media can be prepared for immediate cell survival, prolonged survival, indefinite growth or specialized functions. Preferably, cell culture media capable of supporting prolonged survival of cells are used. Such media specifically comprises a balanced salt solution supplemented with various formulations of organic compounds and/or serum.

[0093] A specific example of media used with the method described herein is serum containing media. Specifically, fetal bovine serum is the most common supplement in animal cell culture media. It is used as a low-cost supplement to provide an optimal culture medium. Serum provides carriers or chelators for labile or water-insoluble nutrients, hormones and growth factors, protease inhibitors, and binds and neutralizes toxic moieties.

[0094] A further specific example of media used with the method described herein is serum-free media. In certain embodiment, presence of serum in the media can have drawbacks for the cell culture. A number of serum-free media have been developed. These media are generally specifically formulated to support the culture of a single cell type, such as Knockout Serum Replacement and Knockout DMEM from Thermo Fisher Scientific for stem cells, and incorporate defined quantities of purified growth factors, lipoproteins, and other proteins, which are otherwise usually provided by the serum. These media are also referred to as‘defined culture media’ since the components in these media are known.

[0095] A further specific example of media used with the method described herein is chemically defined media. Chemically defined medium contains contamination- free pure inorganic and organic ingredients, and may also contain pure protein additives, like growth factors. Specifically, constituents of this type of medium are produced in vertebrate cells, e.g. Chinese hamster ovary cells, bacteria or yeast by genetic engineering with the addition of vitamins, cholesterol, specific amino acids, and fatty acids.

[0096] Yet a further specific example of media used with the method described herein is protein-free media. Specifically, protein-free media do not contain any protein and only contain non-protein constituents. Compared to serum- supplemented media, use of protein-free media promotes superior cell growth and protein expression and facilitates downstream purification of any expressed product. Formulations like M EM, RPM I-1640 are protein-free and protein

supplement is provided when required.

[0097] According to the method provided herein, cells are cultured in the cell culture device for a time sufficient for the formation of cell spheroids. The time sufficient for the formation of cell spheroids depends on the choice of cell line and whether cells are kept in mono culture or co-culture. Specifically, cells are grown in the device described herein for at least 1 day. Preferably, cells are grown for longer than a day, specifically for 2, 3, 4, 5, or 6 days or for about a week or two weeks. Once cell spheroids have formed in the device described herein, they can be harvested, for example by extracting the cell spheroids from the cell culture device in the cell culture medium or in any biologically acceptable solution.

[0098] As described herein, the cell spheroids produced according to the method provided herein can be used in a three-dimensional cell culture. Cell spheroids grown in the cell culture device described herein and embedded in hydrogel or a scaffold can be used for different applications, such as for example regenerative medicine or to study organoids in an in vitro environment mimicking their endogenous surroundings. Specifically, cell spheroids formed for example from stem cells, or differentiated cells in co-culture with stem cells, can be used as pre differentiated seedlings embedded in hydrogel for regenerative medicine

applications, such as in vitro production of cartilage or bone tissue.

[0099] A three-dimensional cell culture is an artificially created environment in which cells are permitted to grow or interact with their surroundings in all three dimensions. Specifically, three-dimensional cell culture as used herein may comprise hydrogel embedding pre-formed cell spheroids. Hydrogels can be broadly classified as either natural or synthetic materials. Hydrogels used herein may be naturally-derived, such as for example collagen, fibrin or alginate or may be synthetic, such as for example polyacrylamide or polyethylene glycol, or may be a hybrid material that combines elements of synthetic and natural polymers, such as for examples hyaluronic acid and polypeptides.

Examples

[00100] The Examples which follow are set forth to aid in the understanding of the invention but are not intended to, and should not be construed to limit the scope of the invention in any way. The Examples do not include detailed descriptions of conventional methods. Such methods are well known to those of ordinary skill in the art.

[00101] Scaffold free high cell density cultures, such as hanging drop cultures of pellet cultures are widely used in various fields of medical science. The main drawbacks of these culture systems however, are often high consumption of time/reagents and few parameters for early readout of treatment outcome, especially for slow processes, e.g. chondrogenic differentiation. While some progress to reduce these drawbacks has been made by automation or simplified spheroid generation most of these methods remain expensive or have tedious preparation procedures. Therefore, the goal of the herein presented work is to establish a new system for spheroid generation which alleviates these problems. To achieve this, conventional cell culture dishes were compartmentalized by laser engraving and adipose derived stem cells (ASC), human articular chondrocytes (HAC) and co-cultures were cultured on the created grid plates in a chondrogenic differentiation setup. Generated pellets were analyzed using light-/electron microscopy, histology, and the kinetics of pellet formation as well as the

controllability of generated size were studied. Additionally, the chondrogenic differentiation capacity was compared to standard pellet culture, and the outgrowth behavior of pellets when embedded into hydrogels was assessed to determine usability as seedlings for cartilage repair. Size of generated pellets was easily controllable by varying grid size and yielded stable sizes (standard deviation < 10%). Formation of mature pellets was completed after 3 weeks and could be reduced to 1-2 weeks in co-culture, with co-cultures showing slightly reduced spheroid diameter, likely due to incomplete monolayer formation. ASC and co culture spheroids showed differences in matrix distribution, likely due to different formation speed, however, both showed better or equal differentiation capacity compared to standard pellet culture. Outgrowth behavior showed to be easily controllable by density of hydrogel and matrix deposition could be observed in all conditions. Thus, the examples herein present a highly interesting system for spheroid generation with promising application in testing of new therapies and differentiation capacity, as well as possible use in cartilage regeneration

applications. Example 1 - Materials & Methods

[00102] Cells and Media. HACs were isolated from human femur heads obtained from joint replacements. The study was approved by the local ethics committee and patient consent was given via a consent form. TERT immortalized ASC

(ASC/TERT1) were obtained from Evercyte GmbH (Austria, Vienna). Chondrocyte expansion media (CM) consisted of DM EM high glucose (Gibco, 41966-029), 10% FCS (PAN Biotech), 2 pg/ml amphotericin B (Gibco), 100 pg/ml gentamicin (Gibco), 50 pg/ml L-ascorbic acid 2-phosphate (Sigma-Aldrich), lOmM HEPES, 5 pg/ml insulin (Sigma-Aldrich) and 2mM L-glutamine (Gibco). EGM-2 for ASC expansion (EBM-2 basal medium + EGM-2 bulletkit) was purchased from Lonza. Hennigs differentiation medium (Hennigs) contained DM EM high glucose (Sigma-Aldrich D6546), lx insulin-transferrin-selenium (Gibco, 41400045), 0.17 mM L-ascorbic acid 2-phosphate, 1 mM sodium pyruvate (Gibco), 0.35 mM L-proline (Sigma- Aldrich, P5607), 1.25 mg/ml bovine serum albumin (Sigma-Aldrich), lx

penicillin/streptomycin (Gibco) and 2 mM L-glutamine (Gibco), and was freshly supplemented with 0.1 pM dexamethasone (Sigma-Aldrich, D4902), lng/ml rhTGF b -3 (R&D Systems, 243-B3/CF) and lng/ml rhBM P-6 (R&D Systems, 507- BP) at use.

[00103] Isolation and cultivation of HACs. For the isolation of primary HACs, cartilage was taken from non-arthritic regions of femoral heads and cut into ~l-2 m³ pieces, followed by 30 min incubation in antibiotic solution (0.5 mg/ml

Gentamicin, 10 pg/ml Amphotericin B). Cartilage was then digested for 30 min in 1 mg/ml Hyaluronidase (Sigma, H3506), 1 h in 1 mg/ml Pronase (Roche,

10165921001) (both in DM EM) and 2 days in 200 U/ml Collagenase II (Gibco, 17101-015) and 1 U/ml Papain (Sigma, P4762) in CM. All digestion steps were done on a roller at 37 ° C and 5% C02. After cartilage digestion the resulting cel l suspension was centrifuged at 300 g, pellet was resuspended in phosphate buffered saline (PBS, Gibco) and cells were counted using CASY1 cell counter (Scharfe Systems). 3xl0⁶ cells were subsequent seeded per T75 flask and cultured in CM at 37 C and 5% C02, changing media twice a week. Cells were passaged when reaching -90% confluency and cultured until the end of passage one for all experiments.

[00104] Transduction of ASC. Adipose derived stromal cells (ASC/TERT1 Cat# CHS-001-0005) immortalized by ectopic expression of the catalytic subunit of human telomerase (hTERT) were provided form the Evercyte GmbH (Evercyte, Vienna, Austria,) and retrovirally infected with the sequence of the red fluorescent protein mCherry using Phoenix-Ampho cells".

[00105] Compartmentalization of plates. Grids were engraved into the growth surface of petri dishes using a T rotec Speedy 300 (Trotec Ltd, Austria) with a standard wavelength of 10.6 pm, set to a pulse duration of 0.2 ms, a power of 12 W and 5000 Hz. Standard petri dishes were used. Such dishes are available for example from Grainer, Corning.

[00106] The petri dishes were lasered with a speed of 33,5 cm/s and in a distance of 1 or 3 mm. An example of the compartmentalized plates used as cell culture devices in the present examples and the incisions is seen in Figures 12 and 13, respectively. Figure 13 shows an SEM image of C02-laser generated incisions within the plastic surface of the cell culture device. The incisions go deep into the material and underneath the cull culture surface level, and are flanked by ridges on the top. Pellet formation kinetics and effects of ASC: H AC ratio. For assessment of pellet formation kinetics and the effects of ASC:HAC co-culture ratios on kinetics and resulting pellet size, ASC alone (n=5), primary HACs (n=3) alone or in co-culture (n=5) were seeded on grid plates with 3 mm grid size at lxlO⁶ cells/plate. HAC cultures were seeded in CM and Hennigs, all other cultures were only cultured in Hennigs. Ratios tested for co-culture were 80:20%, 50:50% and 20:80%. To exclude effects of cellular secretions of HACs on formation kinetics, plates were additionally seeded with a 50:50% mixture of live ASC and HACs fixed with 4% Paraformaldehyde (PFA) and subsequently washed 3 times to remove residual PFA. The number of fully formed pellets was counted manually twice a week. For definition of the start-point for rapid pellet formation a threshold of 25 fully formed pellets was used. This was done as earlier pellet formation in some edge-grids was observed much earlier than for most regions of the plate. The time- point for formation of 25 pellets was calculated by exponential curve fitting (R2 > 0.9) and interpolation. Non-Gaussian distribution was assumed due to too small sample size. Pellet sizes were measured after 5 weeks of culture using TE2000-U and N IS-Elements BP 4.20.03 (Nikon) software and data was tested for normality using D’Agostino & Pearson omnibus normality test (p < 0.0001). Statistical significance of difference in formation speed and pellet size was tested using Kruskal-Wallis and Dunn’s multiple comparison tests. Additionally time-laps imaging of pellet formation was done using Lumascope 620 and Lumaview software (Etaluma).

[00107] Size modulation of ASC pellets. To show the controllability of generated pellet size via variation of grid sizes, ASC were seeded into grid plates with 1 mm and 3 mm grid size at lxlO⁶ cells per plate in duplicates. Cells were cultivated in Hennigs for 3 weeks, until pellet formation was fully concluded. Pellet sizes were analyzed using Eclipse TE2000-U and NIS-Elements BR 4.20.03 software. For 3 mm grid plates all (n=338) and for pellets generated by 1 mm grids 800 pellets were measured. Data was tested for normality using D’Agostino & Pearson omnibus normality test (p < 0.0001) and statistical significance of size difference was tested using two-sided Mann-Whitney test.

[00108] Fibrin embedding of pellets. To assess the possibility of using grid plate generated pellets as seedlings for regenerative medicine applications pellets were embedded into fibrin hydrogels. For ASC only cultures pellets were generated on 1 and 3 mm grids, and 50:50% ASC:HAC were generated on 3 mm grids. All cultures were kept over a course of 3 weeks prior to embedding. Tissucol/Tisseel

(fibrinogen: 72 - 110 mg/ml + 3000 KUI/ml aprotinin; thrombin: 500 U I/m I ; Baxter), was used as stock. Thrombin was diluted 1:8 in CM to reduce reaction speed and fibrinogen was diluted 1:1 or 1:8 with CM containing cell pellets, resulting in highl and low-density fibrin gels respectively. Following embedding pellets were cultivated for 2 weeks. Cultures were imaged twice a week and time-laps of sprouting pellets were taken for the first 3 days of culture using Lumascope 620 and Lumaview software.

[00109] Histology. Histology samples were fixed using 4% formaldehyde overnight at 4 C and subsequently re-buffered to PBS, dehydrated in a graded series of alcohol and embedded in paraffin via xylol (Roth, Switzerland). Sections of 3-4 pm were cut and stained by AZAN for visualization of cell distribution, Alcian blue (0.3% at pH 2.5) to detect glycosaminoglycan and immunostained with collage type I I (Thermo Fisher Scientific, United States) to evaluate the differentiation. For the im mu nohistochemistry endogenous peroxides and alkaline phosphatase were blocked with BIOXALL (Vector Labs, United States) and antigens retrieved with pepsin (pH 2) for collagen type I I staining were performed before incubating with antibody in 1:100 for 1 hour at room temperature and as secondary antibody the polymer labelled system Bright vision poly H RP (VWR, United States) was incubated for 30 minutes at room temperature. A peroxidase substrate kit

(NovaRED™, Vector Labs, United States) was used for development of the color reaction and hematoxylin for counterstaining.

[00110] Scanning electron microscopy (SEM). Samples for SEM were fixed using 4% formaldehyde overnight at 4 C and subsequently re-buffered to PBS.

Dehydration was done using an ethanol series (15%, 30%, 50%, 70%, 80%, 2x96%, 98%, 2x absolute) with 5 min incubation per step and subsequently exchanged in 3 steps to hexamethyldisilazane (Sigma-Aldrich, United States) and left to dry overnight. Samples were sputter coated with gold (Quorum) prior to imaging and were observed using a Zeiss MA-10 SEM at lOkV acceleration voltage.

[00111] Comparison of pellet culture to grid plate culture. For standard pellet culture 96 well round bottom plates were coated with poly(2-hydroxyethyl methacrylate) (poly- HEM A, Sigma, P3932). 0.5 g poly-HEMA was dissolved in 95% ethanol overnight at 38 C while shaking. 50 pi of the solution were added per well and plates were left at 37 C while shaking to evaporate ethanol for at least 8 h. ASC and ASC:HAC (50:50) pellets were created by seeding a total of 5000 cells per well and centrifuging at 650 g for 5 min, yielding pellet of a similar size to grid plates (300-350pm). Cultures on grid plates were seeded at 1 x 106 cells per plate. Cell samples before seeding and pellets after 3 and 5 weeks of cultivation, samples were taken for qRT-PCR and histology.

[00112] mRNA isolation and qRT-PCR. Spheroids and pellets were lysed in RLT lysis buffer containing 10 pl/ml b -mercaptoethanol, frozen and kept at 80 until further processing. RNA was isolated using RNeasy Micro Kit (Qiagen) according to manufacturer protocol, eluting with 14 pi UltraPure DEPC-treated water

(Invitrogen). RNA concentrations were measured using NanoDrop 2000c

spectrophotometer (ThermoScientific). RNA samples were subsequently reverse transcribed using iScript cDNA synthesis kits (Bio-Rad) according to manufacturer protocol using Primus 25 thermal cycler (MWG Biotech). qPCR was performed using SensiMix I I probe kit and TaqMan probes (20pl reactions) for Collal (Applied Biosystems, Hs00164004 ml), Col2al (Eurogentec, forward: 5’-GCC-TGG-TGT- CAT-GGG-TTT-3’, reverse: 5’-GTC-CCT-TCT-CAC-CAG-CTT-TG-3’, probe: 5’-AAA- GGT-GCC-AAC-GGT-GAG-CCT-3’), and H PRT1 (Applied Biosystems,

Hs02800695___ml) as housekeeping gene on a 7500 Fast Real-Time PCR system (Applied Biosystems). Data was analyzed using D A CT - method and are displayed as fold change mean ± SD.

[00113] Statistical analysis. All statistical tests were done using GraphPad Prism 6.01. Groups were seen as significantly different with an a -value < 5% (p < 0.05). Box and whiskers plots are given with whiskers for 1 and 99 percentiles, and non histogram bar charts are given as mean ± standard deviation.

Example 2 - Controllability of pellet size via variation of grid size

[00114] ASC are a promising cell source for tissue regeneration, including regeneration of damaged cartilage, due to them being easily available in high cell numbers from subcutaneous fat, while leading to less donor site morbidity, than the isolation of bone marrow derived stem cells. Therefore, we assessed the ability of ASC to self-assemble into micro-mass pellets, when seeded on

compartmentalized plates, as well as the possibility to control the size of generated pellets by variation of the laser engraved grid size. Fig. 1 depicts the results of pellet generation via 1 mm and 3 mm grid plates. Generated pellet sizes were statistically significantly different (p < 0.0001) with mean diameters of 134.4 pm (1 mm, Fig. lc) and 340.9 pm (3 mm, Fig. Id). Pellets exhibited only small variability in size, with standard deviations around 10% of pellet size or less (1 mm: 13.49 pm, 3 mm: 24.43 pm).

Example 3 - Self-assembling process of micro-mass pellets and effects of coculture

[00115] To shed more light on the self-assembling process of grid plate generated pellets and to assess the ability of HAC and co-culture for pellet formation, pellets were observed in regular intervals (Fig. 2) and time-lapse imaging of the pellet formation process was done. Pure FIAC cultures, when grown in CM only formed pellets after very long cultivation times and this behavior could only be seen in cells isolated from young patients. When cultured in Flennigs however, FIAC cultures assembled into very small pellets without prior formation of a stable cell layer. In all other conditions, before starting of pellet formation, cells created normal monolayers on the untreated surfaces of grid plates. Within the laser engraved grids small pellets formed by aggregation could be observed in the first few days. While speed of pellet self-assembly and to some extent the completeness of monolayer formation varied with different ratios of ASC:PIAC, the overall formation process appeared to have similar progression. Slight detachment and contraction of the cell layer first starts in edge regions compartments, and is most pronounced in the corners (Fig. 2, ASC, Day 12). This is often followed up by rolling up of the cell layer (Fig. 2, ASC, Day 25). At the end of the roll up stage usually 2-3 anchor points to the plate surface are left. Subsequent conversion into pellet form happens rapidly (30-60 min), by loss of an anchor point and collapse into a nob like structure. In the final stage of pellet formation pellets condense fully to assume a spherical shape, displaying a darker core region and a lighter peripheral region in bright field imaging.

[00116] These observations could also be confirmed using SEM imaging. Starting of pellet self-assembly (Fig. 3a), rolling up of cell layer (Fig. 3b), formation of a knob like structure after loss of anchoring point (Fig. 3c) and full condensation into spherical pellets (Fig. 3d). This also shows that loss of anchoring can also occur only for parts of the monolayer forming a knob like structure, while parts of the cell layer remain attached, however this was only observed in compartments without complete monolayer coverage. Additionally, SEM imaging showed more clearly the grid structure created by laser engraving, not only forming valleys, but also notch like edge structures formed by molten or plasticized material.

[00117] In addition to the process of pellet formation, the kinetics of pellet self- assembly and the effect of co-culture were observed by counting fully formed pellets (Fig. 4a). This showed that after starting, pellet formation usually finished within a week. As some pellet in the border regions of the dish formed at much earlier time points, a threshold of 25 pellets was chosen to compare the start of pellet formation. Co-cultures with 50:50% and 20:80% ASC:FIAC were shown to form pellets significantly faster than pure ASC cultures (Fig. 4b, p < 0.05 and <

0.01 respectively). 80:20% ASC:FIAC cultures were not significant, but still showed a trend to be slightly faster in pellet formation then ASC cultures.

[00118] Furthermore, the effects of co-culture ratios on pellet size were observed (Fig. 5). All conditions were significantly different from another (p < 0.0001). Mean sizes ranged from 308 pm to 150 pm for ASC and 20:80% ASC:FIAC respectively, however conditions higher in FIAC content produced increasing numbers of small pellets (< 100 pm), which were below our threshold size. Additionally, in most conditions very large pellets could be observed, which were seen to be formed via not fully compartmentalized areas in the border regions of some plates. [00119] This behavior was also noticeable in histological sections of different conditions (Fig. 6). Additionally, the appearance of formed matrix differed between conditions. Moreover, co-cultures grown on grid plates showed an increasing amount of regions with high collagen I I expressions, in some cases visibly located around a single cell, while ASC cultures showed a more even distribution of staining strength. Further, ASC cultures showed bigger regions of matrix, which were more clustered, while co-culture pellets exhibited a more marbled

appearance of deposited matrix. When cultivating 50:50% ASC:HAC co-cultures on non -compartmentalized dishes, which resulted in very long generation time of a singular pellet, a similar layered appearance of deposited matrix could be observed. Additionally, when sectioned at the correct angle, a spiral like pattern could be observed due to the rolled-up cell layer.

Example 4 - Hydrogel-embedding of grid plate generated pellets

[00120] To test the applicability of grid plate generated pellets as pre

differentiated seedlings embedded in hydrogels for regenerative medicine

applications, pellets of different sizes and composition were embedded into low- and high-density fibrin. All conditions showed sprouting behavior, however it varied strongly between conditions (Fig. 7). Pellets embedded into high density fibrin showed minimal outgrowth after two weeks of culture, while embedding into low density gels yielded a strong sprouting already in the first days after embedding. If pellets were in proximity to each other an increased amount of directed sprouting could be observed. Small pellets exhibited similar behavior, especially in the earlier days of culture, however as cells became more disseminated, they take on a rounder morphology. Co-culture pellets performed the most invasive and while in other conditions a core remained after cellular outgrowth, it was barely visible in this condition. This was also easily observable in fluorescence, where no compact region of fluorescence remained. These differences in behavior could also easily be visualized using time-lapse imaging.

[00121] The behavior observed with bright field and fluorescence imaging was also reflected in histology sections (Fig. 8). Low density gel embedding showed clear migration into the gel, while pellets embedded into high density gels showed a growth zone around the pellet core but little to no migration into the gel. Also, in histology strongest cellular invasion was seen in co-culture pellets. Matrix deposition was visible in all conditions, however, it was more easily detectable in cell-dense growth zones in high-density gels and in zones of strong outgrowth in co-cultures.

Example 5 - Standard pellet culture vs. grid plates

[00122] Clear structural differences were observable between grid plate derived spheroids (spheroids) and standard pellet culture pellets (pellets) (Fig. 9). Pure ASC pellets showed an outer cortex of strongly aligned matrix and cells, while inner regions display a more diffuse matrix distribution and little cell alignment. Between the two regions gradients of matrix distribution are observable. In contrary, ASC spheroids showed regions of clustered matrix, mainly in the inner part of the spheroid. The matrix was most likely formed during monolayer phase and then clustered when the cell layer curled up during spheroid formation. In between these matrix regions cells are distributed in a loose matrix.

[00123] In co-culture pellets (of ASCs and chondrocytes), matrix organization appeared similar as in the ASC pellets, while co-culture spheroids revealed differences to co-culture pellets (Fig. 10). Co-cultured spheroids contained less matrix clusters than ASC spheroids and the matrix was more diffusely distributed throughout the spheroid. However, both pellet and spheroid co-cultures share that often strong Col2 deposition was present in the surrounding of individual or a small group of cells, which are most likely are attributed to especially active

chondrocytes. Additionally, co-cultures usually display stronger Col2 staining than pure ASC cultures.

Discussion

[00124] Pellet culture is one of the standard 3D culturing systems used for differentiation of MSC and re-differentiation of chondrocytes. However, drawbacks of this method are high time and reagent consumption. While some approaches for full automation have tried to alleviate this problem, they usually come with high cost of equipment, often not affordable for many researchers. Therefore, the goal of this study was to establish an easy to manufacture system, for self-assembly of micro-mass pellets allowing reduction of reagent usage and early read-out parameters to assess differentiation potential and therapy effects, while enabling easy harvesting of pellets for therapeutic use and end point analysis.

[00125] When using ASC in a chondrogenic differentiation setup pellets could easily be generated in about 3 weeks, and pellet size could easily be controlled by grid size, with low size variation (standard deviation < 10%) (Fig. 1). Additionally, the generated pellets showed dark areas which have previously been reported to be associated with condensation and matrix deposition of ASC. In this setup due to reduced media consumption, reagent cost reduction up to 10 fold was achievable, assuming generation of 200 pellets with 300pm diameter (3 mm grid), with a break even point at 27 pellets generated. The laser engraving method used is very flexible in regards to the cell culture vessel used as“raw-material”. If less pellets of the same size are needed smaller vessels can be used, thereby further reducing the amount of media consumed and lowering the break-even point further, e.g. when using 12-well plates with 1 ml medium, theoretically yielding 28 pellets, leads to a lowering of the break-even point to 7 pellets. Alternatively, as shown the pellet number can easily be increased by reducing grid size, while in standard pellet culture systems even if small cell numbers would be possible, reduced pellet sizes would not decrease time and space consumption during maintenance, and would only minimally reduce medium consumption.

Therefore, a versatile and easily mass-producible culturing system is herein presented for spheroid generation using readily available cell-culture vessels as “raw material”. Pellet formation was successful using ASC and could be

accelerated using co-culture with HAC. Additionally, generated pellet size is easily controllable by changing grid size allowing for mass-production of spheroids with specific size, while reducing reagent and time consumption. Further, we

demonstrate that comparable or better differentiation capacity can be achieved in a chondrogenic differentiation setup. Finally, generated pellets showed easily controllable outgrowth behavior with matrix deposition when embedded into hydrogel, thus making it a promising platform for the production of building blocks for tissue regeneration.

[00126] Alternative cellular behavior leading to spheroid formation in

compartments of grid plates: Some cell types form spheroids by aggregation and condensation instead of a rolling up from the compartment edges, as observed in the ASCs or co-culture. Depending on the medium, cell type and cellular activity, some cell types form non-detaching nodules (Fig. 11). The formation of these adherent nodules in the shape of half-spheroids provides an easy-to-use and affordable platform to analyze not only cellular interactions during the aggregation phase but also the influences of drugs. Example 6 - Incisions made with scalpels, knives and fraises

Materials & Methods

[00127] Standard petri dish (e.g. from Grainer, Corning) were treated with a scalpel, a Stanley knife or a fraise to generate a grid pattern into the surface and compartment the surface into fields of about 9mm². Plates were imaged in the SEM to visualize the incisions. Before use for cell seeding, dishes were cleaned and sterilizing with 70% alcohol and UV-illumination. Then, 1 Mio. cells (mCherry transduced ASC/TERT1) were seeded and cultivated in Hennigs chondrogenic differentiation medium containing a low dose of growth factors (lng/ml TGF b -3, and lng/ml BM P-6). Cell growth was imaged using light microscopy.

Results

[00128] SEM images revealed the differences of incisions produced with three mechanical method. Scalpel and Stanley knife lead to a steep and quite narrow incision into the material and formed bulges along the cutting line. The fraise formed a flat but broad incision below the level of the growth surface, and no elevated edge (Fig.14).

[00129] After seeding the cells into the compartmented petri dish, they formed a confluent cell layer within one week and started to align and aggregate along the incision border. After another week the cell layer began to retract from the corners, which is the initial stage of spheroid formation by ASC (Fig.15).

[00130] Figure 15 shows bright field images of a scalpel-incised

compartmentation of the growth surface with a cell layer of ASC/TERT1. Left:

After seven days the cells align (arrow) and aggregate (arrow head) along the incision border. Right: After fourteen days a detachment of the cell layer from the corners is visible.

[00131] These results show, that the formation of cell spheroids can be initiated using cell culture devices of the invention, which comprise incisions made by a knife, scalpel or fraise and that spheroid formation is initiated in the same way, regardless of whether incisions are formed using a laser or a knife, scalpel or fraise. References

Babur BK, Ghanavi P, Levett P, Lott WB, Klein T, Cooper-White JJ, Crawford R, Doran M R (2013) The Interplay between Chondrocyte Redifferentiation Pellet Size and Oxygen Concentration. Ed. Xiaoming He. PLoS One 8:

e58865. doi: 10.1371/journal. pone.0058865.

Bruinink A, Wintermantel E (2001) Grooves affect primary bone marrow but not osteoblastic MC3T3-E1 cell cultures. Biomaterials 22: 2465-2473.

doi:10.1016/S0142-9612(00)00434-8.

Dean DM, Napolitano AP, Youssef J, Morgan J R (2007) Rods, tori, and

honeycombs: the directed self-assembly of microtissues with prescribed microscale geometries. FASEB J. 21: 4005-4012. doi:10.1096/fj.07- 8710com.

Desroches BR, Zhang P, Choi B-R, King M E, Maldonado AE, Li W, Rago A, Liu G, Nath N, Hartmann KM, Yang B, Koren G, Morgan J R, Mende U (2012) Functional scaffold-free 3-D cardiac microtissues: a novel model for the investigation of heart cells. AJ P Hear. Circ. Physiol. 302: H2031-H2042. doi:10.1152/ajpheart.00743.2011.

Hardelauf H, Frimat J-P, Stewart J D, Schormann W, Chiang Y-Y, Lampen P,

Franzke J, Hengstler JG, Cadenas C, Kunz-Schughart LA, West J (2011) Microarrays for the scalable production of metabolically relevant tumour spheroids: a tool for modulating chemosensitivity traits. Lab Chip 11: 419- 428. doi:10.1039/C0LC00089B.

Napolitano AP, Chai P, Dean DM, Morgan J R (2007) Dynamics of the Self-

Assembly of Complex Cellular Aggregates on Micromolded Nonadhesive Hydrogels. Tissue Eng. 13: 2087-2094. doi:10.1089/ten.2006.0190.

Tung Y-C, Hsiao AY, Allen SG, Torisawa Y, Ho M, Takayama S (2011) High- throughput 3D spheroid culture and drug testing using a 384 hanging drop array. Analyst 136: 473-478. doi:10.1039/C0AN00609B.

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Claims

Claims

1. A method of producing three-dimensional spheroids of cells, using a two- dimensional cell culture device comprising at least one chamber having an adhesive, flat bottom growth surface which is separated by incisions into compartments, allowing distribution of cell culture media across said at least one chamber, comprising the following steps:

i. introducing cell culture media comprising cells into the cell culture

device,

ii. allowing said cells to settle within the compartments, and

iii. culturing the cells for a time sufficient for the formation of cell spheroids, wherein in at least 80% of the compartments at least one cell spheroid is formed.

2. The method of claim 1, wherein the compartments are separated by incisions or bulges.

3. The method of claim 2, wherein the incisions are formed by a laser, a sharp object or the incisions are elevated incisions.

4. The method of any of claims 1 to 3, wherein the cells are invertebrate cells or mammalian cells, preferably human cells derived from cartilage or fat tissue.

5. The method of claim 4, wherein the cells are adipose derived stem cells (ASC) and/or human articular chondrocytes (HAC).

6. The method of claim 5, wherein both adipose derived stromal/stem cells and human articular chondrocytes are co-cultured in the cell culture device, preferably in a ratio of 80:20, 50:50 or 20:80% ASC:HAC.

7. The method of any one of claims 1 to 6, wherein the compartments comprise a diameter of at least 0.1 mm and up to 15 mm.

8. The method of any one of claims 1 to 7, wherein the at least one chamber

comprises a diameter of about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or 150 mm.

9. The method of any one of claims 1 to 8, wherein the cell culture device

comprises at least 2 chambers, preferably at least 4, 6, 8, 12, 24, 48 or 96 chambers.

10. The method of any one of claims 1 to 9, wherein the cells are cultured for at least a day, specifically 3 days, preferably 1 week.

11. The method of claim 10, comprising the step of harvesting the cell spheroids.

12. The method of any one of claims 1 to 10, wherein the cell spheroids are of uniform size which corresponds to the size of the compartment of the chamber.

13. Use of the cell spheroids produced according to the method of any one of claim 1 to 11 in a three-dimensional cell culture, specifically in a three-dimensional cell culture comprising a hydrogel or a scaffold.