Cell attachment 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 to a surface of a cell culture device. Such peptides can interact with receptors on the surface of the cell while the polymer prevents non-specific binding of the cell to the surface of the device.

Description

Technical Field

[0001] The present invention relates to a cell attachment polymer conjugate comprising an ultra-low attachment (ULA) polymer a) and one or more polypeptides b) and to a cell culture device comprising a surface coated with such a cell attachment polymer. Background Art

[0002] Most cells in higher organisms are embedded in a meshwork of fibrous proteins and carbohydrates, including a large number of soluble signaling molecules, known as the extracellular matrix (ECM). The name "collagen," one of the main components of this complex extracellular network, translates from the French word "colle," meaning "glue," describing one of the functions of the ECM: it serves to secure a cell's proper position within several tissues.

[0003] Over the past few years, it has become clear that the ECM's role extends far beyond this function and is far more complex. Many cells cannot thrive unless surrounded by an ECM. Cells receive signals from the ECM through interactions between cell receptors and proteins, peptides, or soluble molecules that activate cell signaling cascades that control cell behavior. Therefore, it has become crucial to cultivate cells in in vitro systems designed to mimic the cells' natural environment within the human body to ensure similar cellular behavior.

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

[0005] For this purpose, usually The ECM is achieved in culture systems by isolating regulatory components of the ECM and absorbing them onto the surface of cell culture plastics (polystyrene, polyolefins, polyacrylates, etc.), thereby providing stimulatory signals to cells to be cultured in artificial containers. Several commercially available products utilize collagen or other protein-coated surfaces of the ECM of cultured cells to mimic the presence of the ECM.

[0006] Use the complete protein isolate or protein fragment of this type of product from the ECM prepared by enzyme digestion has obvious shortcomings.First, this type of product is not usually xeno-free - this means that the protein isolate originates from animals or humans. This makes these products incompatible with most clinical applications. In addition, the inherent batch difference of separating natural protein preparations may pose a challenge to the use of these products, even for the application outside the clinical field. Secondly, the coating protein not only provides the interaction based on specific receptors with the cell in the culture, but also causes the non-specific interaction between the protein coating of the container and the cell to be cultivated. This interaction can be based on the lipophilic interaction between the hydrophobic part on the surface of the cell and the coating protein or based on other non-covalent and non-specific interactions. This may cause undesirable stimulation to the cell, thereby causing undesirable cell effects, such as stronger or weaker proliferation.

[0007] In addition, the process of coating such proteins onto laboratory vessels such as flasks or microtiter plates is difficult to achieve: small differences in the purity of the peptide or protein, changes in the pH of the coating buffer used, temperature differences during coating, and other process variations can have a significant impact on the stability, charge, and conformation of the protein. This can impair the performance of the coating process itself or the derived product, leading to batch-to-batch variability. Cell culture products coated with such specific proteins can be used to activate specific cell signaling pathways and control cell fate. Therefore, nonspecific binding of cells to undesirable byproducts must be avoided. Such byproducts may be misfolded proteins generated during the separation or coating process.

[0008] Therefore, there is a need to mimic the natural environment of cells in the human body, preferably without the drawbacks mentioned above. Summary of the Invention

[0009] Therefore, the main object of the present invention is to provide an improved possibility of culturing human cells. In particular, one object of the present invention is to provide a coating material for a cell culture device that can advantageously simulate the natural environment of human cells.

[0010] The main object of the present invention is solved by a polymer conjugate comprising or consisting of:

[0011] a) an ultra-low attachment (ULA) polymer carrying one or more functional groups, wherein the one, two, three or more, or all of the functional groups are selected from the group consisting of: a primary amine, a carboxyl group, an aldehyde group, an alkyne, an epoxy group, a hydroxyl group, a ketone, an ester, an amide, a nitrile, a thiol, a hydroxylamine, an imine, an olefin, an isocyanate, a halide, an ether, and an azide. Furthermore, the functional groups of the polymer can be further chemically modified to enable conjugation to one or more polypeptides;

[0012] and

[0013] b) one or more polypeptides, wherein the amount of amino acids in the polypeptide is in the range of 5 to 100 amino acids,

[0014] Preferably, wherein the one, two, three or more or all polypeptides are selected from the group consisting of growth 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,

[0015] wherein said one, two, three or more or all polypeptides b) are conjugated to said ULA polymer a), wherein said conjugation is achieved via said one or more functional groups of polymer a).

[0016] The term "ultra-low attachment (ULA) polymer" as used herein is known to those skilled in the art and is frequently used in the art.

[0017] Preferably, the term "ultra-low attachment (ULA) polymer" as used herein refers to a polymer, preferably a polymeric surfactant consisting of a hydrophobic brush polymer and / or other hydrophobic anchoring groups attached to one or several or many hydrophilic moieties, such as derivatives of polyethylene glycol or polyacrylamide, which provides ultra-low attachment of cells when coated onto a cell culture device.

[0018] Preferably, ultra-low attachment is defined as adherent cells, preferably fibroblasts, particularly preferably mouse fibroblasts, preferably mouse 3T3 line fibroblasts, not adhering to the polymer coated onto the cell culture device.

[0019] Preferably, the term "non-adherent" as used herein is defined as 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 said polymer, preferably said polymer-coated surface.

[0020] Preferably, the term "non-adherent" as used herein is defined as 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 the polymer-coated surface.

[0021] Cell adhesion to the ULA polymer can be determined by cell morphology. For example, non-adherent fibroblasts have a rounded morphology, whereas adherent fibroblasts exhibit several cell processes, increased cell area, and an elongated shape. Non-adherent cells often form spheroids, which can also be observed through morphological characterization.

[0022] 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 hours, further preferably 12 hours, particularly preferably 15 hours, especially preferably 18 hours, even further preferably 21 hours, more preferably 24 hours, and most preferably 30 hours after adding the cells to the cell culture device.

[0023] Advantageously, culturing cells with a cell culture device coated with a conjugate according to the invention provides that the cells do not adhere by nonspecific binding, as they usually do on untreated plastic surfaces. However, they can interact via specific binding of cell receptors to peptides conjugated to a ULA polymer, preferably a surface coated with a ULA polymer. This is significantly different from similar embodiments of the principle of providing peptides, protein fragments or complete proteins on a plastic surface. If the immobilization of specific binding molecules has been achieved on a ULA surface, for example on a plastic surface coated with a ULA polymer, the interaction of the cells with the surface is limited to specific interactions, i.e. interactions between cell receptors and peptides. Any nonspecific interactions are prevented or at least reduced by the ULA coating. Therefore, specific signaling effects can be studied without being covered by nonspecific interactions. Therefore, it is possible to simulate the natural environment of cells using the conjugates according to the invention.

[0024] Preferably, the term "polymer" used to describe the conjugates according to the invention, their characteristics and effects refers to the ULA polymer as described herein.

[0025] It is a further advantage that, because the conjugation procedures employed generally produce stable covalent bonds, the coating conjugated to the peptide can also be sterilized using beta and gamma radiation or x-rays.

[0026] Preferably, the one, two, three or more or all 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 an equal length sequence of an integrin binding protein, preferably collagen, laminin, tenascin, vitronectin or fibronectin.

[0027] It was discovered that the cell-repelling properties of low-NSB surfaces can be combined with the specific signaling properties that characterize the ECM in order to create an environment that prevents nonspecific interactions and allows specific interactions by conjugated polypeptides that perfectly mimic the ECM.

[0028] This desired effect—specific interaction via one or two or more different peptides conjugated to the polymer combined with the non-interactive, cell-repellent properties of the ULA surface—has significant advantages: cultured cells adhere to the coated surface via one or more specific receptors, stimulating the specific receptors to impart 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 unwanted cell adhesion to the surface and the undesirable effects on the cells caused by such nonspecific adhesion.

[0029] The fact that several different peptides can be conjugated to a carrier ULA polymer or to a mixture of ULA polymers provides the following advantage: the specific interaction between the coated surface and the cells can be dispersed over two or more different receptors instead of interacting with only one receptor. If the cells change their properties when specifically binding to the container surface and the receptors are lost or changed during culture, the cells can remain adhered via a second or third receptor. This effect is particularly important when differentiating stem cells when bound to the coated surface: stem cell differentiation is often associated with huge changes in cell properties, including receptor expression. This is why differentiating adherent stem cells are at high risk of detachment of adhered cells from the coated surface during the differentiation process. This may cause cell loss during the subsequent purification process. Having the possibility of adhering cells via more than one receptor increases the chance of not losing such cells.

[0030] Combining ULA polymers with the described stimulatory peptides has another advantage: if cell cultures employing this combination are used to test the properties of small molecules—for example, during experiments to develop new therapeutic drugs—the ULA polymer may reduce the ability of the small molecules to bind to container surfaces—such as plastic surfaces or surfaces coated with proteins that expose a wide range of polar and charged moieties to the solution. ULA surfaces with ULA properties imparted by highly polar polyethylene glycol or polyacrylamide moieties may also prevent or reduce the absorption of small molecules to the surface, maintaining the desired concentration of such small molecules by avoiding loss of material to the surface, and thus providing more realistic results regarding the effect that the desired concentration of such small molecules will have on cells.

[0031] It is thus preferred that the amount of amino acids in the polypeptide b) of the cell attachment polymer conjugate according to the invention 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.

[0032] However, it has also been found that, unlike proteins or protein fragments, smaller oligopeptides or polypeptides cannot usually be coated directly onto plastic surfaces because there are usually no larger hydrophobic moieties that can be directly absorbed onto non-polar plastic surfaces.

[0033] The polymer conjugates according to the present invention address this problem: as described herein, the polypeptides are conjugated to the polymer, thereby anchoring them via the polymer's binding to the surface and reducing nonspecific cell adhesion to the container surface. Furthermore, interaction of the peptides with the cell's surface receptors is possible. This allows for the mimicking of the ECM.

[0034] At the same time, however, it is particularly advantageous to prevent or reduce nonspecific binding as described herein. It is therefore preferred that the ULA polymer a) is selected from the group consisting of: block copolymers; brush polymers (polyethylene glycol-polypropylene oxide or ethylene glycol-propylene oxide; n-butyl methacrylate and 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).

[0035] Compounds particularly useful as ultra-low attachment (ULA) polymers typically have the following structural components: one, several, or many moieties with low polarity: aliphatic chains, siloxanes, or aromatic units on the polymer chain, which typically form or are attached to the polymer's backbone. A 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. Furthermore, the polymer needs to carry chemical groups that allow the peptide to be conjugated to it. If such reactive groups impart a charge to the polymer, either permanent or pH-dependent, such as carboxyl or amino groups, such groups may need to be protected with an excess of a charge neutralizer to preserve the polymer's ULA properties. Using reactive groups, such as hydroxyl groups, to conjugate peptides that do not impart a charge to the polymer may be more suitable to preserve its polarity.

[0036] Useful ULA polymers are siloxane-based anchor polymers with polyethylene glycol or polyethylene glycol and isopropylene glycol side chains, such as Tegoprens (from Evonik, Essen, Germany), Silwet L7200, Silwet L7230, Silwet L7657 (all from Momentive, Albany, NY, USA), oligomeric surfactants such as Brij35 (Merck, Darmstadt, Germany) or graft polymers such as Inutec SL1 (Nordmann, Rassmann GmbH, Hamburg, Germany) or a copolymer prepared from n-butyl methacrylate (nBMA), methacrylic acid and methoxypolyethylene glycol methacrylate (Bisomer S20W) (hereinafter referred to as faCellitate F210).

[0037] All such compounds are amphiphilic, readily available compounds that adsorb to nonpolar surfaces and are endowed with ULA properties by highly polar side chains or highly polar scaffold moieties, where the polymers carry functional groups ready for conjugation to biological receptors such as peptides, proteins or nucleic acids.

[0038] As described herein, the one, two, three or more, or all of the functional groups of the polymer are selected from the group consisting of primary amines, carboxyl groups, aldehyde groups, alkynes, epoxy groups, hydroxyl groups, ketones, esters, amides, nitriles, thiols, hydroxylamines, imines, olefins, isocyanates, halides, ethers, and azides.

[0039] Thus, the term "polymer carrying one or more functional groups" is understood to mean that the functional groups are present in at least one monomer of the polymer or at a terminal end of the polymer or at a branch of the polymer, wherein the functional groups of at least one monomer of the polymer or at a terminal end of the polymer or at a branch of the polymer are not bound to another monomer. Thus, in the polymer, these functional groups or at least one, at least two, at least three or all of these functional groups may be used to bind to another compound, such as one or more polypeptides b).

[0040] The term "wherein the conjugation is at one or more functional groups of polymer a)" describes the conjugation of one or more polypeptides to the polymer. This term should be understood such that the conjugation of the one, two, three or more, or all, polypeptides to the polymer is achieved by binding the one, two, three or more, or all polypeptides to such functional groups of the polymer. Thus, the one, two, three or more, or all, functional groups of the polymer are bound to one or more polypeptides.

[0041] Preferably, the conjugation results in a covalent bond.

[0042] Therefore, in order to provide such conjugation, the polymer needs to carry such functional groups. The one, two, three or more or all functional groups are selected from the group consisting of: primary amines, carboxyl groups, aldehyde groups, alkynes, epoxy groups, hydroxyl groups, ketones, esters, amides, nitriles, thiols, hydroxylamines, imines, olefins, isocyanates, halides, ethers and azides.

[0043] For example, primary amines can be reacted with heterobifunctional linkers such as SMCC or sulfo-SMCC (SMCC: (sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate); Thermo Fisher Scientific Product No. 22322). This procedure can be performed in aqueous solution and converts the primary amine into an amide attached to a maleimide functional group. This maleimide then undergoes a hetero-Michael addition reaction with a thiol group on a cysteine ​​moiety of a peptide designed to bind to a receptor on the cell surface.

[0044] Similarly, carboxyl groups can be activated with carbodiimide derivatives, such as EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide), to form a reactive urea intermediate that can react directly with the free amine in the lysine portion of the peptide to form a stable amide bond.

[0045] Aldehyde groups in the coating polymer can react with amines in the peptide to form a Schiff base, which can then be stabilized by subsequent reduction.

[0046] Epoxides can react with alcohols or primary or secondary amines to form ethers or secondary or tertiary amines - all well suited to forming stable conjugated linkages.

[0047] Alkynes can react with compounds bearing an azide group to produce stable triazole linkages.

[0048] These reactions can be carried out in less than one hour at moderate pH and produce stable products. Conjugates comprising the peptide and the coating polymer or consisting thereof 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 conjugate can be absorbed onto plastic surfaces. Advantageously, the polymer conjugate can also be attached to non-plastic surfaces. In particular, anchoring to metal surfaces can be achieved by thiol-containing moieties or phosphate-containing moieties or phosphonic acid-containing moieties. Binding on glass surfaces characterized by partially deprotonated Si-OH bonds can be achieved by positively charged moieties such as partially protonated amino groups or boronic acid groups.

[0049] In this way the conjugation process can be further controlled and can advantageously provide consistent results.

[0050] Preferably, said one, two, three or more or all polypeptides b) are or correspond to fragments of proteins of the human extracellular matrix (ECM), preferably wherein said one, two, three or more or all 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), PHSRNGGRGD (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, for example RKKGRKKGRKKGRKK (SEQ ID NO. 7) or a peptide according to any one of SEQ ID NOs.: 8 to 18.

[0051] The term "corresponding to a fragment of a protein" is to be understood as meaning that these polypeptides need not be obtained from such proteins. However, it is preferred that the polypeptide comprises or consists 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 a protein.

[0052] Preferably, whenever the disclosure mentions the sequence identity or homology of an amino acid sequence in percentage terms, this type of mentioning is the value calculated for the amino acid sequence using the EMBOSS Water paired sequence alignment (protein) (http: / / www.ebi.ac.uk / Tools / psa / emboss_water / ). The local sequence alignment tool provided by the European Molecular Biology Laboratory (EMBL) European Bioinformatics Institute (EBI) uses an improved Smith-Waterman algorithm (see http: / / www.ebi.ac.uk / Tools / psa / and Smith, TF & Waterman, MS "Identification of common molecular subsequences" Journal of Molecular Biology, 1981 147 (1): 195-197). In addition, at this point, when using the improved Smith-Waterman algorithm to perform the corresponding paired comparison of two sequences, the default parameters currently provided by EMBL-EBI are used. These are for amino acid sequences: matrix=BLOSUM62, gap opening penalty=10, and gap extension penalty=0.5.

[0053] Preferably, the term fragment of a protein refers to a segment of the amino acid sequence of a protein.

[0054] Surprisingly, it was found that the resulting cell-attachment polymer conjugates can be coated onto cell culture devices to provide low-NSB surfaces with signaling properties toward neighboring cells. These signaling properties are highly reproducible because they are conferred by well-characterized synthetic peptides and are not subject to batch-to-batch variability of proteins isolated from natural sources.

[0055] Thus, the presence of an ECM can be advantageously mimicked using the cell attachment polymers according to the present invention.

[0056] The present invention further relates to a cell attachment polymer conjugate, preferably as described herein, obtained or obtainable by a process comprising the steps of:

[0057] i) providing a ULA polymer carrying one or more functional groups, wherein the one, two, three or more, or all of the functional groups are selected from the group consisting of a primary amine, a carboxyl group, an aldehyde group, an epoxy group, a hydroxyl group, a ketone, an ester, an amide, a nitrile, a thiol, a hydroxylamine, an imine, an olefin, an isocyanate, a halide, an ether, and an azide,

[0058] ii) providing one or more polypeptides, wherein the amount of amino acids in the polypeptide is in the range of 5 to 100 amino acids,

[0059] wherein said one, two, three or more or all polypeptides are selected from the group consisting of growth 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,

[0060] iii) conjugating the one, two, three or more or all of the polypeptides provided in step ii) to the polymer provided in step i).

[0061] Preferably, providing the polymer in step i) may comprise or consist of:

[0062] ia) providing one or more monomers preferably carrying one or more functional groups, wherein the one, two, three or more or all functional groups are selected from the group consisting of: a primary amine, a carboxyl group, an aldehyde group, an epoxy group, a hydroxyl group, a ketone, an ester, an amide, a nitrile, a thiol, a hydroxylamine, an imine, an olefin, an isocyanate, a halide, an ether and an azide;

[0063] ib) polymerizing the one, two, three or more or all monomers provided in step ia) to obtain a polymer.

[0064] The present invention further relates to a cell culture device, wherein the device comprises a surface coated with a cell attachment polymer according to the present invention.

[0065] Cell culture devices are known to the skilled person. Such devices may be any devices suitable for culturing cells and / or for processing such cell cultures.

[0066] Such a device can be selected from the group consisting of: a flask (e.g., a round-bottom flask, a spinner flask, an Erlenmeyer flask, a retort, a flat-bottom flask); a cell culture dish; a cell culture flask; a cell culture bag; a pipette tip; a culture dish; a multiwell plate; a reactor, in particular a bioreactor; a test tube; a pipette; a syringe; a chip; a glass slide; and a cell culture support (such as a microcarrier).

[0067] Preferably, the surface of the device is made of a material comprising or consisting of a material selected from the group consisting of glass; quartz; silicon; metal; metal oxide; and organic polymers (e.g., polycarbonate, polystyrene, hydrophilized polystyrene, polyamide, poly(methyl methacrylate), polyester, polyvinyl chloride, polyvinylidene chloride, polymers containing fluorinated monomers such as fluorinated ethylene or fluorinated propylene, polyolefins (such as polyethylene (e.g., low density polyethylene, ultra-low density polyethylene, linear low density polyethylene, high density polyethylene, high molecular weight polyethylene, ultra-high molecular weight polyethylene), polypropylene (e.g., oriented polypropylene, biaxially oriented polypropylene), polynorbornene, cyclic olefin polymer (COP) or cyclic olefin copolymer (COC) (such as a copolymer of ethylene and norbornene)).

[0068] Preferably, said surface of said device is made of 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.

[0069] Preferably, the surface of the device is made of a material comprising or consisting of polystyrene.

[0070] The present invention further relates to a method for producing a device according to the invention, comprising the following steps:

[0071] a) providing cell culture equipment;

[0072] b) providing a polymer conjugate according to the present invention; and

[0073] c) coating said device provided in step a), preferably a surface of said device, with said polymer conjugate provided in step b).

[0074] Preferably, the surface of the device is that part of the device which will come into contact with cells (via the coating) when the cell culture device is used.

[0075] Preferably, the term "coating the device" refers to a typical procedure for coating a device to be used for culturing cells. Such procedures are well known to those skilled in the art. Generally speaking, to coat a device, a coating material is added to the surface to be coated, and after a period of time, preferably in the range of 1 minute to 24 hours, preferably in the range of 5 minutes to 12 hours, preferably in the range of 10 minutes to 4 hours, preferably in the range of 15 minutes to 2 hours, the surface is washed to remove uncoated coating material.

[0076] The present invention further relates to a method for culturing cells, comprising the following steps:

[0077] a) providing a device according to the invention;

[0078] b) adding cells to the device provided in step a); and

[0079] c) culturing the cells added in step b) in the device.

[0080] Furthermore, the present invention relates to the use of the device according to the invention for culturing cells.

[0081] As described herein, the cells to be cultured can be selected from adherent cells and / or non-adherent cells. The term "adherent cells 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 grown from immortalized cell lines, induced pluripotent stem cells, hematopoietic stem cells, and mesenchymal stem cells.

[0082] As described above, it was surprisingly found that the device according to the present invention provides a low NSB surface with signaling properties towards neighboring cells. These signaling properties are highly reproducible because they are conferred by well-characterized synthetic peptides and are not affected by batch-to-batch variability of proteins isolated from natural sources.

[0083] Thus, the presence of an ECM can advantageously be simulated using the device according to the invention.

[0084] The present invention is further characterized below by means of illustrative, non-limiting examples. DETAILED DESCRIPTION

[0085] Example

[0086] Reagents and chemicals were purchased from abcr, Carl Roth, ChemPur, Carbolution, and Merck: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (Carl Roth, order number 2156.2), -hydroxysulfosuccinimide (Carbolution, Order No. CC01035), 1-(2-aminoethyl)maleimide hydrochloride (Chempur, Order No. BD76191-1g), hydrazine monohydrate (Merck, Order No. 207942-5G), Silwet L7657 (Momentive, Albany, NY), Dbco-amine TFA (Abcr, Order No. AB488945), 2-[2-(2-propynyloxy)ethoxy]ethanamine (Abcr, Order No. AB474698). The peptide was synthesized by NovoPro Bioscience Inc and ProteoGenix. Dde refers to ( -(1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl). Unless otherwise stated, all reagents and chemicals were used as purchased.

[0087] Making the Cell-Attachment Polymer Conjugates of the Invention

[0088] Example 1: Manufacturing ULA faCellitate F210

[0089] Ethanol (410.97 g), Bisomer S20W (50% in water, 32.09 g), n-butyl methacrylate (3.29 g), and methacrylic acid (0.66 g) were initially added to a 2 L reactor equipped with an anchor stirrer. The reactor was purged with nitrogen (10 L / min) and stirred at 125 rpm throughout the reaction. At the start of the reaction, the reactor was heated to 80°C over 30 minutes. Once 80°C was reached, t-butyl peroxypivalate (75% in isododecane, 0.27 g) in ethanol (9.73 g) was added one minute later. After stirring for 5 minutes, a mixture of ethanol (125.59 g), Bisomer S20W (50% in water, 288.83 g), n-butyl methacrylate (29.61 g), and methacrylic acid (5.98 g) was added continuously over 3 hours. Simultaneously, tert-butyl peroxypivalate (75% 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-butyl peroxypivalate (75% 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 as quickly as possible to 25°C. The next day, the reactor was heated to 130°C (bath temperature). Simultaneously, steam distillation was performed until all unreacted monomers had been removed. The distillation concluded at an internal temperature of 100°C. The distillation was then stopped, and the reaction mixture was cooled to 25°C as quickly as possible.

[0090] The solvent was removed on a rotary evaporator under reduced pressure. The resulting aqueous slurry was freeze-dried until a colorless to pale yellow solid was obtained.

[0091] Polymer-peptide conjugates for coating surfaces for culturing induced pluripotent stem cells (iPSCs)

[0092] Example 2: Preparation of polymer-peptide conjugates via thiol-maleimide Michael addition

[0093] FaCellitate F210 (500 mg, 643 µmol, 1.00 equiv) was dissolved in PBS buffer (20.0 mL) at 21°C. Subsequently, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (1.95 g, 10.2 mmol, 15.9 equiv) and 1-Hydroxysulfosuccinimide (1.11 g, 5.10 mmol, 7.93 equiv). 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 allowed to warm to 21°C. The mixture was purified by ultrafiltration (Amicon® stirred cell, Ultracel® PL-5 membrane, 5.000 MWCO, wash solution: distilled water, pH 4) and freeze-dried to obtain a colorless solid.

[0094] 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 the addition of peptide 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 (DMSO) (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 freeze-drying, a colorless solid was obtained.

[0095] Example 3: Preparation of polymer-peptide conjugates by direct coupling of peptides to polymers

[0096] FaCellitate F210 (50.0 mg, 64.3 µmol, 1.00 equiv) was dissolved in PBS buffer (5.0 mL) at 21°C. Subsequently, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (195 mg, 1.02 mmol, 15.9 equiv) and -hydroxysulfosuccinimide (111 mg, 510 µmol, 7.93 equiv). The reaction mixture was cooled to 0°C and stirred for 15 minutes, followed by the addition of peptide 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), dissolved in dimethyl sulfoxide / distilled water (1:1, 2.0 mL). The reaction mixture was stirred overnight and allowed to warm to 21°C. After the mixture was purified by ultrafiltration (Amicon® stirred cell, Ultracel® PL-5 membrane, 5.000 MWCO, washing solution: distilled water, pH 7) and freeze-dried, a colorless solid was obtained.

[0097] Then, by applying , The product was deprotected by Dde with a 2% v / v hydrazine monohydrate solution in dimethylformamide and stirred at 21° C. for 30 minutes. The reaction mixture was purified again by ultrafiltration (Amicon® stirred cell, Ultracel® PL-5 membrane, 5.000 MWCO, washing solution: distilled water, pH 7) and freeze-dried to obtain a colorless solid.

[0098] Example 4: Preparation of polymer-peptide conjugates via strain-promoted azide-alkyne cycloaddition

[0099] FaCellitate F210 (50.0 mg, 64.3 µmol, 1.00 equiv) was dissolved in PBS buffer (5.0 mL) at 21°C. Subsequently, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (246 mg, 1.29 mmol, 20.0 equiv) and -hydroxysulfosuccinimide (140 mg, 643 µmol, 10.0 equiv). The reaction mixture was cooled to 0°C and stirred for 15 minutes, after which Dbco-amine TFA (48.0 mg, 122 µmol, 1.90 equiv) dissolved in dimethyl sulfoxide (1.5 mL) was added. The reaction mixture was stirred overnight and allowed to warm to 21°C. The mixture was purified by ultrafiltration (Amicon® stirred cell, Ultracel® PL-5 membrane, 5.000 MWCO, wash solution: distilled water, pH 7) and freeze-dried to obtain a colorless solid. The product (30.0 mg, 28.9 µmol, 1.00 equiv) was dissolved in PBS buffer (5.0 mL) at 21°C, followed by the addition of peptide ring (RGDDYK(N3)) (11.7 mg, 18.1 µmol, 0.625 equiv, SEQ ID NO.: 12), Ac-GK(N3)-WGGPQVTRGDVFTMP-CONH2 (17.2 mg, 9.03 µmol, 0.313 equiv, SEQ ID NO.: 11), and Ac-GK(N3)-WGRKKGRKKGRKKGRKK-CONH2 (4.23 mg, 1.81 µmol, 0.0625 equiv, SEQ ID NO.: 13) dissolved in dimethyl sulfoxide (2.0 mL). The reaction mixture was stirred overnight at 21° C. and purified by ultrafiltration (Amicon® stirred cell, Ultracel® PL-5 membrane, 5.000 MWCO, washing solution: distilled water, pH 7). After freeze-drying, a colorless solid was obtained.

[0100] Example 5: Preparation of polymer-peptide conjugates via thiol-yne reaction

[0101] FaCellitate F210 (300 mg, 386 µmol, 1.00 equiv) was dissolved in PBS buffer (10.0 mL) at 21°C. Subsequently, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (1.48 g, 7.71 mmol, 20.0 equiv) and 2-Hydroxysulfosuccinimide (837 mg, 3.86 mmol, 10.0 equiv). The reaction mixture was cooled to 0°C and stirred for 15 minutes, after which 2-[2-(2-propynyloxy)ethoxy]ethanamine (248 mg, 1.74 mmol, 4.50 equiv) was added. The reaction mixture was stirred overnight and allowed to warm to 21°C. The mixture was purified by ultrafiltration (Amicon® stirred cell, Ultracel® PL-5 membrane, 5.000 MWCO, wash solution: distilled water, pH 7) and freeze-dried to obtain a colorless solid.

[0102] Under argon atmosphere, a portion of this product thus obtained (50.0 mg, 55.3 μmol, 1.00 equiv), 2,2'-azobis(isobutyronitrile) (1.0 mg, 5.53 μmol, 0.100 equiv) and peptide c(RGDyC) (20.6 mg, 34.6 μmol, 0.625 equiv), Ac-GCWGGPQVTRGDVFTMP-CONH2 (32.0 mg, 17.3 μmol, 0.313 equiv, SEQ ID NO.: 8) and Ac-GCWGRKKGRKKGRKKGRKK-CONH2 (7.89 mg, 3.46 μmol, 0.0625 equiv, SEQ ID NO.: 9) were dissolved in anhydrous water. , 5-dimethylformamide (5.0 mL) and stirred at 60° C. for two 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 freeze-dried to obtain a colorless solid.

[0103] Example 6: Preparation of polymer-peptide conjugates via thiol-yne reaction

[0104] Under an argon atmosphere, Silwet L-7657 (1.00 equivalent) and sodium hydride (1.50 equivalent) were dissolved in anhydrous tetrahydrofuran at 21°C. Subsequently, propargyl bromide (2.00 equivalent) was added dropwise over 20 minutes. The reaction mixture was stirred at 21°C for 24 hours, after which distilled water was added at 0°C. The 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 freeze-dried to yield a colorless solid.

[0105] Under argon atmosphere, a portion of this product thus obtained (1.00 equiv.), 2,2'-azobis(isobutyronitrile) (0.100 equiv.) and peptide c(RGDyC) (0.625 equiv.), Ac-GCWGGPQVTRGDVFTMP-CONH2 (0.313 equiv., SEQ ID NO.: 8) and Ac-GCWGRKKGRKKGRKKGRKK-CONH2 (0.0625 equiv., SEQ ID NO.: 9) were dissolved in anhydrous 4% paraformaldehyde. , 5-dimethylformamide (5.0 mL) and stirred at 60° C. for two 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 freeze-dried to obtain a colorless solid.

[0106] Polymer-peptide conjugates for culturing mesenchymal stem cells (MSCs)

[0107] Example 7: Preparation of polymer-peptide conjugates via thiol-maleimide Michael addition

[0108] FaCellitate F210 (500 mg, 643 µmol, 1.00 equiv) was dissolved in PBS buffer (20.0 mL) at 21°C. Subsequently, 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 allowed to warm to 21°C. The mixture was purified by ultrafiltration (Amicon® stirred cell, Ultracel® PL-5 membrane, 5.000 MWCO, wash solution: distilled water, pH 4) and freeze-dried to obtain a colorless solid.

[0109] A portion of this product (200 mg, 222 µmol, 1.00 eq) was dissolved in PBS buffer (10.0 mL) at 21°C, followed by the addition of the peptides Ac-GCWGGRGDSP-CONH2 (160 mg, 155 µmol, 0.700 eq, SEQ ID NO.: 14) or Ac-GCWGGGFOGER-CONH2 (183 mg, 155 µmol, 0.700 eq, 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, wash solution: distilled water, pH 7). After freeze-drying, a colorless solid was obtained.

[0110] Example 8: Preparation of polymer-peptide conjugates by direct coupling of peptides to polymers

[0111] FaCellitate F210 (50.0 mg, 64.3 µmol, 1.00 equiv) was dissolved in PBS buffer (5.0 mL) at 21°C. Subsequently, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (195 mg, 1.02 mmol, 15.9 equiv) and -hydroxysulfosuccinimide (111 mg, 510 µmol, 7.93 equiv). The reaction mixture was cooled to 0°C and stirred for 15 minutes before the 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 allowed to warm to 21°C. The mixture was purified by ultrafiltration (Amicon® stirred cell, Ultracel® PL-5 membrane, 5.000 MWCO, wash solution: distilled water, pH 7) and freeze-dried to obtain a colorless solid.

[0112] Example 9: Preparation of polymer-peptide conjugates via strain-promoted azide-alkyne cycloaddition

[0113] FaCellitate F210 (50.0 mg, 64.3 µmol, 1.00 equiv) was dissolved in PBS buffer (5.0 mL) at 21°C. Subsequently, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (246 mg, 1.29 mmol, 20.0 equiv) and -hydroxysulfosuccinimide (140 mg, 643 µmol, 10.0 equiv). The reaction mixture was cooled to 0°C and stirred for 15 minutes, after which Dbco-amine TFA (48.0 mg, 122 µmol, 1.90 equiv) dissolved in dimethyl sulfoxide (1.5 mL) was added. The reaction mixture was stirred overnight and allowed to warm to 21°C. The mixture was purified by ultrafiltration (Amicon® stirred cell, Ultracel® PL-5 membrane, 5.000 MWCO, wash solution: distilled water, pH 7) and freeze-dried to obtain a colorless solid. The product (30.0 mg, 28.9 µmol, 1.00 equiv) was dissolved in PBS buffer (5.0 mL) at 21°C, followed by the addition of the peptide Ac-GK(N3)-WGGRGDSP-CONH2 (31.3 mg, 28.9 µmol, 1.00 equiv, SEQ ID NO.: 17) or Ac-GK(N3)-WGGGFOGER-CONH2 (36.0 mg, 28.9 µmol, 1.00 equiv), SEQ ID NO.: 18. 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). A colorless solid was obtained after freeze-drying.

[0114] Example 10: Preparation of polymer-peptide conjugates via thiol-yne reaction

[0115] FaCellitate F210 (300 mg, 386 µmol, 1.00 equiv) was dissolved in PBS buffer (10.0 mL) at 21°C. Subsequently, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (1.48 g, 7.71 mmol, 20.0 equiv) and 2-Hydroxysulfosuccinimide (837 mg, 3.86 mmol, 10.0 equiv). The reaction mixture was cooled to 0°C and stirred for 15 minutes, after which 2-[2-(2-propynyloxy)ethoxy]ethanamine (248 mg, 1.74 mmol, 4.50 equiv) was added. The reaction mixture was stirred overnight and allowed to warm to 21°C. The mixture was purified by ultrafiltration (Amicon® stirred cell, Ultracel® PL-5 membrane, 5.000 MWCO, wash solution: distilled water, pH 7) and freeze-dried to obtain a colorless solid.

[0116] A portion of this thus obtained product (50.0 mg, 55.3 μmol, 1.00 equiv.), 2,2′-azobis(isobutyronitrile) (1.0 mg, 5.53 μmol, 0.100 equiv.) and the peptide Ac-GCWGGRGDSP-CONH2 (57.1 mg, 55.3 μmol, 1.00 equiv., SEQ ID NO.: 14) or Ac-GCWGGGFOGER-CONH2 (65.2 mg, 55.3 μmol, 1.00 equiv., SEQ ID NO.: 15) were dissolved in an argon atmosphere. , -dimethylformamide (5.0 mL) and stirred at 60°C for two 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 freeze-dried to obtain a colorless solid.

[0117] Example 11: Preparation of polymer-peptide conjugates via thiol-yne reaction

[0118] Under an argon atmosphere, Silwet L-7657 (1.00 equivalent) and sodium hydride (1.50 equivalent) were dissolved in anhydrous tetrahydrofuran at 21°C. Subsequently, propargyl bromide (2.00 equivalent) was added dropwise over 20 minutes. The reaction mixture was stirred at 21°C for 24 hours, after which distilled water was added at 0°C. The 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 freeze-dried to yield a colorless solid.

[0119] A portion of this thus obtained product (1.00 equivalent), 2,2'-azobis(isobutyronitrile) (0.100 equivalent) and the 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 an argon atmosphere. , -dimethylformamide (5.0 mL) and stirred at 60°C for two 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 freeze-dried to obtain a colorless solid.

[0120] Example 12: Coating of cell culture articles with the cell attachment polymer conjugates of the present invention

[0121] 1. Dissolve the lyophilized polymer-peptide conjugate of any one of Examples 2 to 11 in DPBS buffer to a concentration of 16 mg / ml (10× stock solution)

[0122] 2. Prepare a working solution (working concentration: 1.6 mg / ml) by diluting the stock solution 1:10 in DPBS buffer. Note that the working solution needs to be prepared fresh just before starting surface coating.

[0123] 3. Add 1 ml of working solution to one well of a 6-well plate (Cat. No. 351146, Corning). Other cell culture consumables can be used, such as 24-well plates, 96-well plates, and T75 flasks. If using other formats, adjust the volume of coating solution based on the well / flask size (e.g., 500 µl in one well of a 24-well plate). The surface must be non-tissue culture treated polystyrene.

[0124] 4. Incubate at room temperature for 1 hour.

[0125] 5. Discard the working solution and discard it.

[0126] 6. Wash the surface three times with 1 ml of DPBS buffer.

[0127] 7. Store the coated plates at -18°C or lower; then, culture the cells as described in Example 13.

[0128] Example 13: Cultivation of cells using the cell culture preparation according to the invention

[0129] Cell suspensions were prepared by resuspending BIONI010-B cells (human induced pluripotent stem cell line) in 2 ml of mTeSR+ medium (Stemcell Technologies).

[0130] 1. Add 250,000 cells to one well of a 6-well plate coated according to Example 12; for control experiments, use an uncoated polystyrene cell culture plate. Incubate the cells at 37°C and 5% CO2.

[0131] 2. After 24 hours, cell attachment was monitored using bright field microscopy.

[0132] 3. Monitor cell confluence (as an indirect measure of cell proliferation) and cell morphology daily using bright-field microscopy until the culture reaches 60%-80% confluence (usually after 7 days).

[0133] 4. Use CTS according to the manufacturer's instructions. TM VerseneTM Cells were detached using 4% paraformaldehyde (PAGE) buffer (No. 15040066, Thermo Fisher).

[0134] 5. Determination of pluripotency marker expression:

[0135] - hNANOG and hTRA1-81 at the protein level by flow cytometry

[0136] - hOCT4, hSOX2 and hNANOG at the transcriptional level by RT-PCR

[0137] result: All seeded cells adhered specifically to the peptides presented on the coated surface. The cells appeared round and grew in colonies with clearly defined edges. Seven days after seeding, the culture reached 70% confluence and 2,000,000 cells were counted. At both the protein and transcript levels, 90%-100% of the cells were positive for the pluripotency markers analyzed.

[0138] Comparison: BIONI010-B cells were seeded onto tissue culture treated polystyrene surfaces without coating with a polymer-peptide conjugate. None of the seeded cells adhered to the surface. The cells formed multiple spheroids that floated within the wells. Most spheroids were lost during media changes. After 7 days of culture, the few remaining spheroids were dissociated to measure the expression of pluripotency markers. Less than 30% of the cells were positive for the analyzed markers.

Claims

1. A cell attachment polymer conjugate comprising: a) an ultra-low attachment (ULA) polymer carrying one or more functional groups, wherein the one, two, three or more, or all of the functional groups are selected from the group consisting of a primary amine, a carboxyl group, an aldehyde group, an alkyne, an epoxy group, a hydroxyl group, a ketone, an ester, an amide, a nitrile, a thiol, a hydroxylamine, an imine, an olefin, an isocyanate, a halide, an ether, and an azide; and b) one or more polypeptides, wherein the amount of amino acids in the polypeptide is in the range of 5 to 100 amino acids, wherein said one, two, three or more or all polypeptides are selected from the group consisting of growth 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, wherein said one, two, three or more or all polypeptides b) are conjugated to said polymer a), wherein said conjugation is achieved by linking at said one or more functional groups of polymer a).

2. The cell attachment 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 an equal length sequence of an integrin binding protein, preferably collagen, laminin, tenascin, vitronectin or fibronectin.

3. The cell attachment polymer conjugate according to claim 1 or 2, wherein the amount of amino acids of 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. The cell attachment 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, copolymers of n-butyl methacrylate and methacrylic acid or alkyl-containing brush polymers such as poly-(ethylene glycol)-methyl ether-methacrylate and propargyl acrylate; alternating styrene maleic anhydride copolymers such as esterified block copolymers. 5 . The cell attachment polymer conjugate according to any one of the preceding claims, wherein the cell attachment polymer conjugate comprises a covalent bond between the ultra low attachment (ULA) polymer (a) and the one or more polypeptides (b). 6 . The cell attachment polymer conjugate of any preceding claim, wherein the one or more polypeptides are or correspond to fragments of proteins of the human extracellular matrix (ECM).

7. A method for producing the cell attachment polymer conjugate according to any one of claims 1 to 6, comprising the following steps: i) providing at least one ULA polymer carrying one or more functional groups, wherein the one, two, three or more, or all, functional groups are selected from the group consisting of primary amines, carboxyl groups, aldehyde groups and epoxy groups, hydroxyl groups, ketones, esters, amides, nitriles, thiols, hydroxylamines, imines, olefins, isocyanates, halides, ethers, and azides, ii) providing one or more polypeptides, wherein the amount of amino acids in the polypeptide is in the range of 5 to 100 amino acids, wherein the one or more polypeptides are selected from the group consisting of growth 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 the one or more polypeptides provided in step ii) to the at least one ULA polymer provided in step i).

8. A cell culture device, wherein the device comprises a surface coated with the cell attachment polymer according to any one of claims 1 to 6.

9. The cell culture device 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 flat-bottom flasks; cell culture dishes; cell culture bottles; cell culture bags; pipette tips; culture dishes; multiwell plates; reactors, in particular bioreactors; test tubes; pipettes; syringes; chips; slides; and cell culture supports, such as microcarriers.

10. The cell culture device 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 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 fluorinated 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. The cell culture device 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 the following steps: a) providing cell culture equipment; b) providing a polymer conjugate according to any one of claims 1 to 6; and c) coating said device provided in step a), preferably a surface of said device, with said polymer conjugate provided in step b).

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 fluorinated propylene, polyolefins such as polyethylene, polypropylene, polynorbornene, cyclic olefin polymers or cyclic olefin copolymers such as copolymers of ethylene and norbornene, or mixtures thereof.