Synthetic hyaluronic acid-dopamine coatings

Abstract

The disclosure provides for compositions and methods of preserving lymphatic phenotype where the composition comprises 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.

Description

[0001] SYNTHETIC HYALURONIC ACID-DOPAMINE COATINGS

[0002] RELATED APPLICATIONS

[0003] This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 63 / 516,330, filed July 28, 2023, which is incorporated herein by reference.

[0004] STATEMENT REGARDING FEDERAL FUNDING

[0005] This invention was made with government support under grant R35 GM 143055 awarded by the National Institutes of Health and grant 2047903 awarded by the National Science Foundation. The government has certain rights in the invention.

[0006] BACKGROUND OF THE INVENTION

[0007] Lymphatic endothelial cells (LECs) 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.

[0008] Another important aspect of having a robust in vitro culture system for LECs is tissue engineering. Nowadays there have been tremendous efforts to generate lymphatic vessels using natural and synthetic materials. Often it is not realized that just having a more complex and physiologically relevant 3D system will not result in better tissue engineering unless rudimentary 2D culture is optimized as well. Therefore, it is important to be able to culture LECs on a coating that supports their proliferation, survival, and functional activity over extended periods of time. For decades, the isolation and growth of cells in vitro under controlled conditions has been one of the most utilized experimental approaches in the field of cell biology. As a result, various extra cellular matrix (ECM) coatings both natural and synthetic have been established.

[0009] Currently, 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. Previous studies revealed that the ligand for integrin a5pi, selectively promoted the growth of LECs through vascular endothelial growth factor receptor-3 (VEGFR- 3), which is one of the transmembrane receptors responsible for lymphatic endothelial migration, survival and proliferation. Another study showed that the adhesion and migration of LECs stimulated by vascular endothelial growth factor-C (VEGF-C), or VEGF-D are a9pi- dependent, which is present in some mutant versions of fibronectin. Similarly, Collagen-I and Collagen-IV have been used in some studies for culturing LECs as a monolayer or as an embryoid body (EB) culture. Though it was observed that Collagen-IV induces migration, cell alignment, proliferation, and differentiation into mature lymphatic capillaries in vivo, another study found that Collagen-IV did not favor LEC differentiation in vitro. Collagen-I has been used for monolayer culture of primary LECs and supporting lymphatic vessel like structure. 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.

[0010] Compared to ECM proteins, the use of hyaluronic acid or hyaluronan (HA) has been shown to hold promise in culture of blood endothelial cells (ECs). HA is an abundant component of the ECM that binds to various receptors and influences activities of ECs. Low molecular weight (LMW) HA was proved to have the ability to interact with its receptors, such as CD44 or receptor for hyaluronan-mediated motility (RHAMM), triggering series of intracellular signal transduction and promoting angiogenesis. Although 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 is thus likely to play a major role in the regulation of HA on biological behaviors of LECs. Indeed, it was shown in several studies that LMW HA induces lymphangiogenesis through LYVE-1 mediated signaling pathways. One recent study also shows that HA binding peptide modulates EC spreading and migration through focal adhesion kinase (FAK). Despite the unique expression of HA receptors by LECs, the effect of HA on the regulation of lymphatic characteristic markers has not been explored yet.

[0011] Thus, there is a need for robust and effective compositions and methods of culturing and maintaining LECs that maintain their lymphatic phenotype over multiple cell passages. The current invention satisfies these needs. SUMMARY OF THE INVENTION

[0012] 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. Compared to other conventional ECM based coating with fibronectin, we demonstrated that HA-DP can preserve lymphatic phenotypes over several passages of LECs culture in vitro. Moreover, LECs cultured on HA-DP exhibited reduced FAK, which may be responsible for the maintenance of the lymphatic characteristics.

[0013] In some aspects, the disclosure provides for certain 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. Preferably, about 40% to about 50% of the HA is functionalized with dopamine, or about 40% to about 45% of the HA is functionalized with dopamine.

[0014] In some aspects, the average molecular weight of the HA is about 55 kDa to about 85 kDa.

[0015] In some aspects, 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).

[0016] In other aspects, 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.

[0017] In some aspects, the surface comprises a polymer surface, a plastic surface, or a glass surface. Preferably, the surface comprises a cell culture plate or well.

[0018] In some aspects, 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.

[0019] In some aspects, 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.

[0020] In some aspects, 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).

[0021] In some aspects, 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.

[0022] These and other features and advantages of this invention will be more fully understood from the following detailed description of the invention taken together with the accompanying claims. It is noted that the scope of the claims is defined by the recitations therein and not by the specific discussion of features and advantages set forth in the present description.

[0023] BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The following drawings form part of the specification and are included to further demonstrate certain embodiments or various aspects of the invention. In some instances, embodiments of the invention can be best understood by referring to the accompanying drawings in combination with the detailed description presented herein. The description and accompanying drawings may highlight a certain specific example, or a certain aspect of the invention. However, one skilled in the art will understand that portions of the example or aspect may be used in combination with other examples or aspects of the invention.

[0025] 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. Representative flow cytometry analysis indicating Prox-1 expression by LECs (white histogram) at (A) P.5, (B) P.6, and (C) P.7 compared to isotype control (gray histogram). Flow cytometry data for LECs at P.5, P.6, and P.7 were analyzed for percentage of (D) Proxl+ , (E) Podoplanin+ , and (F) LYVE-1+ cells population. Data represents mean ± stdev., n=4 per condition. Significance levels were set at: *P <0.001.

[0026] 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.

[0027] 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. Three biological replicates (n=3) were collected per condition and analyzed with real-time qRT-PCR with triplicate readings. Flow cytometry analysis for LECs (P.5-7) cultured on tissue culture plate (black), fibronectin-coated plate (dotted), and HA- DP (checkered) coated plate indicating percentage of cells that are (I) LYVE-1 + and (J) Podoplanin+ . Data represents mean ± stdev., n=4 per condition. Significance levels were set at: *P <0.01.

[0028] 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. (B) The orientation of F-actin was analyzed using angle variation (SD) for LECs cultured on fibronectin and HA-DP coated plates. Data represents mean ± stdev., n=4 per condition. Significance levels were set at: **P. Dotted = Fibronectin; cross-hatched = HA-DP.

[0029] FIG. 5A-F. YAP / TAZ Expression for LECs Cultured on HA-DP and Fibronectin Coated Plates. Real-time qRT-PCR data for (A) CTGF, (B) MYC, (C) YAP, and (D) TAZ expressed by LECs (P.7) cultured on HA-DP and fibronectin coated plates. Three biological replicates (n=3) were collected per condition and analyzed with real-time qRT-PCR with triplicate readings fluorescent intensity quantification demonstrates (E) an increase in cytoplasmic localization for YAP and (F) an increase in cytoplasmic localization of TAZ for LECs cultured on HA-DP compared to fibronectin. CTCF, corrected total cell fluorescence. Data represents mean ± stdev., n=4 per condition. Significance levels were set at: **P <0.001. Dotted = Fibronectin; cross-hatched = HA-DP.

[0030] 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. When 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. In contrast, 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.

[0031] DETAILED DESCRIPTION OF THE INVENTION Definitions.

[0032] The following definitions are included to provide a clear and consistent understanding of the specification and claims. As used herein, the recited terms have the following meanings. All other terms and phrases used in this specification have their ordinary meanings as one of skill in the art would understand. Such ordinary meanings may be obtained by reference to technical dictionaries, such as Hawley’s Condensed Chemical Dictionary 14thEdition, by R.J. Lewis, John Wiley & Sons, New York, N.Y., 2001 or Singleton, et al., Dictionary of Microbiology and Molecular Biology, 2d ed., John Wiley and Sons, New York (1994), and Hale & Markham, The Harper Collins Dictionary of Biology. Harper Perennial, N.Y. (1991). General laboratory techniques (DNA extraction, RNA extraction, cloning, PCR amplification, cell culturing, etc.) are known in the art and described, for example, in Molecular Cloning: A Laboratory Manual, J. Sambrook et al., 4th edition, Cold Spring Harbor Laboratory Press, 2012.

[0033] 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.

[0034] The singular forms "a," "an," and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to "a compound" includes a plurality of such compounds, so that a compound X includes a plurality of compounds X. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for the use of exclusive terminology, such as "solely," "only," and the like, in connection with any element described herein, and / or the recitation of claim elements or use of "negative" limitations.

[0035] The term "and / or" means any one of the items, any combination of the items, or all of the items with which this term is associated. The phrases "one or more" and "at least one" are readily understood by one of skill in the art, particularly when read in context of its usage. For example, the phrase can mean one, two, three, four, five, six, ten, 100, or any upper limit approximately 10, 100, or 1000 times higher than a recited lower limit. For example, one or more substituents on a phenyl ring refers to one to five substituents on the ring.

[0036] As will be understood by the skilled artisan, all numbers, including those expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth, are approximations and are understood as being optionally modified in all instances by the term "about." These values can vary depending upon the desired properties sought to be obtained by those skilled in the art utilizing the teachings of the descriptions herein. It is also understood that such values inherently contain variability necessarily resulting from the standard deviations found in their respective testing measurements. When values are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value without the modifier "about" also forms a further aspect.

[0037] The terms "about" and "approximately" are used interchangeably. Both terms can refer to a variation of ± 5%, ± 10%, ± 20%, or ± 25% of the value specified. For example, "about 50" percent can in some embodiments carry a variation from 45 to 55 percent, or as otherwise defined by a particular claim. For integer ranges, the term "about" can include one or two integers greater than and / or less than a recited integer at each end of the range. Unless indicated otherwise herein, the terms "about" and "approximately" are intended to include values, e.g., weight percentages, proximate to the recited range that are equivalent in terms of the functionality of the individual ingredient, composition, or embodiment. The terms "about" and "approximately" can also modify the endpoints of a recited range as discussed above in this paragraph.

[0038] As will be understood by one skilled in the art, for any and all purposes, particularly in terms of providing a written description, all 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) includes each specific value, integer, decimal, or identity within the range. 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. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art, 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. In the same manner, 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.

[0039] This disclosure provides ranges, limits, and deviations to variables such as volume, mass, percentages, ratios, etc. It is understood by an ordinary person skilled in the art that a range, such as “number 1” to “number 2”, implies a continuous range of numbers that includes the whole numbers and fractional numbers. For example, 1 to 10 means 1, 2, 3, 4, 5, ... 9, 10. It also means 1.0, 1.1, 1.2. 1.3, ..., 9.8, 9.9, 10.0, and also means 1.01, 1.02, 1.03, and so on. If the variable disclosed is a number less than “number 10”, it implies a continuous range that includes whole numbers and fractional numbers less than numberlO, as discussed above. Similarly, if the variable disclosed is a number greater than “numberlO”, it implies a continuous range that includes whole numbers and fractional numbers greater than numberlO. These ranges can be modified by the term “about”, whose meaning has been described above.

[0040] One skilled in the art will also readily recognize that where members are grouped together in a common manner, such as in a Markush group, the invention encompasses not only the entire group listed as a whole, but each member of the group individually and all possible subgroups of the main group. Additionally, for all purposes, the invention encompasses not only the main group, but also the main group absent one or more of the group members. The invention therefore envisages the explicit exclusion of any one or more of members of a recited group. Accordingly, provisos may apply to any of the disclosed categories or embodiments whereby any one or more of the recited elements, species, or embodiments, may be excluded from such categories or embodiments, for example, for use in an explicit negative limitation. The term "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.

[0041] The term “substantially” as used herein, is a broad term and is used in its ordinary sense, including, without limitation, being largely but not necessarily wholly that which is specified. For example, 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%.

[0042] Wherever the term “comprising” is used herein, options are contemplated wherein the terms “consisting of’ or “consisting essentially of’ are used instead. As used herein, “comprising” is synonymous with "including," "containing," or "characterized by," and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. As used herein, "consisting of" excludes any element, step, or ingredient not specified in the aspect element. As used herein, "consisting essentially of" does not exclude materials or steps that do not materially affect the basic and novel characteristics of the aspect. In each instance herein any of the terms "comprising", "consisting essentially of" and "consisting of" may be replaced with either of the other two terms. The disclosure illustratively described herein may be suitably practiced in the absence of any element or elements, limitation, or limitations not specifically disclosed herein.

[0043] As used herein, the term, “hyaluronic acid (HA)” 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. where n may be an integer ranging from 50 to 10,000.

[0044] In some embodiments of the present invention, 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. For example, 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. In the present invention, as the salt of hyaluronic acid, a tetrabutylammonium salt of hyaluronic acid (HA-TB A) may be used.

[0045] 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.

[0046] The terms “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.

[0047] In some contexts, 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.

[0048] Embodiments of the invention.

[0049] Lymphatic endothelial cells (LECs) 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. However, LECs are often difficult to study in vivo and in vitro models that accurately mimic their behaviors and phenotypes are limited. In particular, 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. Dopamine conjugated hyaluronic acid (HA-DP) was synthesized with 42% degree of substitution to enable surface modification and conjugation onto standard tissue culture plates. Compared to fibronectin coating and tissue culture plate controls, surface coating with HA-DP was able to preserve lymphatic markers, such as prospero homeobox protein 1 (Proxl), podoplanin (PDPN), and LYVE-1, over several passages in vitro. LECs cultured on HA-DP expressed lower level of focal adhesion kinase (FAK) and YAP / TAZ, which may be responsible for the maintenance of the lymphatic characteristics. Collectively, the HA-DP coating may be useful for culture of human LECs in vitro toward their applications in basic lymphatic biology and lymphatic regeneration.

[0050] Accordingly, the disclosure provides for compositions that may comprise a polymer of hyaluronic acid (HA) that is functionalized with a chemical group such as, for example, dopamine. Preferably, 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. Generally, 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.

[0051] In some embodiments, the HA polymer may be functionalized with one or more chemical groups to a certain degree of saturation. The term “degree of saturation” as used herein 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.

[0052] In some embodiments, 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%. For example, in some embodiments, the degree of saturation of HA functionalized with dopamine (i.e., HA-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%. In some embodiments, about 40% to about 50% of the hyaluronic acid is functionalized with the dopamine. In some embodiments, about 40% to about 45% of the hyaluronic acid is functionalized with the dopamine. In some embodiments, the degree of saturation of HA functionalized with dopamine (i.e., HA-dopamine) may be about 42%. In some embodiments, 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. In some embodiments, 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. In other embodiments, 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. In other embodiments, an average molecular weight of the HA is about 55 kDa to about 85 kDa.

[0053] In some embodiments, 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.

[0054] In some embodiments, a composition also may comprise a lymphatic endothelial cell growth stimulating agent. In some embodiments, 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). In some embodiments, 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. In some embodiments, the concentration of the lymphatic endothelial cell growth stimulating agent may be about 0.1 ng / mL to about 100 ng / mL. For example, a concentration of VEGF-C may be about 5 ng / mL to about 60 ng / mL or about 50 ng / mL. In another example, lymphatic endothelial cell growth stimulating agent comprises angiopoietin- 2 in an amount of about 100 ng / mL to about 1000 ng / mL.

[0055] In some embodiments, 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. For example, the surface may comprise a polymer surface, a plastic surface, or a glass surface. In other embodiments, 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.

[0056] 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.

[0057] In some embodiments, the surface may be the surface of a cell culture vessel. The term “culture vessel” as used herein 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. According to the present invention, “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. Such culture carriers are preferably formed of materials including, for example, polystyrene, polypropylene, acrylate polymers, nylon, nitrocellulose, sepharose, and so forth. Accordingly, 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.

[0058] 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. As noted above, the surface may be a surface or culture vessel as described above. In some embodiments, the surface comprises a cell culture plate or well. In some embodiments, 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.

[0059] In some embodiments, 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. In some embodiments, a cell culture medium may comprise serum and leukemia inhibitory factor (LIF).

[0060] In some embodiments, 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. In some embodiments, 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. Conditioned medium includes undefined components from cultured cells and is not defined.

[0061] 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. 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. Invitrogen; Sigma Aldrich Inc.). 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. Biol (2010) 24130- 137), N2 (Lee et al., (2000) Nat Biotechnol 18(6) 675-679; Lumelsky et al., (2001) Science 292(5520): 1389-1394) and NS21 (Chen etalJ., NeurosciMeths (2008) 171 239-247). In some embodiments, a suitable defined medium may comprise N2 and B27.

[0062] A cell culture system as described herein may comprise a surface as described herein, cell culture medium, and mammalian cells. As used herein, the term “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.

[0063] In some embodiments, 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. In some embodiments, cell samples can be isolated from placenta, umbilical cord, bone marrow, liver, blood, including cord blood, or any other suitable tissue. In some embodiments, 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.

[0064] In some embodiments, cells may be isolated from tissues or biological samples for ex vivo culture. In some embodiments, cells are released from tissues or biological samples using physical and / or enzymatic disruption. In some embodiments, one or more enzymes such as collagenase, trypsin, or proteinase are used to digest the extracellular matrix. In some embodiments, 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.

[0065] 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).

[0066] In some embodiments, the mammalian cells are endothelial cells. In some embodiments, the endothelial cells are human endothelial cells. In some embodiments, 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. In preferred embodiments, the cells comprise lymphatic endothelial cells. In some contexts, the term “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.” 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.

[0067] In some embodiments, 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.

[0068] In some embodiments, the HA-dopamine composition may be stored as a lyophilized powder. Upon use, the HA-dopamine lyophilized powder may be reconstituted in deionized water at a concentration of about 1 mg / mL to about 5 mg / mL. Prior to coating a surface, 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.

[0069] The disclosure also provides for methods of preserving a lymphatic phenotype of lymphatic endothelial cells. For example, such 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. Preferably, 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. In other embodiments, 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.

[0070] In some embodiments, 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). For example, 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.

[0071] In some embodiments, 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. In other embodiments, 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).

[0072] 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.

[0073] EXAMPLES

[0074] Example 1.

[0075] LECs exhibit a decrease in lymphatic markers in vitro.

[0076] While previous studies have reported that blood endothelial cells lose their characteristics over time, little is known about the phenotypes of LECs during in vitro culture. Therefore, we aimed to characterize the phenotypes of LECs during in vitro culture. Human dermal LECs from single donor (n=3) were cultured on tissue culture plates from passage 5 to 7 and their lymphatic phenotypes were characterized using FACS, real-time qRT-PCR, and immunofluorescent image analysis. We confirmed using immunofluorescent and FACS analysis (Figure 1A-C) that LECs consistently express Prox-1 from passage 5 to 7. Since Prox- 1 is known to be the master lymphatic regulator, these observations confirmed that LECs maintained their lymphatic identity (Figure ID). However, we observed a gradual decrease in the expression of key lymphatic markers PDPN (Figure IE) and LYVE-1 (Figure IF). Overall, these results suggest that while LECs maintained their identity in culture by expressing transcription factor Proxl, they started to lose their lymphatic phenotypes by gradual decrease in key lymphatic markers PDPN and LYVE-1.

[0077] Synthesis and Characterization of HA-DP.

[0078] After validating the heterogeneity and plasticity of LECs, we hypothesized that the conventional coating system might not be optimized for culturing LECs. Since LECs uniquely express LYVE-1 to bind to HA and activate intracellular signaling to promote lymphangiogenesis, we postulated that tunable synthetic HA coating can serve as a supportive matrix for LEC culture in vitro. Since HA is a negatively charged polysaccharide, we have to modify HA with dopamine group to enable its conjugation to the surface of the tissue culture plate (Lee et al., Science, 2007, 318, 426-430). The dopamine group was conjugated with the carboxyl group of tHA to generate HA-DP polymer (Figure 2A).1H-NMR indicated dopamine peaks at 7.47-7.21 ppm and N-acetyl groups of HA peaks at 1.99 ppm, which confirmed the conjugation of dopamine group to HA. We synthesized HA-DP polymer with various degree of substitution (DS) and discovered that HA-DP with 42% of DS to be optimum for surface coating (data not shown). Therefore, HA-DP with 42% DS was used to coat tissue culture plate for our subsequent studies. Toluidine blue assay was used to quantify the amount HA-DP conjugated to the tissue culture plate. The presence of HA-DP was evident through toluidine blue assay in HA-DP coated plate compared to the control (Figure 2B). Quantification using a standard curve revealed 9.5 ± 0.2 pg / cm2 was present in the HA-DP coated plate (Figure 2C).

[0079] Preservation of LEC phenotype though HA-DP coating.

[0080] To test the hypothesis that HA-DP can preserve lymphatic phenotypes, we cultured LECs on HA-DP, as well as tissue culture plates and fibronectin-coated plates as controls. LECs were analyzed for their lymphatic phenotypes using FACS and real-time qRT-PCR analysis. To evaluate lymphatic phenotypes at gene expression levels among different coating conditions, we decided to evaluate them at early passage (P.5) and late passage (P.7), where their gene expression levels were known to be different. At early passage (P.5), we noticed that LECs cultured on HA-DP expressed 1.70 ± 0.18 -fold more expression of LYVE-1 and 2.24 ± 0.19 -fold more expression of PDPN compared to LECs cultured on tissue culture plates (Figure 3A-B). LECs cultured on fibronectin coated plates expressed 1.39 ± 0.20 - fold more expression of PDPN compared to LECs cultured on tissue culture plate (Figure 3B). While the expression levels of other lymphatic markers Proxl and VEGFR3 were not significantly different among different coating conditions (Figure 3C-D). At higher passage (P.7), compared to LECs cultured on tissue culture plate, 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). Overall, LECs cultured on HA-DP express higher lymphatic markers compared to LECs cultured on tissue culture plates and fibronectin coated plates. To further validate this trend at the protein level, we performed FACS analysis on LECs cultured on different coating conditions from passage 5 to 7. For LECs cultured on tissue culture plates, we noticed a decrease in the percentage of LYVE-1+ cells (Figure 31) and PDPN+ cells (Figure 3 J) from passage 5 to 7. LECs cultured on fibronectin coated plates also demonstrated a decrease in the percentage of LYVE-1+ cells (Figure 31) and PDPN+ cells (Figure 3J) at a lesser extent. Interestingly, LECs cultured on HA-DP plates maintained the percentage of LYVE-1 + cells (Figure 31) and demonstrated less decrease in the percentage of PDPN+ cells (Figure 4J). Overall, these results suggested that HA-DP coating preserves lymphatic phenotypes during in vitro culture of LECs for at least until passage 7.

[0081] Differences in mechanotransduction on fibronectin and HA-DP.

[0082] Since 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. However, at late passage (P.7) LECs cultured on fibronectin-coated plate express higher FAK (0.03 ± 0.004 count / pm2 ) compared to LECs cultured on HA-DP (0.013 ± 0.0004 count / pm2 ) . Similarly, F-actin density and alignment was more pronounced in LECs cultured on fibronectin compared to HA-DP (Figure 4A-B). Since FAK can also activate the “Hippo Pathway,” we also investigated YAP / TAZ and its downstream genes CTGF and MYC. At low passage (P.5), though not significant, LECs cultured on HA-DP express lower CTGF, YAP, and TAZ. But, at high passage (P.7), LECs cultured on HA-DP express lower TAZ (0.71 ± 0.1-fold), CTGF (0.34 ± 0.12- fold), and MYC (0.76 ± 0.07-fold) compared to LECs cultured on fibronectin coated plates (Figure 5A-D). These observations were also confirmed using immunofluorescent imaging of YAP / TAZ . Furthermore, quantification of the fluorescent signal indicates that culturing LECs on HA-DP led to a more cytoplasmic localization of YAP / TAZ compared to LECs cultured on fibronectin coated plates (Figure 5E-F). Therefore, culturing LECs on HA-DP enabled cytoplasmic degradation of YAP / TAZ, which subsequently enhance transcription of lymphatic master regulator Proxl, including its targets LYVE-1, Podoplanin, and VEGFR3. This mechanistic study explains our results and suggests that HA- DP coated plates preserve lymphatic phenotypes by downregulating FAK and YAP / TAZ pathways (Figure 6). Overall, these observations are consistent with previous studies that show YAP / TAZ negatively regulate Proxl during development and in vitro culture.

[0083] The discovery of unique 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. However, they started to lose other key lymphatic markers, such as LYVE-1 and PDPN. Both 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. We demonstrated that a simple coating with HA-DP can effectively preserve key lymphatic markers over several passages in vitro. In fact, key lymphatic makers were maintained on high passage (P.7) of LECs cultured on HA-DP, compared to LECs cultured on fibronectin coated plates. LECs uniquely express LYVE-1, a specific receptor for HA, and provide a unique advantage for engineered matrices containing HA. Other than LYVE-1, VEGFR3 is the only receptor that can preserve lymphatic phenotypes in the presence of VEGF-C. But 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.

[0084] Moreover, we showed that 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. Consistent to previous findings, our data also suggest that culturing LECs on 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. Collectively, we demonstrated that a simple HA-DP can be used to preserve lymphatic phenotypes during in vitro culture of primary LECs. Mechanistically, HA-DP caused downregulation of YAP / TAZ, which upregulates Proxl and therefore maintains lymphatic phenotypes, consistent with previous findings. Overall, 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.

[0085] Example 2. Material and Methods.

[0086] Synthesis of dopamine-conjugated hyaluronic acid (HA-DP). HA (1.026 g, 2.56 mmol disaccharide unit) was dissolved in 85.5 mL MES buffer (0.1 M, pH 5.5) and 28.5 mL ethanol was added. Equilibrium the reaction mixture to room temperature, DMTMM (2.9 g, 4 equivalents) was added to activate HA at room temperature. After 30 minutes of incubation, dopamine (484.7 mg, 1 equivalent) was added to the reaction mixture, followed by stirring at room temperature overnight. HA-DP was purified by dialysis against deionized water at 4 °C for 4 days, lyophilized, and stored at -20 °C until use. HA-DP was characterized by 1H NMR in D2O showing peaks at 1.99 ppm ((C=O)CH3 in HA and 7.3-7.4 (aromatic protons in DP). The degree of substitution was calculated as 42%.

[0087] Coating of hyaluronic acid-dopamine (HA-DP). HA-DP was coated on tissue culture plates through polymerization of dopamine under basic conditions. HA-DP was dissolved in deionized water at 5 mg / ml. Prior adding HA-DP to wells for coating, 20 pL of 10 M NaOH was added per 1 mL of HA-DP. After incubating at 37 °C overnight, the wells were vigorously washed with deionized water and cell culture medium for cell culture.

[0088] Characterization of HA coating. 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. At this stage, a picture was taken to show TBO staining of coated wells compared to non-coated ones. Then, 50% acetic was added to the samples and incubated at room temperature for 20 minutes. Finally, the solutions were collected and ran for absorbance in microplate reader at 634 nm. Absorbance of free HA solutions of different concentration were used to create standard curve, then absorbance of HA-DP coated sample was fit to the standard curve to quantify amount of HA in the coating.

[0089] Cell Culture. Human juvenile lymphatic endothelial cells (C-12216) of four donors (PromoCell, Heidelberg, Germany) were expanded and used for experiments between passages 4 and 8, as described in Alderfer et al., FASEB J, 2021, 35, e21498.; Jeong et al., Cellular and Molecular Bioengineering, 2022, 15, 467-478. Briefly, LECs were maintained at 37°C with 5% CO2 in Endothelial Cell Growth Medium (EGM-MV2, C-22022, PromoCell). To keep the cell passaging constant throughout experiments, cells were passaged every 5 days at a 1 to 3 ratio. Human LECs were characterized for the positive expression of CD31, LYVE-1, Proxl, and podoplanin throughout the experiments. All cell lines were routinely tested for mycoplasma contamination and were negative throughout this study.

[0090] 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 (IxlO6cells) 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. For intracellular staining, 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.

[0091] Gene Expression. To analyze the effect of different ECM on lymphatic phenotypes, LECs were cultured on tissue culture plastic, fibronectin, and HA-DP coatings for 5 days in EGM-MV2 media. Three biological replicates (n = 3) were collected per condition and analyzed with real-time qRT-PCR with triplicate readings as described in Alderfer et al., FASEB J, 2021, 35, e21498. RNA was reverse transcribed using a high-capacity cDNA reverse transcription kit (Thermo Fisher) according to the manufacturer’s protocol. 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.).

[0092] Immunofluorescence. To visualize the lymphatic protein expression, LECs were seeded on tissue culture plastic, fibronectin, and HA-DP coatings for 5 days. Samples were fixed with 4% paraformaldehyde, blocked with 1% BSA, permeabilized with 0.1% Triton- X, and stained for LYVE- 1, Proxl, CD144, ERG and podoplanin. To visualize focal adhesion kinase and F-actin distribution FAK antibody (Sigma, 2 pg / ml) and phalloidin (Abeam, 1:1000) were used. Samples were rinsed twice in PBS and counterstained with DAPI (Thermo Fischer, 300 nM). All samples were imaged in Nikon AX-R confocal at 40X magnification.

[0093] FAK and F-actin quantification. The thresholding tool in ImageJ was used to identify the FAKs. Then analyze particles was used with size restricted to 4-20 pm to count the number of focal adhesions. Ten fluorescent images were taken per coating at 40x using the Nixon AXR confocal microscope and analyzed with the FIJI Directionality Plug-in (Varberg et al., American Journal of Physiology-Cell Physiology, 2018, 315, C502-C515.). A statistical analysis of the dispersion was performed using GraphPad Prism 9 (GraphPad Software Inc., Fa Jolla, CA).

[0094] Lymphangiogenesis assay. Human EECs were seeded on firm, medium, and soft hydrogels at a density of 100,000 cells / cm2, which was consistent to our previous studies, and cultured for 12 hours in EGM MV2 media supplemented with either 0.5 or 50ng / mE recombinant human VEGF-C (R&D Systems). Images were acquired from the middle of each well (n=10 per condition) at 4x using an inverted light microscope (ECHO Revolve, San Diego, CA). After an initial screening at time intervals of 3, 6, 9, 12, and 15 hours, the 12 hours timepoint was determined to be the ideal endpoint for imaging as it not only allowed for differences between conditions to develop, but also allowed for images to be captured before some tube contraction occurred.

[0095] Statistical analysis. Statistical analysis was performed with GraphPad Prism. For each coating condition, at least three independent experiments were performed with three biological replicates. Statistical comparisons were made using Student’s t test for paired data, analysis of variance (ANOVA) for multiple comparisons, and with Tukey post hos analysis for parametric data. Specifically, Student’s t test was used to analyze differences between protein expression and gene expression on different coatings. Significance levels were set at the following: *P < .05, **P < .01, ***P < .001, ****P < .0001.

[0096] All publications, patents, and patent documents cited herein are incorporated by reference as though individually incorporated by reference, and in particular, Alderfer et al., FASEB J. 2021 May; 35(5): e21498.; Saha et al., Biomater. Sci., 2023, 11, 7346-7357; Fan et al., ACS Appl. Mater. Interfaces 2023, 15, 50, 58181-58195. No limitations inconsistent with this disclosure are to be understood therefrom. The invention has been described with reference to various specific and preferred embodiments and techniques. However, many variations and modifications may be made while remaining within the spirit and scope of the invention.

[0097] While specific embodiments have been described above with reference to the disclosed embodiments and examples, such embodiments are only illustrative and do not limit the scope of the invention. Changes and modifications can be made in accordance with ordinary skill in the art without departing from the invention in its broader aspects as defined in the following claims.

Claims

What is claimed is:

1. A composition comprising: a polymer comprising hyaluronic acid and dopamine, wherein about 20% to about 60% of the hyaluronic acid (HA) is functionalized with the dopamine, wherein the dopamine is covalently bonded to the hyaluronic acid.

2. The composition of claim 1, wherein about 40% to about 50% of the HA is functionalized with the dopamine.

3. The composition of claim 2, wherein about 40% to about 45% of the HA is functionalized with the dopamine.

4. The composition of claim 1, further comprising a lymphatic endothelial cell growth stimulating agent.

5. The composition of claim 4, wherein 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).

6. The composition of claim 1, wherein an average molecular weight of the HA is about 55 kDa to about 85 kDa.

7. A cell culture system comprising a surface coated with the composition of any one of claims 1-6, wherein the dopamine is further conjugated to the surface; and a cell culture medium.

8. The cell culture system of claim 7, wherein the surface comprises a polymer surface, a plastic surface, or a glass surface.

9. The cell culture system of claim 7, wherein the surface comprises a cell culture plate or well.

10. The cell culture system of claim 7, wherein the cells comprise lymphatic endothelial cells.

11. 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 (HA) 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.

12. The vessel of claim 11, wherein about 40% to about 45% of the HA is functionalized with the dopamine.

13. The vessel of claim 11, wherein the surface comprises a polymer surface, a plastic surface, or a glass surface.

14. The vessel of claim 13, wherein the surface comprises a cell culture plate or well.

15. A method of preserving a lymphatic phenotype of lymphatic endothelial cells comprising; contacting a surface that is coated with the composition of claim 1 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.

16. The method of claim 15, further comprising 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).

17. The method of claim 15, wherein about 40% to about 45% of the hyaluronic acid is functionalized with the dopamine.

18. The method of claim 15, wherein the surface comprises a cell culture plate or well.

19. The method of claim 15, wherein the lymphatic endothelial cells grown on the surface 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.

20. A lymphatic endothelial cell obtained by the method of any one of claims 15-19.

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