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WO2025029744A2 - Revêtements synthétiques d'acide hyaluronique-dopamine - Google Patents

Revêtements synthétiques d'acide hyaluronique-dopamine Download PDF

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Publication number
WO2025029744A2
WO2025029744A2 PCT/US2024/040065 US2024040065W WO2025029744A2 WO 2025029744 A2 WO2025029744 A2 WO 2025029744A2 US 2024040065 W US2024040065 W US 2024040065W WO 2025029744 A2 WO2025029744 A2 WO 2025029744A2
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dopamine
lymphatic
cell culture
composition
lecs
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WO2025029744A3 (fr
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Donny Hanjaya-Putra
Fei FAN
Sanjoy Saha
Laura ALDERFER
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University of Notre Dame
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University of Notre Dame
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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
    • C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
    • C08B37/0072—Hyaluronic acid, i.e. HA or hyaluronan; Derivatives thereof, e.g. crosslinked hyaluronic acid (hylan) or hyaluronates
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09D—COATING COMPOSITIONS, e.g. PAINTS, VARNISHES OR LACQUERS; FILLING PASTES; CHEMICAL PAINT OR INK REMOVERS; INKS; CORRECTING FLUIDS; WOODSTAINS; PASTES OR SOLIDS FOR COLOURING OR PRINTING; USE OF MATERIALS THEREFOR
    • C09D105/00—Coating compositions based on polysaccharides or on their derivatives, not provided for in groups C09D101/00 or C09D103/00
    • C09D105/08—Chitin; Chondroitin sulfate; Hyaluronic acid; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/069—Vascular Endothelial cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/80—Neurotransmitters; Neurohormones
    • C12N2501/815—Dopamine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/50—Proteins
    • C12N2533/52—Fibronectin; Laminin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/70—Polysaccharides
    • C12N2533/80—Hyaluronan

Definitions

  • Lymphatic endothelial cells play a vital role in the immune system, serving as the gatekeepers for lymphocyte trafficking and the maintenance of immune homeostasis. They are also important for the removal of interstitial fluid and waste products from tissues. In a variety of diseases, such as cancer and chronic inflammation, the integrity of LECs is disrupted, leading to lymphatic dysfunction and immune suppression. To understand many of these mechanisms and pathophysiology, it is imperative to have a reliable in vitro culture system that can preserve the phenotype and characteristics of LECs.
  • LECs are cultured on tissue culture plastics or on conventional coatings like fibronectin or collagen.
  • Fibronectin is one of the most used ECMs for in vitro culture; it was shown that LECs adhered and proliferated differently on a fibronectin coated plate compared to tissue culture plastic.
  • VEGFR- 3 vascular endothelial growth factor receptor-3
  • VEGF-C vascular endothelial growth factor-C
  • VEGF-D vascular endothelial growth factor-D
  • EB embryoid body
  • Laminin among the natural ECMs, is the least utilized coating for LECs. It is reported that a4- laminins, such as 411 (formerly laminin-8), 421 (formerly laminin-9), and 423 (formerly laminin- 14), are expressed by vascular and lymphatic endothelial cells. Overall, the outlook on using collagen or laminin as coating for culturing LEC is still in contention, while fibronectin is considered as a benchmark in this regard.
  • HA hyaluronic acid or hyaluronan
  • ECs blood endothelial cells
  • LMW Low molecular weight
  • RHAMM receptor for hyaluronan-mediated motility
  • CD44 and RHAMM are reported as the main receptors on vascular ECs, they are mostly absent from lymphatic vessels; wherein the only known receptor for HA is lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1), a homolog of CD44.
  • LYVE-1 lymphatic vessel endothelial hyaluronan receptor 1
  • LYVE-1 lymphatic vessel endothelial hyaluronan receptor 1
  • This disclosure describes the development of a novel synthetic coating based on HA to preserve the phenotypes and characteristics of LECs.
  • the present disclosure uses synthesized dopamine-conjugated HA (HA-DP), which can be conjugated onto the surface of tissue culture plates.
  • HA-DP can preserve lymphatic phenotypes over several passages of LECs culture in vitro.
  • LECs cultured on HA-DP exhibited reduced FAK, which may be responsible for the maintenance of the lymphatic characteristics.
  • compositions that may be useful, for example, in cell culturing and cell culture systems, in which the compositions comprise a polymer comprising hyaluronic acid and dopamine, wherein about 20% to about 60% of the hyaluronic acid is functionalized with the dopamine, wherein the dopamine is covalently bonded to the hyaluronic acid.
  • the compositions comprise a polymer comprising hyaluronic acid and dopamine, wherein about 20% to about 60% of the hyaluronic acid is functionalized with the dopamine, wherein the dopamine is covalently bonded to the hyaluronic acid.
  • the compositions comprise a polymer comprising hyaluronic acid and dopamine, wherein about 20% to about 60% of the hyaluronic acid is functionalized with the dopamine, wherein the dopamine is covalently bonded to the hyaluronic acid.
  • about 40% to about 50% of the HA is functionalized with dopamine, or
  • the average molecular weight of the HA is about 55 kDa to about 85 kDa.
  • the HA-dopamine may be supplemented with a growth stimulating agent comprising one or more of vascular endothelial growth factor C (VEGF-C), angiopoietin- 2, VEGF-A, VEGF-D, fibroblast growth factor-2 (FGF-2), angiopoietin-1, angiopoietin-3, endothelian-1, endothelian-3, hepatocyte growth factor (HGF), semaphorin-3A, collagen and calcium-binding EGF domain-containing protein 1 (CCBE1), Sphingosine 1-phosphate (SIP), and bone morphogenetic protein-9 (BMP-9).
  • VEGF-C vascular endothelial growth factor C
  • FGF-2 fibroblast growth factor-2
  • angiopoietin-1 angiopoietin-3
  • HGF hepatocyte growth factor
  • semaphorin-3A collagen and calcium-binding EGF domain-containing protein 1 (CCBE1), Sphin
  • the disclosure also provides for a cell culture system comprising a surface coated with the HA-dopamine composition, wherein the dopamine is further conjugated to the surface; and a cell culture medium.
  • the surface comprises a polymer surface, a plastic surface, or a glass surface.
  • the surface comprises a cell culture plate or well.
  • embodiments of the disclosure provide for a vessel for mammalian cell culture comprising a surface coated with a composition, the composition comprising hyaluronic acid and dopamine, wherein about 20% to about 60% of the hyaluronic acid is functionalized with the dopamine, wherein the dopamine is covalently bonded to the hyaluronic acid, and wherein the dopamine is further conjugated to the surface.
  • the disclosure provides for methods of preserving a lymphatic phenotype of lymphatic endothelial cells comprising; contacting a surface that is coated with an HA-dopamine composition with lymphatic endothelial cells; culturing the lymphatic endothelial cells on the surface in the presence of a suitable cell culture medium; wherein the lymphatic endothelial cells have a higher expression of lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1) and podoplanin after about 5 to about 7 cell culture passages as compared to lymphatic endothelial cells cultured on a fibronectin coated surface after about 5 to about 7 cell culture passages, thereby preserving the lymphatic phenotype; and wherein the HA-dopamine composition comprises a polymer comprising HA and dopamine, wherein about 20% to about 60% of the HA is functionalized with the dopamine, wherein the dopamine is covalently bonded to the hyaluronic acid.
  • the method may further comprise supplementing at least one of the composition or the cell culture medium with one or more agents that stimulate lymphatic endothelial cells growth, the agent comprising one or more of vascular endothelial growth factor C (VEGF-C), angiopoietin-2, VEGF-A, VEGF-D, fibroblast growth factor-2 (FGF-2), angiopoietin-1, angiopoietin-3, endothelian-1, endothelian-3, hepatocyte growth factor (HGF), semaphorin-3A, collagen and calcium-binding EGF domain-containing protein 1 (CCBE1), Sphingosine 1-phosphate (SIP), and bone morphogenetic protein-9 (BMP-9).
  • VEGF-C vascular endothelial growth factor C
  • FGF-2 fibroblast growth factor-2
  • angiopoietin-1 angiopoietin-3
  • HGF hepatocyte growth factor
  • semaphorin-3A collagen and
  • the lymphatic endothelial cells grown on a surface coated with the HA-DP composition express about 2-fold to about 3-fold less focal adhesion kinase at cell culture passages number 7 compared to the lymphatic endothelial cells cultured on the fibronectin coated surface at cell culture passage number 7.
  • FIG. 1A-F Expression of Lymphatic Markers by LECs cultured on tissue culture plates.
  • LECs cultured on tissue culture plates were analyzed for Prox-1 using flow cytometry.
  • FIG. 2A-C Synthesis and Characterization of HA-DP.
  • A Schematic of chemical reaction to generate HA-DP. The dopamine was conjugated with the carboxyl group of HA using DMTMM, MES buffer (pH 5.5).
  • B Representative photo taken after TBO staining and washing as described in characterization method.
  • C The difference between amount of bound HA on the surface between two coating method. The left column of the graph represents the coating with HA-DP and right one represents coating with free HA solution.
  • FIG. 3A-J Protein and Gene Expression of LEC Cultured on Different Coatings.
  • Realtime qRT-PCR for early passage P.5 LECs cultured on tissue culture plate (black), fibronectin- coated plate (dotted), and HA-DP (checkered) coated plate for (A) LYVE-1, (B) PDPN, (C) Proxl, and (D) VEGFR3.
  • Real-time qRT-PCR for late passage P.7 LECs cultured on tissue culture plate (black), fibronectin-coated plate (dotted), and HA-DP (checkered) coated plate for (E) LYVE-1, (F) PDPN, (G) Proxl, and (H) VEGFR3.
  • FIG. 4A-B Focal Adhesion Kinase (FAK) Expression of LECs Cultured on Fibronectin and HA-DP Coated Plates
  • A The number of FAK per surface area was analyzed for LECs cultured on fibronectin and HA-DP coated plates.
  • FIG. 5A-F YAP/TAZ Expression for LECs Cultured on HA-DP and Fibronectin Coated Plates.
  • FIG. 6 Schematic Diagram Depicting the Role of HA-DP in Preserving Lymphatic Phenotypes.
  • HA-DP was able to preserve key lymphatic markers, including Proxl, LYVE-1, Podoplanin, and VEGFR3.
  • LECs are cultured on fibronectin coated plates, YAP/TAZ enter the nucleus and bind to the PROX-1 promoter, inhibiting its transcription, including its targets, such as LYVE-1, Podoplanin, and VEGFR3.
  • culturing LECs on HA-DP coated plates enables YAP/TAZ to undergo cytoplasmic degradation, which subsequently enhance transcription of Proxl, including its targets, such as LYVE-1, Podoplanin, and VEGFR3.
  • references in the specification to "one embodiment”, “an embodiment”, etc., indicate that the embodiment described may include a particular aspect, feature, structure, moiety, or characteristic, but not every embodiment necessarily includes that aspect, feature, structure, moiety, or characteristic. Moreover, such phrases may, but do not necessarily, refer to the same embodiment referred to in other portions of the specification. Further, when a particular aspect, feature, structure, moiety, or characteristic is described in connection with an embodiment, it is within the knowledge of one skilled in the art to affect or connect such aspect, feature, structure, moiety, or characteristic with other embodiments, whether or not explicitly described.
  • ranges recited herein also encompass any and all possible sub-ranges and combinations of sub-ranges thereof, as well as the individual values making up the range, particularly integer values. It is therefore understood that each unit between two particular units is also disclosed. For example, if 10 to 15 is disclosed, then 11, 12, 13, and 14 are also disclosed, individually, and as part of a range.
  • a recited range e.g., weight percentages or carbon groups
  • any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, or tenths.
  • each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc.
  • all language such as “up to”, “at least”, “greater than”, “less than”, “more than”, “or more”, and the like include the number recited and such terms refer to ranges that can be subsequently broken down into sub-ranges as discussed above.
  • all ratios recited herein also include all sub-ratios falling within the broader ratio. Accordingly, specific values recited for radicals, substituents, and ranges, are for illustration only; they do not exclude other defined values or other values within defined ranges for radicals and substituents. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint.
  • contacting refers to the act of touching, making contact, or of bringing to immediate or close proximity, including at the cellular or molecular level, for example, to bring about a physiological reaction, a chemical reaction, or a physical change, e.g., in a solution, in a reaction mixture, in vitro, or in vivo.
  • substantially is a broad term and is used in its ordinary sense, including, without limitation, being largely but not necessarily wholly that which is specified.
  • the term could refer to a numerical value that may not be 100% the full numerical value.
  • the full numerical value may be less by about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, or about 20%.
  • hyaluronic acid refers to a polymer having a repeat unit represented by the following General Formula 1 unless otherwise indicated and is used with a meaning encompassing a salt or derivative of hyaluronic acid.
  • n may be an integer ranging from 50 to 10,000.
  • the “hyaluronic acid derivative” refers to all of the modified forms of hyaluronic acid based on the basic structure of hyaluronic acid of General Formula 1, into which a functional group such as an amine group, an aldehyde group, a vinyl group, a thiol group, an allyloxy group, N-succinimidyl-3- (2-pyridyldithio)propionate (SPDP), N-hydroxysuccinimide (NHS), or the like is introduced.
  • a functional group such as an amine group, an aldehyde group, a vinyl group, a thiol group, an allyloxy group, N-succinimidyl-3- (2-pyridyldithio)propionate (SPDP), N-hydroxysuccinimide (NHS), or the like is introduced.
  • the hyaluronic acid derivative HA-diaminobutane, HA-hexamethylenediamine, HA-aldehyde, HA-adipic acid dihydrazide (HA-ADH), HA-2- aminoethyl methacrylate hydrochloride, HA-spermine, HA-spermidine, HA-SPDP, HA-NETS, or the like may be used.
  • HA-TB A tetrabutylammonium salt of hyaluronic acid
  • Hyaluronic acid is present in most animals and is a linear polysaccharide polymer with biodegradability, biocompatibility, and no immune responses, and thus may be safely applied to the human body. Since hyaluronic acid plays a number of different roles in the body depending on its molecular weight, it may be used for a variety of uses.
  • coating and “coated” as used herein refer to applying a biological material to a surface of the culture carrier by known methods in the field of the art, for example, but not limited to, an application method, an immersion method or the like.
  • the term “expansion,” is used to encompass repair, regeneration, proliferation, differentiation, migration, survival, or any growth parameter of any lymphatic structure, including lymphatic endothelial cells and any structures composed in whole or in part of lymphatic endothelial cells.
  • Cells that enhance expansion of the lymphatic system are cells that enhance expansion of the lymphatic system by any mechanism, either direct or indirect. “Modulation of expansion” is meant to encompass an influencing expansion in either a stimulatory or inhibitory manner, as is necessary for treating a disorder characterized by anomalous, abnormal, undesirable, or insufficient lymphatic function. It is understood that the various functions or components of the lymphatic system can become more or less active, and therefore can require different levels of modulation, at different times, even within the same patient. These requirements are affected, e.g., by disease type, disease stage, patient variation due to age, gender, health status, genetic factors, environmental factors, drugs and combinations of drugs administered currently or formerly to the patient, etc.
  • Lymphatic endothelial cells play a critical role in the formation and maintenance of the lymphatic vasculature, which is essential for the immune system, fluid balance, and tissue repair.
  • LECs are often difficult to study in vivo and in vitro models that accurately mimic their behaviors and phenotypes are limited.
  • LECs have been shown to lose their lymphatic markers over time while being cultured on 2D tissue culture plate. Since LECs uniquely express lymphatic vessel endothelial cell hyaluronan receptor- 1 (LYVE-1), we hypothesized that surface coating with hyaluronic acid (HA) can preserve LEC phenotypes and functionalities.
  • LYVE-1 lymphatic vessel endothelial cell hyaluronan receptor- 1
  • HA-DP Dopamine conjugated hyaluronic acid
  • compositions may comprise a polymer of hyaluronic acid (HA) that is functionalized with a chemical group such as, for example, dopamine.
  • HA hyaluronic acid
  • the composition comprising the HA-dopamine may be used to coat a surface for culturing mammalian cells, such as, but not limited to, lymphatic endothelial cells.
  • a composition of the disclosure comprises a polymer comprising hyaluronic acid and dopamine, wherein about 20% to about 60% of the HA is functionalized with the dopamine, and the dopamine is covalently bonded to the HA.
  • the HA polymer may be functionalized with one or more chemical groups to a certain degree of saturation.
  • degree of saturation refers to the extent of modification of the hyaluronic acid polymer backbone. The degree of substitution may be calculated, for example, as the number of repeat hyaluronic acid units functionalized with a target chemical grouper every 100 units and is typically expressed as a percentage.
  • the degree of saturation of an HA polymer may be about 15% to about 70%. In some embodiments, the degree of saturation of an HA polymer may be about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, or about 70%.
  • the degree of saturation of HA functionalized with dopamine i.e., HA-dopamine
  • the degree of saturation of HA functionalized with dopamine may be about 20% to about 60%, or about 30% to about 50%, about 40% to about 50%, or about 40% to about 45%.
  • about 40% to about 50% of the hyaluronic acid is functionalized with the dopamine.
  • HA-dopamine HA-dopamine
  • the HA polymer may have average molecular weight of about 5 kDa to about 750 kDa, or about 10 kDa, about 25 kDa, about 50 kDa, about 75 kDa, about 100 kDa, about 125 kDa, about 150 kDa, about 175 kDa, about 200 kDa, about 225 kDa, about 250 kDa, about 275 kDa, about 300 kDa, about 325 kDa, about 350 kDa, about 375 kDa, about 400 kDa, about 425 kDa, about 450 kDa, about 475 kDa, about 500 kDa, about 550 kDa, about 600 kDa, about 650 kDa, about 700 kDa, or a range in between any two of the aforementioned integers.
  • the HA average molecular weight is about 50 kDa to about 90 kDa, or about 50 kDa, about 55 kDa, about 60 kDa, about 65 kDa, about 70 kDa, about 75 kDa, about 80 kDa, about 85 kDa, or about 90 kDa.
  • the HA average molecular weight is about 60 kDa to about 80 kDa, or about 60 kDa, about 62 kDa, about 64 kDa, about 66 kDa, about 68 kDa, about 70 kDa, about 72 kDa, about 74 kDa, about 76 kDa, about 78 kDa, or about 80 kDa.
  • an average molecular weight of the HA is about 55 kDa to about 85 kDa.
  • a composition may comprise about 0.1% to about 10% wt/wt of HA-functionalized polymer, or about 0.5% to about 8% wt/wt of HA-functionalized polymer, or about 1% to about 5% wt/wt of HA-functionalized polymer, or about 1.5% to about 3% wt/wt.
  • a composition also may comprise a lymphatic endothelial cell growth stimulating agent.
  • the growth stimulating agent comprises one or more of vascular endothelial growth factor C (VEGF-C), angiopoietin-2, VEGF-A, VEGF- D, fibroblast growth factor-2 (FGF-2), angiopoietin-1, angiopoietin-3, endothelian-1, endothelian-3, hepatocyte growth factor (HGF), semaphorin-3A, collagen and calcium-binding EGF domain-containing protein 1 (CCBE1), Sphingosine 1-phosphate (SIP), and bone morphogenetic protein-9 (BMP-9).
  • VEGF-C vascular endothelial growth factor C
  • FGF-2 fibroblast growth factor-2
  • angiopoietin-1 angiopoietin-3
  • HGF hepatocyte growth factor
  • semaphorin-3A collagen and calcium-binding EGF domain-containing protein 1 (
  • a composition may include a lymphatic endothelial cell growth stimulating agent at a concentration of about 0.1 ng/mL to about 1000 ng/mL.
  • the concentration of the lymphatic endothelial cell growth stimulating agent may be about 0.1 ng/mL to about 100 ng/mL.
  • a concentration of VEGF-C may be about 5 ng/mL to about 60 ng/mL or about 50 ng/mL.
  • lymphatic endothelial cell growth stimulating agent comprises angiopoietin- 2 in an amount of about 100 ng/mL to about 1000 ng/mL.
  • the composition may be spread onto or otherwise deposited on a surface that may be used, for example, for culturing or growth of mammalian cells.
  • the surface may comprise a polymer surface, a plastic surface, or a glass surface.
  • the surface may be any material suitable for culturing cells, including a ceramic substance, a glass, a plastic, a polymer or co-polymer, any combinations thereof, or a coating of one material on another.
  • Such surfaces may include glass materials such as soda-lime glass, pyrex glass, vycor glass, quartz glass; silicon; plastics or polymers, including dendritic polymers, such as poly(vinyl chloride), poly(vinyl alcohol), poly(methyl methacrylate), poly(vinyl acetate-maleic anhydride), poly(dimethylsiloxane) monomethacrylate, cyclic olefin polymers, fluorocarbon polymers, polystyrenes, polypropylene, polyethyleneimine; copolymers such as poly(vinyl acetate-co-maleic anhydride), poly(styrene-co-maleic anhydride), poly(ethylene-co-acrylic acid) or derivatives of these or the like.
  • dendritic polymers such as poly(vinyl chloride), poly(vinyl alcohol), poly(methyl methacrylate), poly(vinyl acetate-maleic anhydride), poly(dimethylsiloxane) mono
  • Examples of surfaces suitable for cell culture include single and multi-well plates, such as 6, 12, 96, 384, and 1536 well plates, jars, petri dishes, flasks, beakers, plates, roller bottles, slides, such as chambered and multichambered culture slides, tubes, cover slips, cups, spinner bottles, perfusion chambers, bioreactors, and fermenters.
  • the surface may be the surface of a cell culture vessel.
  • culture vessel refers to an element that can serve as a carrier or support during cell culture, and this term should not be construed in any limiting way.
  • “culture vessel” should be understood as including, but not limited to, conventional culture vessels such as stirring flasks, stirred tank reactors, petri dishes, multiwell plates, microtiter plates, test tubes and culture flasks, cover glass, or the like.
  • culture carriers are preferably formed of materials including, for example, polystyrene, polypropylene, acrylate polymers, nylon, nitrocellulose, sepharose, and so forth.
  • a vessel for mammalian cell culture comprising a surface coated with a composition, the composition comprising HA and dopamine, wherein about 20% to about 60% of the HA is functionalized with the dopamine, wherein the dopamine is covalently bonded to the HA, and wherein the dopamine is further conjugated to the surface.
  • the disclosure also describes a cell culture system comprising a surface coated with an HA-dopamine composition, wherein the composition comprises a polymer comprising HA and dopamine, wherein about 20% to about 60% of the HA is functionalized with the dopamine, wherein the dopamine is covalently bonded to the HA; and wherein the dopamine is further conjugated to the surface; and a cell culture medium.
  • the surface may be a surface or culture vessel as described above.
  • the surface comprises a cell culture plate or well.
  • the surface may be immersed in cell culture medium and used for cell culture or stored for subsequent use.
  • a cell culture medium is a nutritive solution that supports the growth and proliferation of mammalian cells.
  • the substrates are not limited to any specific cell culture medium and any media may be used to culture cells on the substrate. Suitable cell culture media are well known in the art.
  • the cell culture medium may be an undefined medium.
  • An undefined medium may contain one or more undefined components or constituents, such as feeder cells, stromal cells, serum, matrigel, serum albumin and complex extracellular matrices.
  • a cell culture medium may comprise serum and leukemia inhibitory factor (LIF).
  • LIF leukemia inhibitory factor
  • the cell culture medium may be a defined medium.
  • a defined medium contains only specified components, preferably components of known chemical structure and is devoid of undefined components or constituents, such as feeder cells, stromal cells, serum, matrigel, serum albumin and complex extracellular matrices.
  • the defined medium is humanized.
  • a humanized defined medium is devoid of components or supplements derived or isolated from non-human animals, such as Fetal Bovine Serum (FBS) and Bovine Serum Albumin (BSA), and mouse feeder cells.
  • FBS Fetal Bovine Serum
  • BSA Bovine Serum Albumin
  • Conditioned medium includes undefined components from cultured cells and is not defined.
  • a medium may comprise a defined basal medium supplemented with a serum-free media supplement and/or one or more additional components, for example transferrin, 1- thioglycerol, 2-mercaptoethanol, FGF2, defined lipids, L-Gln, non-essential amino acids, and optionally polyvinyl alcohol; polyvinyl alcohol and insulin; serum albumin; or serum albumin and insulin.
  • additional components for example transferrin, 1- thioglycerol, 2-mercaptoethanol, FGF2, defined lipids, L-Gln, non-essential amino acids, and optionally polyvinyl alcohol; polyvinyl alcohol and insulin; serum albumin; or serum albumin and insulin.
  • Suitable chemically defined basal medium such as Advanced Dulbecco's modified eagle medium (DMEM) (Price et al., Focus (2003) 25 3-6), Knockout Dulbecco's Modified Eagle's Medium (KO-DMEM), DMEM/F12, Iscove's Modified Dulbecco's medium (IMDM) and RPMI-1640 (Moore, G. E. and Woods L. K., (1976) Tissue Culture Association Manual. 3, 503-508) are known in the art and available from commercial sources (e.g. Sigma-Aldrich MI USA; Life Technologies USA). Serum-free media supplements, such as N2, B27 and N21, are well known in the art and widely available commercially (e.g.
  • Suitable serum-free media supplements include B27 (Brewer etal., Brain Res (1989) 494 65-74; Brewer et al., J. Neurosci Res 35 567-576 (1993); Brewer et al., Focus 161 6-9; Brewer et al., (1995) J. Neurosci. Res. 42:674-683; Roth et al., J Trace Elem Med.
  • a suitable defined medium may comprise N2 and B27.
  • a cell culture system as described herein may comprise a surface as described herein, cell culture medium, and mammalian cells.
  • mammalian cell sample refers to any cell obtained from a mammalian subject. Non-limiting examples of mammalian subjects include humans, non-human primates, mice, rats, horses, dogs, cats, and guinea pigs. In some embodiments, the mammalian cell sample is obtained from a human.
  • a cell sample is isolated from a tissue or organ (e.g., a human tissue or organ), including but not limited to solid tissues and organs.
  • cell samples can be isolated from placenta, umbilical cord, bone marrow, liver, blood, including cord blood, or any other suitable tissue.
  • patient-specific cell samples are isolated from a patient for culture (e.g., for cell expansion and optionally differentiation) and subsequent re-implantation into the same patient or into a different patient.
  • cells may be isolated from tissues or biological samples for ex vivo culture.
  • cells are released from tissues or biological samples using physical and/or enzymatic disruption.
  • one or more enzymes such as collagenase, trypsin, or proteinase are used to digest the extracellular matrix.
  • tissue or biological samples are placed in culture medium (e.g., with or without physical or enzymatic disruption), and cells that are released and that grow in the culture medium can be isolated for further culture.
  • the methods described herein are suitable for culturing a variety of mammalian cell types.
  • Examples of cells include, but are not limited to, stem cells (e.g., hematopoietic stem cells, somatic stem cells, totipotent stem cells, pluripotent stem cells, fetal stem cells, embryonic stem cells, mesenchymal stem cells, and induced pluripotent stem cells), progenitor cells (e.g., satellite cells, neural progenitor cells, bone marrow stromal cells, pancreatic progenitor cells, angioblasts and endothelial progenitor cells), immune cells (e.g., T- lymphocytes, dendritic cells) and differentiated cells (e.g., epithelial cells, endothelial cells, cardiomyocytes, fibroblasts, and chondrocytes).
  • stem cells e.g., hematopoietic stem cells, somatic stem cells, totipotent stem cells, pluripotent stem cells
  • the mammalian cells are endothelial cells.
  • the endothelial cells are human endothelial cells.
  • suitable endothelial cells originated from tissue including, by way of non-limiting example, blood, blood vessel, lymphatic vessel, tissue of the digestive tract, tissue of the genitourinary tract, adipose tissue, tissue of the respiratory tract, tissue of the reproductive system, bone marrow, and umbilical tissue.
  • the cells comprise lymphatic endothelial cells.
  • lymphatic endothelial cell also referred to as a LEC refers to endothelial cells that line lymph vessels and that are related to, but distinct from, those endothelial cells that line blood vessels which are referred to as “blood endothelial cells” or “BECs.”
  • BECs blood endothelial cells
  • Other examples of endothelial cells and cull culture methods are described, for example, in Schrepfer et al., U.S. Patent Publication No. US 2021/0292715.
  • LECs may be seeded on a surface coated with an HA-DP composition in an amount of about 500 cells to about 10,000,000 cells, or about 10,000 cells to about 9,000,000 cells, or about 100,000 cells to about 8,000,000 cells, or about 500,000 cells to about 5,000,000 cells, or about 1,000,000 cells to about 3,000,000 cells.
  • the HA-dopamine composition may be stored as a lyophilized powder.
  • the HA-dopamine lyophilized powder may be reconstituted in deionized water at a concentration of about 1 mg/mL to about 5 mg/mL.
  • a strong base such as 10 molar sodium hydroxide, may be added to the mixture to polymerize the HA-dopamine composition, and then the mixture may be coated onto the surface.
  • the disclosure also provides for methods of preserving a lymphatic phenotype of lymphatic endothelial cells.
  • a method may comprise contacting a surface that is coated with a HA-dopamine composition with lymphatic endothelial cells; and culturing the lymphatic endothelial cells on the surface in the presence of a suitable cell culture medium; wherein the lymphatic endothelial cells have a higher expression of lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1) and podoplanin after about 5 to about 7 cell culture passages as compared to lymphatic endothelial cells cultured on a fibronectin coated surface after about 5 to about 7 cell culture passages, thereby preserving the lymphatic phenotype.
  • LYVE-1 lymphatic vessel endothelial hyaluronan receptor 1
  • the HA-dopamine composition comprises about 40% to about 45% of the hyaluronic acid functionalized with the dopamine, and optionally, one or more agents that stimulate lymphatic endothelial cells growth.
  • the lymphatic endothelial cells grown on a surface coated with the HA-DP composition have a higher expression of lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1) and podoplanin after about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, or about 15 cell culture passages as compared to lymphatic endothelial cells cultured on a fibronectin coated surface after about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, or about 15 cell culture passages, thereby preserving the lymphatic phenotype.
  • LYVE-1 lymphatic vessel endothelial hyaluronan receptor 1
  • a method for preserving a lymphatic phenotype of lymphatic endothelial cells supplementing at least one of the composition or the cell culture medium with one or more agents that stimulate lymphatic endothelial cells growth, the agent comprising one or more of vascular endothelial growth factor C (VEGF-C), angiopoietin-2, VEGF-A, VEGF- D, fibroblast growth factor-2 (FGF-2), angiopoietin-1, angiopoietin-3, endothelian-1, endothelian-3, hepatocyte growth factor (HGF), semaphorin-3A, collagen and calcium-binding EGF domain-containing protein 1 (CCBE1), Sphingosine 1-phosphate (SIP), and bone morphogenetic protein-9 (BMP-9).
  • VEGF-C vascular endothelial growth factor C
  • FGF-2 fibroblast growth factor-2
  • angiopoietin-1 angiopoietin-3
  • an HA-dopamine composition may include about 40% to about 45% of the HA functionalized with the dopamine, and one or more of VEGF-C in an amount of about 5 ng/mL to about 60 ng/mL and angiopoietin-2 in an amount of about 100 ng/mL to about 1000 ng/mL.
  • the lymphatic endothelial cells grown on the surface comprising the HA-DP composition express about 2-fold to about 3-fold less focal adhesion kinase at cell culture passages number 7 compared to the lymphatic endothelial cells cultured on the fibronectin coated surface at cell culture passage number 7.
  • the lymphatic endothelial cells grown on the surface comprising the HA-DP composition express about 2-fold to about 3-fold less focal adhesion kinase after about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, or about 15 cell culture passages compared to the lymphatic endothelial cells cultured on the fibronectin coated surface after about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, or about 15 cell culture passages (z.e., the comparison is made after the same number of culture passages).
  • the disclosure also provides for a lymphatic endothelial cell obtained by the methods for preserving the lymphatic phenotype of lymphatic endothelial cells as disclosed herein.
  • LECs exhibit a decrease in lymphatic markers in vitro.
  • lymphatic phenotypes were analyzed for their lymphatic phenotypes using FACS and real-time qRT-PCR analysis.
  • FACS fluorescence-activated cell sorting
  • LECs cultured on HA-DP express higher levels of lymphatic markers LYVE-1 (4.67 ⁇ 0.56 -fold), PDPN (4.33 ⁇ 0.33 -fold), Proxl (3.64 ⁇ 0.30 - fold), and VEGFR3 (2.0 ⁇ 0.64 -fold) (Figure 3E-H).
  • LECs cultured on fibronectin coated plates also expressed higher lymphatic markers LYVE-1 (3.85 ⁇ 0.57 -fold), PDPN (1.94 ⁇ 0.38 -fold), Proxl (2.63 ⁇ 0.49 -fold), and VEGFR3 (1.28 ⁇ 0.63 -fold) compared to LECs cultured on tissue culture plates (Figure 3E-H).
  • LECs cultured on HA-DP express higher lymphatic markers compared to LECs cultured on tissue culture plates and fibronectin coated plates.
  • FACS analysis FACS analysis on LECs cultured on different coating conditions from passage 5 to 7.
  • HA-DP can preserve lymphatic phenotypes better than the standard of culture on fibronectin-coated plates, we decided to further investigate the differences in mechanotransduction among culture conditions. Difference in ECM component often induces mechanotransduction on the cells independent of matrix stiffness. Though there might not be much difference in stiffness between fibronectin and HA-DP, we were interested in to investigate focal adhesion kinase (FAK) and F-actin. At low passage (P.5), we did not observe significant expression of FAK on LECs cultured on fibronectin and HA-DP.
  • FAK focal adhesion kinase
  • lymphatic markers such as Proxl, LYVE-1, and PDPN have allowed the isolation and culture of LECs, which paved the way for many studies for better understanding of lymphatic biology. While it is challenging to isolate and maintain murine LECs in culture, human primary LECs can be isolated from adult and juvenile tissues. Once isolated, these LECs can be cultured on regular tissue culture plates, as well as on plates coated with fibronectin or collagen. While there is no agreement on the best culture condition to culture LECs in vitro, it has been recognized that LECs may start to lose their lymphatic expression over time during culture. In this study, we confirmed that LECs can be cultured from P.5-7, while maintaining their Proxl expression.
  • LYVE-1 and PDPN are surface receptor uniquely expressed by LECs.
  • LYVE-1 is important for leukocytes trafficking and highly abundant in lymphatic capillaries.
  • PDPN is responsible for blood- lymphatic separation and highly abundant in lymphatic collecting vessels. Therefore, preserving both LYVE-1 and PDPN expression on LECs is crucial for their lymphatic identity and function.
  • HA-DP high passage
  • LECs uniquely express LYVE-1, a specific receptor for HA, and provide a unique advantage for engineered matrices containing HA.
  • VEGFR3 is the only receptor that can preserve lymphatic phenotypes in the presence of VEGF-C.
  • a growth factor independent, ECM-based interaction can be modulated through HA-DP coating, which opens many opportunities to modify culture media based on different applications.
  • HA-DP coating causes less mechanotransduction on LECs resulted in reduced FAK and F-actin stress fibres.
  • the “Hippo Pathway” YAP/TAZ is critically involved in initial LEC specification, differentiation, and sprouting during early lymphatic development and in maintaining lymphatic integrity during adulthood. Recent studies described that YAP/TAZ work as stress-mediated mechanotransducers in LECs like that of BECs but different in that YAP/TAZ regulate Proxl transcriptional activity in LECs. Strikingly, lymphatic YAP/TAZ negatively regulates Proxl transcription, and they modulate Proxl activity and lymphatic plasticity.
  • HA-DP coated plates causes cytoplasmic degradation of YAP/TAZ, which subsequently enhances transcription of lymphatic master regulator Proxl, including its targets LYVE-1, PDPN, and VEGFR3.
  • a simple HA-DP can be used to preserve lymphatic phenotypes during in vitro culture of primary LECs.
  • HA-DP caused downregulation of YAP/TAZ, which upregulates Proxl and therefore maintains lymphatic phenotypes, consistent with previous findings.
  • this simple yet effective HA-DP coating may be useful for culturing human LECs in vitro for applications in basic lymphatic biology and lymphatic regeneration.
  • HA-DP dopamine-conjugated hyaluronic acid
  • HA-DP hyaluronic acid-dopamine
  • HA-DP coating was characterized and quantified using toluidine blue assay as described in Lee et al., International Journal of Biological Macromolecules, 2020, 151, 1314-1321. Briefly, serial dilution of free HA in DI water was prepared to create a standard curve. Then, both free HA solution and HA-DP coated plate were incubated with 1 ml Toluidine blue O (TBO, Sigma 198161-5G). In the case of free HA solution, supernatant was washed off carefully after centrifugation. For coated six- wells plate, the supernatant was aspirated directly. Both samples were washed properly with lOmM NaOH.
  • FACS Analysis Human LECs were analyzed for lymphatic markers using flow cytometry (FACS) following standard procedure (e.g., Bui et al., Communications Biology, 2022, 5, 635). Briefly, cells were trypsinized and centrifuged following resuspension in FACS buffer. Suspended cells (IxlO 6 cells) were stained with the antibodies (Ipg/mL) for 30 minutes at room temperature: Anti-LYVE-1 antibody (R&D systems, FAB20892A), Anti-PDPN antibody APC (Biolegend, 337004), as well as their corresponding IgG isotype controls.
  • FACS flow cytometry
  • the cells were fixed and permeabilized with Foxp3 / Transcription Factor Staining Buffer Set (Thermo, 00-5523-00) and then incubated with Anti-Proxl antibody FITC (Novus Biologicals, NBP1-30045AF488) for 30 minutes. The cells were washed twice and resuspended in FACS buffer for analysis. Then, the cells were analyzed using flow cytometry (BD LSR FortessaX-20) and the metadata were analyzed using FlowJo.
  • BD LSR FortessaX-20 flow cytometry
  • cDNA was then used with the TaqMan Universal PCR Master Mix and Gene Expression Assays for LYVE- 1, Proxl, PDPN, VEGFR3, YAP, TAZ, MYC, CTGF, and GAPDH. Each sample was prepared in triplicate and the relative expression was normalized to GAPDH and analyzed using the AACt method (Livak et al., Methods. 2001 Dec;25(4):402-8.).

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Abstract

La divulgation prévoit des compositions et des procédés de conservation de phénotype lymphatique, la composition comprenant un polymère comprenant de l'acide hyaluronique et de la dopamine, environ 20 % à environ 60 % de l'acide hyaluronique étant fonctionnalisé avec la dopamine, la dopamine étant liée de manière covalente à l'acide hyaluronique.
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