Cell-adhesive polymer conjugates

Abstract

The present invention relates to a polymer conjugate comprising a ULA polymer a) and one or more polypeptides b), wherein the polypeptides are conjugated to the ULA polymer, and the conjugate is coated on the surface of a device for culturing cells. The peptides can interact with receptors on the surface of the cells, while the polymer prevents non-specific binding of the cells to the surface of the device.

Description

[Technical Field]

[0001] The present invention relates to cell adhesive polymer conjugates comprising an ultra-low attachment (ULA) polymer a) and one or more polypeptides b), and to devices for culturing cells, the devices comprising a surface coated with such cell adhesive polymers. [Background technology]

[0002] Most cells in higher organisms are embedded in a meshwork of fibrous proteins and carbohydrates containing large amounts of soluble signaling molecules, called the extracellular matrix (ECM). The name "collagen," one of the main components of this complex extracellular network, can be translated from the French word "colle" for "glue," which refers to one of the functions of the ECM: ensuring the correct positioning of cells in several tissues.

[0003] In recent years, it has become clear that the role of the ECM extends far beyond this function and is considerably more complex. Many cells cannot thrive without being surrounded by an ECM. Cells receive signals from the ECM through the interaction of cellular receptors with proteins, peptides, or soluble molecules that activate cell signaling cascades that control cell behavior. Therefore, for culturing cells in in vitro systems, it is important to mimic the cells' natural environment in the human body to ensure similar cellular behavior.

[0004] Plastic surfaces used for cell culture are typically treated with plasma or corona discharge to increase the hydrophilicity of the plastic surface. This modulates the tendency of cell surface proteins to adhere to the plastic surface. More advanced cell culture materials use polymers to coat the plastic surface to reduce nonspecific binding (NSB). In particular, coatings with polymeric surfactants, such as hydrophobic brush polymers or other hydrophobic anchor groups linked to hydrophilic moieties, such as derivatives of polyethylene glycol or polyacrylamide, often minimize NSB. When dissolved in water, the polymers can coat the plastic surface of the container within seconds, creating a highly polar surface, allowing even adherent cells to be cultured in suspension and form spheroids in plastic containers, such as the cavities of microtiter plates. This indicates that cells essentially have no affinity for such coated containers. These coatings are generally quite stable and can even be irradiated with hard beta, gamma, or X-ray radiation to create a sterile environment. Furthermore, these surfaces do not tend to interact with cellular receptors in a manner similar to that of ECM components. Therefore, surfaces with low NSB are far from suitable for mimicking the effects of ECM on cultured cells.

[0005] To this end, ECM is often realized in in vitro culture systems by isolating regulatory components of the ECM and absorbing them onto the surface of cell culture plastics (polystyrene, polyolefin, polyacrylate, etc.), thereby providing stimulatory signals to cells to be cultured within the artificial container. Some commercially available products use surfaces coated with collagen or other proteins of the ECM for cultured cells to mimic the presence of the ECM.

[0006] These products, which use isolated whole proteins or ECM-derived protein fragments prepared by enzymatic cleavage, have obvious disadvantages. First, these products are often not xeno-free, meaning that the isolated proteins are of animal or human origin. This makes them unsuitable for most clinical applications. In addition, the lot-to-lot variations inherent in the preparation of isolated native proteins can pose challenges for using these products for non-clinical applications. Second, coated proteins not only provide specific receptor-based interactions with cells in culture, but also cause non-specific interactions between the protein coating of the container and the cultured cells. These interactions can be based on lipophilic interactions between the cell surface and hydrophobic moieties on the coated protein, or other non-covalent, non-specific interactions. This can lead to unwanted stimulation of cells, resulting in undesirable effects on the cells, such as increased or decreased proliferation.

[0007] Furthermore, the coating process of such proteins onto laboratory equipment, such as flasks or microtiter plates, is difficult to carry out. Slight variations in the purity of the peptide or protein, variations in the pH of the coating buffer used, differences in temperature during coating, and other process variations can significantly affect the stability, charge, and conformation of the protein. This can impair the performance of the coating process itself or the resulting product, leading to lot-to-lot variations. Such cell culture products coated with specific proteins can be used to activate specific cell signaling pathways to control cell fate. Therefore, it is essential to avoid nonspecific binding of cells to undesired by-products. These by-products can be misfolded proteins generated during the isolation or coating process.

[0008] Thus, there is a need to mimic the natural environment of cells in the human body, preferably without the disadvantages mentioned above. Summary of the Invention [Means for solving the problem]

[0009] It was therefore an initial object of the present invention to provide improved possibilities for culturing human cells. In particular, it is an object of the present invention to provide a coating material for cell culture devices that can advantageously mimic the natural environment of human cells.

[0010] This first object of the present invention is solved by a polymeric conjugate comprising or consisting of: a) an ultra-low adhesion (ULA) polymer having one or more functional groups, wherein one, two, three or more, or all of the functional groups are selected from the group consisting of primary amines, carboxyl groups, aldehyde groups, alkynes, epoxide groups, hydroxyl groups, ketones, esters, amides, nitriles, thiols, hydroxylamines, imines, alkenes, isocyanates, halides, ethers, and azides; In addition, the functional groups of the polymer may be further chemically modified to allow for conjugation of one or more polypeptides; and b) one or more polypeptides, wherein the amount of amino acids in the polypeptide(s) ranges from 5 to 100 amino acids; Preferably, one, two, three or more or all of the polypeptides are selected from the group consisting of proliferation peptides, peptides that stimulate cell differentiation, preferably stem cell differentiation, peptides that prevent cell differentiation, preferably stem cell differentiation, peptides that promote cell adhesion, peptides that promote cell survival, peptides that promote cell proliferation, peptides that promote cell expansion, peptides that support migration and / or motility, and peptides that promote cell cycle progression; One, two, more than two, or all of the polypeptides b) are conjugated to the ULA polymer a), the conjugation being achieved through one or more functional groups of the polymer a).

[0011] The term "ultra-low adhesion (ULA) polymer" as used herein is known to those skilled in the art and is frequently used in the state of the art.

[0012] Preferably, the term "ultra-low adhesion (ULA) polymer" as used herein refers to a polymer, preferably a polymeric surfactant, comprising a hydrophobic brush polymer and / or other hydrophobic anchor group linked to one, several or many hydrophilic moieties, such as derivatives of polyethylene glycol or polyacrylamide, that provides ultra-low adhesion of cells when coated onto a cell culture device.

[0013] Preferably, ultra-low adhesion is defined by the failure of adherent cells, preferably fibroblasts, particularly preferably mouse fibroblasts, preferably mouse fibroblasts of the 3T3 strain, to adhere to the polymer coated on the cell culture device.

[0014] Preferably, the term "non-adherent" as used herein defines that at most 20%, preferably at most 19%, preferably at most 18%, preferably at most 17%, preferably at most 16%, preferably at most 15%, preferably at most 14%, preferably at most 13%, preferably at most 12%, preferably at most 11%, preferably at most 10%, preferably at most 9%, preferably at most 8%, preferably at most 7%, preferably at most 6%, preferably at most 5%, preferably at most 4%, preferably at most 3%, preferably at most 2%, preferably at most 1% of the number of cells added to the cell culture device adhere to the polymer, preferably to the polymer-coated surface.

[0015] Preferably, the term "non-adherent" as used herein defines that at least 80%, preferably at least 81%, preferably at least 82%, preferably at least 83%, preferably at least 84%, preferably at least 85%, preferably at least 86%, preferably at least 87%, preferably at least 88%, preferably at least 89%, preferably at least 90%, preferably at least 91%, preferably at least 92%, preferably at least 93%, preferably at least 94%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% of the number of cells added to the cell culture device do not adhere to the polymer, preferably to the polymer coated surface.

[0016] Whether cells adhere to ULA polymers can be determined by cell morphology. For example, non-adherent fibroblasts have a rounded morphology, whereas adherent fibroblasts exhibit several cell processes, an increased cell area, and an elongated shape. Non-adherent cells often form spheroids, which can also be observed by morphological characterization of the cells.

[0017] Whether cells adhere to a ULA polymer, preferably a ULA polymer-coated surface, is preferably determined within 24 hours after adding the cells to the cell culture device. Whether cells adhere to a ULA polymer is preferably determined 6, more preferably 12, particularly preferably 15, particularly preferably 18, even more preferably 21, more preferably 24, and most preferably 30 hours after adding the cells to the cell culture device.

[0018] Advantageously, culturing cells on a cell culture device coated with the conjugates of the present invention prevents cells from adhering to untreated plastic surfaces through nonspecific binding. Furthermore, the cells can interact with the ULA polymer, preferably the peptide conjugated to the ULA polymer-coated surface, through specific cell receptor binding. This is a clear difference from similar embodiments that rely on the principle of providing peptides, protein fragments, or whole proteins on a plastic surface. When immobilization of specific binding molecules is achieved on a ULA surface, such as a ULA polymer-coated plastic surface, cell-surface interactions are limited to specific interactions, i.e., cell receptor-peptide interactions. The ULA coating prevents or at least reduces any nonspecific interactions. Thus, specific signaling effects can be investigated without being obscured by nonspecific interactions. Thus, the conjugates of the present invention allow mimicking the natural environment of cells.

[0019] Preferably, the term "polymer" used to describe the conjugates according to the present invention, their characteristics and effects, refers to the ULA polymers described herein.

[0020] Further advantageously, the peptide-conjugated coatings can also be sterilized by beta and gamma radiation or x-rays, since the conjugation procedures used generally result in stable covalent bonds.

[0021] Preferably, one, two, three or more, or all of the polypeptides exhibit at least 70%, preferably at least 80%, preferably at least 90%, preferably at least 91%, preferably at least 92%, preferably at least 93%, preferably at least 94%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% sequence homology to the sequence of an integrin binding protein of equal length, preferably collagen, laminin, tenascin, vitronectin, or fibronectin.

[0022] It has been discovered that the cell-repellent properties of low-NSB surfaces can be combined with the specific signaling properties that characterize the ECM to create an environment that prevents non-specific interactions and allows specific interactions through conjugated polypeptides that perfectly mimic the ECM.

[0023] The combination of these desired effects, i.e., specific interaction through one or more different peptides conjugated to a polymer, with the non-interaction cell-repellent properties of the ULA surface, has a distinct advantage: cultured cells adhere to the coated surface through one or several specific receptors, stimulating the specific receptors to provide the desired signaling to the cultured cells, while avoiding or reducing nonspecific interactions between the cells and the surface of the culture vessel. The latter effect prevents undesired adhesion of cells to the surface and the undesirable effects on the cells that result from such nonspecific adhesion.

[0024] The fact that several different peptides can be conjugated to a carrier ULA polymer or a mixture of ULA polymers offers the advantage that the specific interaction between the coated surface and cells can be broadened beyond interaction with just one receptor to two or more different receptors. Cells can change their properties while specifically bound to the container surface, so that if a receptor is lost or altered during culture, the cells can continue to adhere through a second or third receptor. This effect is particularly important when differentiating stem cells while they are bound to the coated surface. Stem cell differentiation is often associated with significant changes in cellular properties, including receptor expression. Therefore, differentiating adhered stem cells carries a high risk of detachment of the adhered cells from the coated surface during the differentiation process, which can result in cell loss during the subsequent purification process. The possibility of cell adhesion through more than one receptor increases the likelihood of not losing these cells.

[0025] Combining ULA polymers with stimulatory peptides as described has another advantage. For example, when cell cultures using such combinations are used to test the properties of small molecules, such as in experiments to develop new therapeutic agents, such ULA polymers can reduce the ability of small molecules to bind to container surfaces, such as plastic surfaces or protein-coated surfaces that expose a wide range of different polarities and differently charged moieties to solution. ULA surfaces with ULA properties imparted by highly polar polyethylene glycol or polyacrylamide moieties can also prevent or reduce absorption of small molecules to the surface, maintaining a desired concentration of such small molecules by avoiding material loss to the surface, thereby providing more realistic results for the effect that a desired concentration of such small molecules has on cells.

[0026] Therefore, the amount of amino acids in the polypeptide(s) b) of the cell-adhesive polymer conjugate according to the present invention is preferably within the range of 5 to 95 amino acids, preferably within the range of 5 to 90 amino acids, preferably within the range of 5 to 85 amino acids, preferably within the range of 5 to 80 amino acids, preferably within the range of 5 to 75 amino acids, preferably within the range of 5 to 70 amino acids, preferably within the range of 6 to 65 amino acids, preferably within the range of 6 to 60 amino acids, preferably within the range of 6 to 55 amino acids, preferably within the range of 6 to 50 amino acids, preferably within the range of 6 to 45 amino acids, preferably within the range of 6 to 40 amino acids, preferably within the range of 7 to 35 amino acids, preferably within the range of 7 to 30 amino acids.

[0027] However, unlike proteins or protein fragments, it has also been found that smaller oligopeptides or polypeptides often cannot be directly coated onto plastic surfaces because there are often no larger hydrophobic moieties that can be directly absorbed onto non-polar plastic surfaces.

[0028] The polymer conjugate according to the present invention solves this problem. As described herein, by conjugating a polypeptide (or polypeptides) with a polymer, these polypeptides are anchored through the binding of the polymer to the surface, reducing the non-specific adhesion of cells to the surface of the container. Furthermore, the peptide can interact with cell surface receptors. This means that this implementation can mimic ECM.

[0029] At the same time, however, it is particularly advantageous to prevent or reduce nonspecific binding as described herein. Thus, the ULA polymer a) is preferably selected from the group consisting of block copolymers, brush polymers (polyethylene glycol-polypropylene oxide (oxie) or ethylene glycol-propylene oxide, n-butyl methacrylate with methacrylic acid or alkyl-containing brush polymers (copolymers of poly(ethylene glycol)-methyl ether-methacrylate and propargyl acrylate)), alternating styrene-maleic anhydride copolymers (esterified block copolymers).

[0030] Compounds that are particularly useful for serving as ultra-low adhesion (ULA) polymers typically have the following structural components: one, several, or many low-polarity moieties: aliphatic chains, siloxanes, or aromatic units in the polymer chain, often forming the polymer backbone or attached to it. The second component is a highly polar moiety, such as polyethylene glycol or polyacrylamide, attached to the end of the hydrophobic backbone or grafted alongside it. In addition, the polymer must have chemical groups that allow for conjugation of peptides to the polymer. When such reactive groups impart a charge to the polymer, whether permanent or pH-dependent, such as carboxy or amino groups, it may be necessary to protect these groups with excess charge-neutralizing agents to preserve the polymer's ULA properties. Conjugating peptides that do not impart a charge to the polymer using reactive groups, such as hydroxy groups, may be better suited to preserve their polarity.

[0031] Useful ULA polymers include siloxane-based anchor polymers with polyethylene glycol or polyethylene glycol and isopropylene glycol side chains, such as Tegopren (Evonik, Essen, Germany), Silwet L7200, Silwet L7230, Silwet L7657 (all Momentive, Albany, NY, USA), oligomeric surfactants such as Brij 35 (Merck, Darmstadt, Germany), or graft polymers such as Inutec SL1 (Nordmann, Rassmann GmbH, Hamburg, Germany), or hereafter referred to as faCellitate F210, is a copolymer prepared from n-butylmethacrylate (nBMA), methacrylic acid, and methoxypolyethylene glycol methacrylate (Bisomer S20W).

[0032] All such compounds are amphiphilic, readily available compounds that can be absorbed onto non-polar surfaces and have ULA properties conferred by highly polar side chains or highly polar scaffold moieties with polymeric functional groups that can be conjugated to biological receptors such as peptides, proteins, or nucleic acids.

[0033] As described herein, one, two, more than two, or all of the functional groups of the polymer are selected from the group consisting of primary amines, carboxyl groups, aldehyde groups, alkynes, epoxide groups, hydroxyl groups, ketones, esters, amides, nitriles, thiols, hydroxylamines, imines, alkenes, isocyanates, halides, ethers, and azides.

[0034] Thus, the term "polymer having one or more functional groups" should be understood as a polymer in which the functional group(s) are present on at least one of the monomer(s) of the polymer, or at the end of the polymer, or at a branch of the polymer, and the functional group on at least one of the monomers of the polymer, or at the end of the polymer, or at a branch of the polymer, is not attached to another monomer, such that the functional group(s) or at least one, at least two, at least three, or all of the functional groups in the polymer are available for attachment to another compound, such as, for example, one or more polypeptides b).

[0035] The term "conjugation is located at one or more functional groups of polymer a)" describes the conjugation of one or more polypeptides to a polymer. This term should be understood as the conjugation of one, two, three or more, or all of the polypeptide(s) to the polymer by binding one, two, three or more, or all of the polypeptide(s) to such functional groups of the polymer. Thus, one, two, three or more, or all of said functional groups of the polymer are bound to one or more polypeptides.

[0036] Preferably, the conjugation results in a covalent bond.

[0037] Thus, to provide such conjugation, the polymer must have such functional group(s), where one, two, more than two, or all of the functional groups are selected from the group consisting of primary amines, carboxyl groups, aldehyde groups, alkynes, epoxide groups, hydroxyl groups, ketones, esters, amides, nitriles, thiols, hydroxylamines, imines, alkenes, isocyanates, halides, ethers, and azides.

[0038] For example, primary amines can be reacted with heterobifunctional linkers such as SMCC or sulfo-SMCC (SMCC: (Sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexan-1-carboxylate); Thermo Fisher Scientific Product No. 22322). This procedure can be carried out in aqueous solution, and the primary amine is converted to an amide linked to a maleimide functionality. This maleimide then reacts in a hetero-Michael addition with a thiol group on a cysteine ​​moiety of a polypeptide tailored to bind to a cell surface receptor.

[0039] Similarly, carboxy groups can be activated by derivatives of carbodiimides, such as EDC (1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide), to form reactive urea intermediates that can react directly with free amines on lysine moieties of peptides to form stable amide bonds.

[0040] Aldehyde groups of the coating polymer can react with amines of the peptide to form Schiff bases, which can then be stabilized by subsequent reduction.

[0041] Epoxides can react with alcohols or primary or secondary amines to form ethers or secondary or tertiary amines, all of which are perfectly suitable for forming stable conjugation bonds.

[0042] Alkynes can react with compounds bearing an azide group to give a stable triazole linkage.

[0043] These reactions can be carried out at moderate pH for less than an hour and result in stable products. Conjugates containing or consisting of peptides and coating polymers can be purified by ultrafiltration, size-exclusion chromatography, dialysis, or other procedures based on the different sizes or charges of the different components in the reaction. The resulting polymer conjugates can be absorbed onto plastic surfaces. Advantageously, polymer conjugates can also be attached to non-plastic surfaces. In particular, tethering to metal surfaces can be achieved through thiol-, phosphate-, or phosphonate-containing moieties. Binding to glass surfaces, which are typically characterized by partially deprotonated Si—OH bonds, can be achieved through positively charged moieties, such as partially protonated amino or boronic acid groups.

[0044] In this manner, the conjugation process may be more controlled and may advantageously provide consistent results.

[0045] Preferably, one, two, three or more, or all of the polypeptides b) are fragments of or correspond to proteins of the human extracellular matrix (ECM), preferably one, two, three or more, or all of the proteins of the human extracellular matrix are selected from the group consisting of integrin-binding proteins, such as collagen, laminin, vitronectin, tenascin, fibronectin, collagen I, fibronectin-RGD (linear and cyclic), PRARI (SEQ ID NO.:1), PHSRNGRGD (SEQ ID NO.:2), laminin (SEQ ID NO.:3), KAFDITYVRLKF (SEQ ID NO.:4), vitronectin (SEQ ID NO.:5), and bone sialoprotein (SEQ ID NO.:6). Preferably, other protein or peptide sequences are also of interest, such as RKKGRKKGRKKGRKK (SEQ ID NO.:7) or a peptide according to any one of SEQ ID NOs.:8-18.

[0046] The term "corresponding to a fragment of a protein" should be understood as not requiring that the polypeptide(s) be derived from such protein, but preferably the polypeptide(s) comprise or consist of an amino acid sequence that is identical to or has at least 60%, preferably at least 65%, preferably at least 70%, preferably at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 91%, preferably at least 92%, preferably at least 93%, preferably at least 94%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% sequence identity to the amino acid sequence of a fragment of such protein.

[0047] Preferably, whenever the present disclosure indicates sequence identity or homology of amino acid sequences by percentage, such reference is to the value that may be calculated using EMBOSS Water Pairwise Sequence Alignments (Proteins) (http: / / www.ebi.ac.uk / Tools / psa / emboss_water / ) for the respective amino acid sequences. The local sequence alignment tool provided by the European Molecular Biology Laboratory (EMBL), European Bioinformatics Institute (EBI) uses a modified Smith-Waterman algorithm (see http: / / www.ebi.ac.uk / Tools / psa / and Smith, T.F. and Waterman, M.S., "Identification of common molecular subsequences," Journal of Molecular Biology, 1981, 147(1):195-197). Further reference is made here to the default parameters currently provided by EMBL-EBI when performing pairwise alignments of two sequences using the modified Smith-Waterman algorithm. For amino acid sequences, these parameters are: matrix = BLOSUM62, gap open penalty = 10, and gap extend penalty = 0.5.

[0048] Preferably, the term fragment of a protein refers to a part of the amino acid sequence of the protein.

[0049] Surprisingly, it has been found that the resulting cell-adhesive polymer conjugates can be coated onto devices for cell culture to provide a low-NSB surface with signaling properties toward neighboring cells. These signaling properties are conferred by well-characterized synthetic peptides and are therefore fully reproducible because they are not affected by lot-to-lot variations of proteins isolated from natural sources.

[0050] Thus, the presence of ECM can be advantageously mimicked by the cell adhesive polymer according to the present invention.

[0051] The present invention further relates to a cell-adhesive polymer conjugate, preferably as described herein, obtained or obtainable by a method comprising the steps of: i) providing a ULA polymer having one or more functional groups, wherein one, two, more than two, or all of the functional groups are selected from the group consisting of primary amines, carboxyl groups, aldehyde groups, epoxide groups, hydroxyl groups, ketones, esters, amides, nitriles, thiols, hydroxylamines, imines, alkenes, isocyanates, halides, ethers, and azides; ii) providing one or more polypeptides, wherein the amount of amino acids in the polypeptide(s) is in the range of 5 to 100 amino acids; wherein one, two, three or more or all of the polypeptides are selected from the group consisting of proliferation peptides, peptides that stimulate cell differentiation, preferably stem cell differentiation, peptides that prevent cell differentiation, preferably stem cell differentiation, peptides that promote cell adhesion, peptides that promote cell survival, peptides that promote cell proliferation, peptides that promote cell expansion, peptides that support migration and / or motility, and peptides that promote cell cycle progression; iii) conjugating one, two, three or more or all of the polypeptides provided in step ii) with the polymer provided in step i).

[0052] Preferably, the step of providing a polymer in step i) may comprise or consist of: ia) providing one or more monomers preferably having one or more functional groups, wherein one, two, more than two, or all of the functional groups are selected from the group consisting of primary amines, carboxyl groups, aldehyde groups, epoxide groups, hydroxyl groups, ketones, esters, amides, nitriles, thiols, hydroxylamines, imines, alkenes, isocyanates, halides, ethers, and azides; ib) polymerizing one, two, more than two or all of the monomer(s) provided in step ia) to obtain a polymer.

[0053] The present invention further relates to a device for culturing cells, which comprises a surface coated with a cell adhesive polymer according to the present invention.

[0054] Devices for culturing cells are known to those skilled in the art and may be any device suitable for culturing cells and / or handling such cell cultures.

[0055] Such devices may be selected from the group consisting of include flasks (e.g., round-bottom flasks, spinner flasks, Erlenmeyer flasks, retorts, Florence flasks), cell culture dishes, cell culture bottles, cell culture bags, pipette tips, Petri dishes, multi-well plates, reactors, particularly bioreactors, tubes, pipettes, syringes, chips, slides, and cell culture scaffolds (such as microcarriers).

[0056] Preferably, the surface of the device comprises or is made of a material selected from the group consisting of glass, quartz, silicon, metal, metal oxide, and organic polymer (e.g., polycarbonate, polystyrene, hydrophilized polystyrene, polyamide, poly(methyl methacrylate), polyester, polyvinyl chloride, polyvinylidene chloride, polymers containing fluorinated monomers such as fluorinated ethylene or propylene, polyolefin (e.g., polyethylene (e.g., low-density polyethylene, very-low-density polyethylene, linear low-density polyethylene, high-density polyethylene, high-molecular-weight polyethylene, ultra-high-molecular-weight polyethylene, etc.), polypropylene (e.g., oriented polypropylene, biaxially oriented polypropylene, etc.), polynorbornene, cyclic olefin polymers (COP), or cyclic olefin copolymers (COC), such as copolymers of ethylene and norbornene)).

[0057] Preferably, the surface of the device comprises or consists of a material selected from the group consisting of polyolefins, in particular polystyrene, polyethylene, polypropylene, and (partially) fluorinated polyolefins, such as fluorinated ethylene propylene.

[0058] Preferably, the surface of the device is made of a material comprising or consisting of polystyrene.

[0059] The present invention further relates to a method for manufacturing a device according to the invention, the method comprising the steps of: a) providing a device for culturing cells; b) providing a polymeric conjugate according to the present invention; c) coating the device provided in step a), preferably the surface of the device, with the polymeric conjugate provided in step b).

[0060] Preferably, the surface of the device refers to the or part of the device that comes into contact with cells (via the coating) when the device is used to culture cells.

[0061] Preferably, the term "coating a device" refers to a typical procedure for coating a device used to culture cells. Such procedures are well known to those skilled in the art. Typically, to coat a device, a coating material is applied to the surface to be coated, and after a period preferably within a range of 1 minute to 24 hours, preferably within a range of 5 minutes to 12 hours, preferably within a range of 10 minutes to 4 hours, preferably within a range of 15 minutes to 2 hours, the surface is washed to remove any uncoated coating material.

[0062] The present invention further relates to a method for culturing cells, the method comprising: a) providing a device according to the present invention; b) adding cells to the device provided in step a); and c) culturing the cells added in step b) in the device.

[0063] Furthermore, the present invention relates to the use of the device according to the invention for culturing cells.

[0064] The cells cultured as described herein may be selected from adherent cells and / or non-adherent cells. The term "adherent and non-adherent cells" refers to a mixture of adherent and non-adherent cells. Preferably, the cells are selected from eukaryotic cells, somatic cells, primary cells, cells propagated from immortalized cell lines, induced pluripotent stem cells, hematopoietic stem cells, and mesenchymal stem cells.

[0065] As noted above, it has surprisingly been found that devices according to the present invention provide low NSB surfaces with signaling properties directed to neighboring cells, and these signaling properties are fully reproducible because they are conferred by well-characterized synthetic peptides and are not subject to lot-to-lot variations of proteins isolated from natural sources.

[0066] Thus, the presence of ECM can be advantageously mimicked by a device according to the invention.

[0067] In the following, the invention will be further characterized by illustrative, non-limiting examples. [Example]

[0068] Reagents and chemicals were purchased from abcr, Carl Roth, ChemPur, Carbolution, and Merck: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (Carlos, order number 2156.2), N-hydroxysulfo-succinimide (Carbolution, order number CC01035), 1-(2-aminoethyl)maleimide hydrochloride (Chempur, order number BD76191-1g), hydrazine monohydrate (Merck, order number 207942-5G), Silwet L7657 (Momentive, Albany, NY), Dbco-amine tfa (abcr, order number AB488945), 2-[2-(2-propynyloxy)ethoxy]ethylamine (abcr, order number AB474698). Peptides were synthesized by NovoPro Bioscience Inc. and ProteoGenix. Dde denotes (N-(1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl). All reagents and chemicals were used as purchased unless otherwise stated.

[0069] Preparation of the cell adhesive polymer conjugate of the present invention Example 1

[0070] Manufacturing of ULA Facelitate F210 For the first batch, ethanol (410.97 g), Bisomer S20W (50% aqueous solution, 32.09 g), n-butyl methacrylate (3.29 g), and methacrylic acid (0.66 g) were added to a 2 L reactor equipped with an anchor stirrer. The reactor was purged with nitrogen (10 L / min) throughout the reaction and stirred at 125 rpm. At the beginning of the reaction, the reactor was heated to 80°C over 30 minutes. When 80°C was reached, a solution of tert-butylperoxypivatate (75% solution in isododecane, 0.27 g) in ethanol (9.73 g) was added after 1 minute. After stirring for 5 minutes, a mixture of ethanol (125.59 g), Bisomer S20W (50% aqueous solution, 288.83 g), n-butyl methacrylate (29.61 g), and methacrylic acid (5.98 g) was added continuously over a period of 3 hours. Simultaneously, a solution of tert-butylperoxypivatate (75% solution in isododecane, 2.40 g) in ethanol (87.60 g) was added over a period of 3.5 hours. The reaction mixture was then allowed to polymerize for an additional 2 hours. Ethanol (20.33 g) and tert-butylperoxypivatate (75% solution in isododecane, 2.67 g) were then added continuously over a period of 1 hour. Finally, polymerization was continued for an additional 2 hours, after which the reaction mixture was cooled to 25°C as quickly as possible. The next day, the reactor was heated to 130°C (bath temperature). Simultaneously, steam distillation was carried out until the unreacted monomer was removed. The distillation was complete at an internal temperature of 100° C. The distillation was then stopped and the reaction mass was cooled to 25° C. as quickly as possible.

[0071] The solvent was removed by rotary evaporation under reduced pressure, and the resulting aqueous slurry was freeze-dried to give a colorless to yellowish solid.

[0072] Polymer-peptide conjugates for coating surfaces for the culture of induced pluripotent stem cells (iPSCs) Example 2

[0073] Preparation of polymer-peptide conjugates via thiol-maleimide Michael addition At 21°C, phaselitate F210 (500 mg, 643 μmol, 1.00 equiv.) was dissolved in PBS buffer (20.0 mL). Then, at 21°C, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (1.95 g, 10.2 mmol, 15.9 equiv.) and N-hydroxysulfosuccinimide (1.11 g, 5.10 mmol, 7.93 equiv.) were added. The reaction mixture was cooled to 0°C and stirred for 15 minutes, after which 1-(2-aminoethyl)maleimide hydrochloride (450 mg, 2.55 mmol, 3.97 equiv.) was added. The reaction mixture was stirred overnight and warmed to 21°C. The mixture was purified by ultrafiltration (Amicon® stirred cell, Ultracel® PL-5 membrane, 5.000 MWCO, washing solution: distilled water, pH 4), and a colorless solid was obtained after lyophilization.

[0074] A portion of this product (200 mg, 222 μmol, 1.00 equiv.) was dissolved in PBS buffer (10.0 mL) at 21° C., followed by addition of peptides c(RGDyC) (52.4 mg, 88.1 μmol, 0.397 equiv.), Ac-GCWGGPQVTRGDVFTMP-CONH2 (81.5 mg, 44.1 μmol, 0.199 equiv., SEQ ID NO.: 8), and Ac-GCWGRKKGRKKGRKKGRKK-CONH2 (20.1 mg, 8.80 μmol, 0.0396 equiv., SEQ ID NO.: 9) dissolved in dimethyl sulfoxide (3.0 mL). The reaction mixture was stirred overnight at 21° C. and purified by ultrafiltration (Amicon® stirred cell, Ultracel® PL-5 membrane, 5.000 MWCO, wash solution: distilled water, pH 7). After lyophilization, a colorless solid was obtained. Example 3

[0075] Preparation of polymer-peptide conjugates by direct peptide binding to polymers Phaselitate F210 (50.0 mg, 64.3 μmol, 1.00 equiv.) was dissolved in PBS buffer (5.0 mL) at 21° C. Then, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (195 mg, 1.02 mmol, 15.9 equiv.) and N-hydroxysulfosuccinimide (111 mg, 510 μmol, 7.93 equiv.) were added at 21° C. The reaction mixture was cooled to 0°C and stirred for 15 min, after which a solution of the peptides c(RGDyK) (30.0 mg, 48.4 μmol, 0.753 equiv.), Ac-GKWGGPQVTRGDVFTMP-CONH2 (45.0 mg, 23.9 μmol, 0.372 equiv., SEQ ID NO.: 8), and Ac-GKWGRK(Dde)K(Dde)GRK(Dde)K(Dde)GRK(Dde)K(Dde)GRK(Dde)K(Dde)-CONH2 (17.0 mg, 4.69 μmol, 0.0729 equiv., SEQ ID NO.: 10) in dimethyl sulfoxide / distilled water (1:1, 2.0 mL) was added. The reaction mixture was stirred overnight and warmed to 21°C. The mixture was purified by ultrafiltration (Amicon® stirred cell, Ultracel® PL-5 membrane, 5.000 MWCO, washing solution: distilled water, pH 7) and a colorless solid was obtained after lyophilization.

[0076] The product was then deprotected by Dde by applying hydrazine monohydrate in N,N-dimethylformamide (2% v / v) and stirring for 30 minutes at 21° C. The reaction mixture was again purified by ultrafiltration (Amicon® stirred cell, Ultracel® PL-5 membrane, 5.000 MWCO, washing solution: distilled water, pH 7) and lyophilized to give a colorless solid. Example 4

[0077] Preparation of polymer-peptide conjugates via strain-promoted azide-alkyne cycloaddition Phaselitate F210 (50.0 mg, 64.3 μmol, 1.00 equiv.) was dissolved in PBS buffer (5.0 mL) at 21 °C. Then, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (246 mg, 1.29 mmol, 20.0 equiv.) and N-hydroxysulfosuccinimide (140 mg, 643 μmol, 10.0 equiv.) were added at 21 °C. The reaction mixture was cooled to 0 °C and stirred for 15 min. Dbco-amine tfa (48.0 mg, 122 μmol, 1.90 equiv.) dissolved in dimethyl sulfoxide (1.5 mL) was then added. The reaction mixture was stirred overnight and warmed to 21 °C. The mixture was purified by ultrafiltration (Amicon® stirred cell, Ultracel® PL-5 membrane, 5.000 MWCO, washing solution: distilled water, pH 7) and a colorless solid was obtained after lyophilization. The product (30.0 mg, 28.9 μmol, 1.00 equivalent) was dissolved in PBS buffer (5.0 mL) at 21° C., and then the peptides cyclo(RGDDYK(N3)) (11.7 mg, 18.1 μmol, 0.625 equivalent, SEQ ID NO.: 12), Ac-GK(N3)-WGGPQVTRGDVFTMP-CONH2 (17.2 mg, 9.03 μmol, 0.313 equivalent, SEQ ID NO.: 11), and Ac-GK(N3)-WGRKKGRKKGRKKGRKK-CONH2 (4.23 mg, 1.81 μmol, 0.0625 equivalent, SEQ ID NO.: 13) dissolved in dimethyl sulfoxide (2.0 mL) were added. The reaction mixture was stirred overnight at 21° C. and purified by ultrafiltration (Amicon® stirred cell, Ultracel® PL-5 membrane, 5.000 MWCO, washing solution: distilled water, pH 7). After lyophilization, a colorless solid was obtained. Example 5

[0078] Preparation of polymer-peptide conjugates via thiol-yne reaction Phaselitate F210 (300 mg, 386 μmol, 1.00 equiv.) was dissolved in PBS buffer (10.0 mL) at 21°C. Then, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (1.48 g, 7.71 mmol, 20.0 equiv.) and N-hydroxysulfosuccinimide (837 mg, 3.86 mmol, 10.0 equiv.) were added at 21°C. The reaction mixture was cooled to 0°C and stirred for 15 minutes, after which 2-[2-(2-propynyloxy)ethoxy]ethylamine (248 mg, 1.74 mmol, 4.50 equiv.) was added. The reaction mixture was stirred overnight and warmed to 21°C. The mixture was purified by ultrafiltration (Amicon® stirred cell, Ultracel® PL-5 membrane, 5.000 MWCO, washing solution: distilled water, pH 7) and a colorless solid was obtained after lyophilization.

[0079] A portion of this product (50.0 mg, 55.3 μmol, 1.00 equivalent), 2,2′-azobis(isobutyronitrile) (1.0 mg, 5.53 μmol, 0.100 equivalent), and peptides c(RGDyC) (20.6 mg, 34.6 μmol, 0.625 equivalent), Ac-GCWGGPQVTRGDVFTMP-CONH (32.0 mg, 17.3 μmol, 0.313 equivalent, SEQ ID NO.: 8), and Ac-GCWGRKKGRKKGRKKGRKK-CONH (7.89 mg, 3.46 μmol, 0.0625 equivalent, SEQ ID NO.: 9) were dissolved in anhydrous N,N-dimethylformamide (5.0 mL) under an argon atmosphere and stirred at 60° C. for 2 days. The mixture was then purified by ultrafiltration (Amicon® stirred cell, Ultracel® PL-5 membrane, 5.000 MWCO, washing solution: distilled water, pH 7) and lyophilized to give a colorless solid. Example 6

[0080] Preparation of polymer-peptide conjugates via thiol-yne reaction Under an argon atmosphere, Silwet L-7657 (1.00 equivalents) and sodium hydride (1.50 equivalents) were dissolved in anhydrous tetrahydrofuran at 21°C. Propargyl bromide (2.00 equivalents) was then added dropwise over the course of 20 minutes. The reaction mixture was stirred at 21°C for 24 hours, after which distilled water was added at 0°C. Tetrahydrofuran was then removed under reduced pressure, and the remaining mixture was purified by ultrafiltration (Amicon® stirred cell, Ultracel® PL-5 membrane, 5.000 MWCO, wash solution: distilled water, pH 7) and lyophilized to yield a colorless solid.

[0081] A portion of this product (1.00 equivalent), 2,2'-azobis(isobutyronitrile) (0.100 equivalent), and peptides c(RGDyC) (0.625 equivalent), Ac-GCWGGPQVTRGDVFTMP-CONH (0.313 equivalent, SEQ ID NO.: 8), and Ac-GCWGRKKGRKKGRKKGRKK-CONH (0.0625 equivalent, SEQ ID NO.: 9) were dissolved in anhydrous N,N-dimethylformamide (5.0 mL) under an argon atmosphere and stirred for 2 days at 60°C. The mixture was then purified by ultrafiltration (Amicon® stirred cell, Ultracel® PL-5 membrane, 5.000 MWCO, washing solution: distilled water, pH 7) and lyophilized to obtain a colorless solid.

[0082] Polymer-peptide conjugates for culturing mesenchymal stem cells (MSCs) Example 7

[0083] Preparation of polymer-peptide conjugates via thiol-maleimide Michael addition At 21°C, phaselitate F210 (500 mg, 643 μmol, 1.00 equiv.) was dissolved in PBS buffer (20.0 mL). Then, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (1.95 g, 10.2 mmol, 15.9 equiv.) and N-hydroxysulfosuccinimide (1.11 g, 5.10 mmol, 7.93 equiv.) were added at 21°C. The reaction mixture was cooled to 0°C and stirred for 15 minutes, after which 1-(2-aminoethyl)maleimide hydrochloride (450 mg, 2.55 mmol, 3.97 equiv.) was added. The reaction mixture was stirred overnight and warmed to 21°C. The mixture was purified by ultrafiltration (Amicon® stirred cell, Ultracel® PL-5 membrane, 5.000 MWCO, washing solution: distilled water, pH 4), and a colorless solid was obtained after lyophilization.

[0084] A portion of the resulting product (200 mg, 222 μmol, 1.00 equivalents) was dissolved in PBS buffer (10.0 mL) at 21° C., followed by the addition of peptide Ac-GCWGGRGDSP-CONH2 (160 mg, 155 μmol, 0.700 equivalents, SEQ ID NO.: 14) or Ac-GCWGGGFOGER-CONH2 (183 mg, 155 μmol, 0.700 equivalents, SEQ ID NO.: 15). The reaction mixture was stirred overnight at 21° C. and purified by ultrafiltration (Amicon® stirred cell, Ultracel® PL-5 membrane, 5.000 MWCO, washing solution: distilled water, pH 7). After lyophilization, a colorless solid was obtained. Example 8

[0085] Preparation of polymer-peptide conjugates by direct peptide binding to polymers Phaselitate F210 (50.0 mg, 64.3 μmol, 1.00 equiv.) was dissolved in PBS buffer (5.0 mL) at 21°C. Then, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (195 mg, 1.02 mmol, 15.9 equiv.) and N-hydroxysulfosuccinimide (111 mg, 510 μmol, 7.93 equiv.) were added at 21°C. The reaction mixture was cooled to 0°C and stirred for 15 minutes, after which peptide Ac-GKWGGRGDSP-CONH2 (81.6 mg, 77.2 μmol, 1.20 equiv., SEQ ID NO.: 16) or Ac-GKWGGGFOGER-CONH2 (93.0 mg, 77.2 μmol, 1.20 equiv., SEQ ID NO.: 15) was added. The reaction mixture was stirred overnight and warmed to 21°C. The mixture was purified by ultrafiltration (Amicon® stirred cell, Ultracel® PL-5 membrane, 5.000 MWCO, washing solution: distilled water, pH 7) and a colorless solid was obtained after lyophilization. Example 9

[0086] Preparation of polymer-peptide conjugates via strain-promoted azide-alkyne cycloaddition Phaselitate F210 (50.0 mg, 64.3 μmol, 1.00 equiv.) was dissolved in PBS buffer (5.0 mL) at 21 °C. Then, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (246 mg, 1.29 mmol, 20.0 equiv.) and N-hydroxysulfosuccinimide (140 mg, 643 μmol, 10.0 equiv.) were added at 21 °C. The reaction mixture was cooled to 0 °C and stirred for 15 min. Dbco-amine tfa (48.0 mg, 122 μmol, 1.90 equiv.) dissolved in dimethyl sulfoxide (1.5 mL) was then added. The reaction mixture was stirred overnight and warmed to 21 °C. The mixture was purified by ultrafiltration (Amicon® stirred cell, Ultracel® PL-5 membrane, 5.000 MWCO, washing solution: distilled water, pH 7) and lyophilized to obtain a colorless solid. The product (30.0 mg, 28.9 μmol, 1.00 equivalent) was dissolved in PBS buffer (5.0 mL) at 21 °C, and then peptide Ac-GK(N3)-WGGRGDSP-CONH2 (31.3 mg, 28.9 μmol, 1.00 equivalent, SEQ ID NO.: 17) or Ac-GK(N3)-WGGGFOGER-CONH2 (36.0 mg, 28.9 μmol, 1.00 equivalent), SEQ ID NO.: 18, was added. The reaction mixture was stirred overnight at 21° C. and purified by ultrafiltration (Amicon® stirred cell, Ultracel® PL-5 membrane, 5.000 MWCO, washing solution: distilled water, pH 7). After lyophilization, a colorless solid was obtained. Example 10

[0087] Preparation of polymer-peptide conjugates via thiol-yne reaction Phaselitate F210 (300 mg, 386 μmol, 1.00 equiv.) was dissolved in PBS buffer (10.0 mL) at 21°C. Then, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (1.48 g, 7.71 mmol, 20.0 equiv.) and N-hydroxysulfosuccinimide (837 mg, 3.86 mmol, 10.0 equiv.) were added at 21°C. The reaction mixture was cooled to 0°C and stirred for 15 minutes, after which 2-[2-(2-propynyloxy)ethoxy]ethylamine (248 mg, 1.74 mmol, 4.50 equiv.) was added. The reaction mixture was stirred overnight and warmed to 21°C. The mixture was purified by ultrafiltration (Amicon® stirred cell, Ultracel® PL-5 membrane, 5.000 MWCO, washing solution: distilled water, pH 7) and a colorless solid was obtained after lyophilization.

[0088] A portion of this product (50.0 mg, 55.3 μmol, 1.00 equivalent), 2,2′-azobis(isobutyronitrile) (1.0 mg, 5.53 μmol, 0.100 equivalent), and peptide Ac-GCWGGRGDSP-CONH (57.1 mg, 55.3 μmol, 1.00 equivalent, SEQ ID NO.: 14) or Ac-GCWGGGFOGER-CONH (65.2 mg, 55.3 μmol, 1.00 equivalent, SEQ ID NO.: 15) were dissolved in N,N-dimethylformamide (5.0 mL) under an argon atmosphere and stirred for 2 days at 60° C. The mixture was then purified by ultrafiltration (Amicon® stirred cell, Ultracel® PL-5 membrane, 5.000 MWCO, washing solution: distilled water, pH 7) and lyophilized to obtain a colorless solid. Example 11

[0089] Preparation of polymer-peptide conjugates via thiol-yne reaction Under an argon atmosphere, Silwet L-7657 (1.00 equivalents) and sodium hydride (1.50 equivalents) were dissolved in anhydrous tetrahydrofuran at 21°C. Propargyl bromide (2.00 equivalents) was then added dropwise over the course of 20 minutes. The reaction mixture was stirred at 21°C for 24 hours, after which distilled water was added at 0°C. Tetrahydrofuran was then removed under reduced pressure, and the remaining mixture was purified by ultrafiltration (Amicon® stirred cell, Ultracel® PL-5 membrane, 5.000 MWCO, wash solution: distilled water, pH 7) and lyophilized to yield a colorless solid.

[0090] A portion of this product (1.00 equivalent), 2,2'-azobis(isobutyronitrile) (0.100 equivalent), and peptide Ac-GCWGGRGDSP-CONH2 (1.00 equivalent, SEQ ID NO.: 14) or Ac-GCWGGGFOGER-CONH2 (1.00 equivalent, SEQ ID NO.: 15) were dissolved in N,N-dimethylformamide (5.0 mL) under an argon atmosphere and stirred for 2 days at 60°C. The mixture was then purified by ultrafiltration (Amicon® stirred cell, Ultracel® PL-5 membrane, 5.000 MWCO, washing solution: distilled water, pH 7) and lyophilized to obtain a colorless solid. Example 12

[0091] Coating of cell culture articles with the cell adhesive polymer conjugates of the present invention 1. Lyophilized polymer-peptide conjugate in the form of any one of Examples 2-11 is dissolved in DPBS buffer to reach a concentration of 16 mg / ml (10X stock solution).

[0092] 2. Prepare a working solution by diluting the stock solution 1:10 with DPBS buffer (working concentration: 1.6 mg / ml). Note that the working solution needs to be freshly prepared just before starting the surface coating.

[0093] 3. Add 1 ml of working solution to one well of a 6-well plate (#351146, Corning). Other cell culture consumables can be used, such as 24-well plates, 96-well plates, and T75 flasks. When using other formats, adapt the volume of coating solution according to the well / flask dimensions (e.g., 500 μl for one well of a 24-well plate). Note that the surface must be non-tissue culture treated polystyrene.

[0094] 4. Incubate at room temperature for 1 hour.

[0095] 5. Discard the working solution and discard it.

[0096] 6. Wash the surface three times with 1 ml of DPBS buffer.

[0097] 7. Store the coated plates at -18°C or below; then culture the cells as described in Example 13. Example 13

[0098] Cultivation of cells with the cell culture article according to the invention A cell suspension is prepared by resuspending BIONI010-B cells (human induced pluripotent stem cell line) in 2 ml of mTeSR+ medium (Stemcell Technologies).

[0099] 1. Add 250,000 cells to one well of a 6-well plate coated according to Example 12; for control experiments, use uncoated polystyrene cell culture plates. Incubate the cells at 37°C with 5% CO2.

[0100] 2. After 24 hours, cell adhesion is monitored using bright field microscopy.

[0101] 3. Monitor cell confluency (as an indirect measure of cell proliferation) and cell morphology using brightfield microscopy daily until the cultures reach 60-80% confluency (usually after 7 days).

[0102] 4. Detach cells using CTS™ Versene™ solution (#15040066, Thermo Fisher) according to the manufacturer's instructions.

[0103] 5. Determine the expression of the following pluripotency markers: - hNANOG and hTRA1-81 at the protein level by flow cytometry - hOCT4, hSOX2, and hNANOG at the transcriptional level by RT-PCR

[0104] result All seeded cells adhered specifically to the peptides present on the coated surface. The cells had a round appearance and grew as colonies with a clearly defined periphery. Seven days after seeding, the cultures reached 70% confluence, and 2,000,000 cells were counted. 90-100% of the cells were positive for the analyzed pluripotency markers, both at the protein and transcriptional levels.

[0105] control BIONI010-B cells were seeded onto tissue-culture-treated polystyrene surfaces without a coating of polymer-peptide conjugate. The seeded cells did not adhere to the surface. The cells formed multiple spheroids that floated within the well. Most spheroids were lost during medium changes. The few remaining spheroids were isolated after 7 days of culture to determine the expression of pluripotency markers. Fewer than 30% of the cells were positive for the markers analyzed.

Claims

1. a) an ultra-low adhesion (ULA) polymer having one or more functional groups, wherein one, two, more than two, or all of the functional groups are selected from the group consisting of primary amines, carboxyl groups, aldehyde groups, alkynes, epoxide groups, hydroxyl groups, ketones, esters, amides, nitriles, thiols, hydroxylamines, imines, alkenes, isocyanates, halides, ethers, and azides; and b) a cell-adhesive polymer conjugate of one or more polypeptides, wherein the amount of amino acids in said polypeptide(s) is in the range of 5 to 100 amino acids; one, two, three or more or all of said polypeptides are selected from the group consisting of proliferation peptides, peptides that stimulate cell differentiation, preferably stem cell differentiation, peptides that prevent cell differentiation, preferably stem cell differentiation, peptides that promote cell adhesion, peptides that promote cell survival, peptides that promote cell proliferation, peptides that promote cell expansion, peptides that support migration and / or motility, and peptides that promote cell cycle progression; A cell adhesive polymer conjugate, wherein one, two, three or more or all of the polypeptides b) are conjugated to the polymer a), and the conjugation is achieved by binding at one or more functional groups of the polymer a).

2. 2. The cell-adhesive polymer conjugate of claim 1, wherein the one or more polypeptides exhibit at least 70%, preferably at least 80%, preferably at least 90%, preferably at least 91%, preferably at least 92%, preferably at least 93%, preferably at least 94%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% sequence identity to the sequence of an integrin-binding protein of equal length, preferably collagen, laminin, tenascin, vitronectin, or fibronectin.

3. 3. The cell-adhesive polymer conjugate of claim 1 or 2, wherein the amount of amino acids in the one or more polypeptides is in the range of 5 to 95 amino acids, preferably in the range of 5 to 90 amino acids, preferably in the range of 5 to 85 amino acids, preferably in the range of 5 to 80 amino acids, preferably in the range of 5 to 75 amino acids, preferably in the range of 5 to 70 amino acids, preferably in the range of 6 to 65 amino acids, preferably in the range of 6 to 60 amino acids, preferably in the range of 6 to 55 amino acids, preferably in the range of 6 to 50 amino acids, preferably in the range of 6 to 45 amino acids, preferably in the range of 6 to 40 amino acids, preferably in the range of 7 to 35 amino acids, preferably in the range of 7 to 30 amino acids.

4. 10. The cell adhesive polymer conjugate of any one of the preceding claims, wherein the ULA polymer a) is selected from the group consisting of block copolymers, brush polymers, such as polyethylene glycol-polypropylene oxide or ethylene glycol-propylene oxide, n-butyl methacrylate or alkyl-containing brush polymers with methacrylic acid, such as copolymers of poly-(ethylene glycol)-methyl ether-methacrylate and propargyl acrylate, alternating styrene-maleic anhydride copolymers, such as esterified block copolymers.

5. The cell-adhesive polymer conjugate of any one of the preceding claims, wherein the cell-adhesive polymer conjugate comprises a covalent bond between the ultra-low adhesion (ULA) polymer (a) and the one or more polypeptides (b).

6. 10. The cell-adhesive polymer conjugate of any one of the preceding claims, wherein the one or more polypeptides are fragments of or correspond to proteins of the human extracellular matrix (ECM).

7. A method for producing the cell-adhesive polymer conjugate of any one of claims 1 to 6, comprising: i) providing at least one ULA polymer having one or more functional groups, wherein one, two, more than two, or all of said functional groups are selected from the group consisting of primary amines, carboxyl groups, aldehyde groups, and epoxide groups, hydroxyl groups, ketones, esters, amides, nitriles, thiols, hydroxylamines, imines, alkenes, isocyanates, halides, ethers, and azides; ii) providing one or more polypeptides, wherein the amount of amino acids in said polypeptide(s) is in the range of 5 to 100 amino acids; wherein said one or more polypeptides are selected from the group consisting of proliferation peptides, peptides that stimulate cell differentiation, preferably stem cell differentiation, peptides that prevent cell differentiation, preferably stem cell differentiation, peptides that promote cell adhesion, peptides that promote cell survival, peptides that promote cell proliferation, peptides that promote cell expansion, peptides that support migration and / or motility, and peptides that promote cell cycle progression, iii) conjugating said one or more polypeptides provided in step ii) with said at least one ULA polymer provided in step i).

8. A device for culturing cells, said device comprising a surface coated with the cell adhesive polymer according to any one of claims 1 to 6.

9. 9. The device for culturing cells according to claim 8, wherein the device is selected from the group consisting of flasks, such as round-bottom flasks, spinner flasks, Erlenmeyer flasks, retorts, and Florence flasks, cell culture dishes, cell culture bottles, cell culture bags, pipette tips, Petri dishes, multi-well plates, reactors, in particular bioreactors, tubes, pipettes, syringes, chips, slides, and cell culture scaffolds, such as microcarriers.

10. 10. The device for culturing cells according to claim 8 or 9, wherein the device is made of a material selected from the group consisting of glass, quartz, silicon, metal, metal oxide, and organic polymer such as polycarbonate, polystyrene, hydrophilized polystyrene, polyamide, poly(methyl methacrylate), polyester, polyvinyl chloride, polyvinylidene chloride, polymers containing fluorinated monomers such as fluorinated ethylene or propylene, polyolefins such as polyethylene, polypropylene, polynorbornene, cyclic olefin polymers, or cyclic olefin copolymers such as copolymers of ethylene and norbornene, or mixtures thereof.

11. 11. The device for culturing cells according to any one of claims 8 to 10, wherein the device is made of a material selected from the group consisting of polyolefins, in particular polystyrene, polyethylene, polypropylene, and (partially) fluorinated polyolefins, such as fluorinated ethylene propylene.

12. A method for manufacturing a cell culture device, preferably a cell culture device according to any one of claims 8 to 10, comprising: a) providing a device for culturing cells; b) providing a polymer conjugate according to any one of claims 1 to 6; c) coating the device provided in step a), preferably the surface of the device, with the polymer conjugate provided in step b).

13. 13. The method of claim 12, wherein the device is made of a material selected from the group consisting of glass, quartz, silicon, metal, metal oxide, and organic polymers such as polycarbonate, polystyrene, hydrophilized polystyrene, polyamide, poly(methyl methacrylate), polyester, polyvinyl chloride, polyvinylidene chloride, polymers containing fluorinated monomers such as fluorinated ethylene or propylene, polyolefins such as polyethylene, polypropylene, polynorbornene, cyclic olefin polymers or cyclic olefin copolymers such as copolymers of ethylene and norbornene, or mixtures thereof.

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