WO2020156907A1

WO2020156907A1 - Biocompatible device with an adsorbed layer of acrylic comb copolymer

Info

Publication number: WO2020156907A1
Application number: PCT/EP2020/051519
Authority: WO
Inventors: Rupert Konradi; Veronique Schwartz; Tobias MENTZEL; Patrick WILKE; Peter Stengel
Original assignee: Basf Se
Priority date: 2019-01-30
Filing date: 2020-01-22
Publication date: 2020-08-06

Abstract

The present invention relates to a biocompatible device which comprises on its surface an adsorbed layer of a polymer P which is a copolymer of at least one macromonomer selected from an ester E of (meth)acrylic acid and polyethylene oxide or an polyethylene glycol (meth)acrylamide, and at least one monomer M selected from alkyl (meth)acrylate, aryloxyalkyl (meth)acrylate, alkyl (meth)acrylamide or aryl (meth)acrylamide. The invention further relates to a process for making the biocompatible device comprising the following steps: providing the biocompatible device, and applying to the surface of the biocompatible device a solution S of the polymer P in a solvent L; and it relates to a process for cultivating cells, comprising the following steps: providing the biocompatible device which is a device for cultivating cells, and cultivating the cells in the supernatant medium above the surface of the biocompatible device.

Description

Biocompatible device with an adsorbed layer of acrylic comb copolymer

The present invention relates to a biocompatible device which comprises on its surface an adsorbed layer of a polymer P which is a copolymer of at least one macromonomer selected from an ester E of (meth)acrylic acid and polyethylene oxide or an polyethylene glycol (meth acrylamide, and at least one monomer M selected from alkyl (meth)acrylate, aryloxyalkyl (meth)acrylate, alkyl (meth)acrylamide or aryl (meth)acrylamide. The invention further relates to a process for making the biocompatible device comprising the following steps: providing the biocompatible device, and applying to the surface of the biocompatible device a solution S of the polymer P in a solvent L; and it relates to a process for cultivating cells, comprising the following steps: providing the biocompatible device which is a device for cultivating cells, and cultivating the cells in the supernatant medium above the surface of the biocompatible device. Combinations of preferred embodiments with other preferred embodiments are within the scope of the present invention.

Biocompatible devices such as biosensors and devices for cultivation of cells play an important role in many technologies. An important issue with the application of biocompatible devices is the unwanted deposition of biological or organic material on a surface. For example when cultivating cells, some types of cells tend to attach to the surface. For biosensors, one undesired side effect is that the signal-to-noise ratio of the detected signals and the limit of detection (LOD) is worsened. Several approaches have been tried to solve these problems and to prevent the formation and deposition of organic or biological materials on a surface of biocompatible devices.

This objective was achieved by a biocompatible device which comprises on its surface an adsorbed layer of a polymer P which is a copolymer of at least one macromonomer selected from an ester E of (meth)acrylic acid and polyethylene oxide or an polyethylene glycol (meth)acrylamide, and at least one monomer M selected from alkyl (meth)acrylate, aryloxyalkyl (meth)acrylate, alkyl (meth)acrylamide or aryl (meth)acrylamide.

The object was also achieved by a process for making the biocompatible device comprising the following steps: A) providing the biocompatible device, and

B) applying to the surface of the biocompatible device a solution S of the polymer P in a

solvent L.

The object was also achieved by a process for cultivating cells, comprising the following steps: a) providing the biocompatible device which is a device for cultivating cells, and

b) cultivating the cells in the supernatant medium above the surface of the biocompatible device.

The object was also achieved by a process for cultivating cells, comprising the following steps: a) providing the biocompatible device which is a device for cultivating cells where the

biocompatible device is obtainable by the process for making the biocompatible device, and b) cultivating the cells in the supernatant medium above the surface of the biocompatible device.

A suitable biocompatible device is a biosensor or a device for cultivating cells.

A device for cultivating cells can for example be any device that is suitable for cultivating cells or for handling such cell. In another form devices can for example be any device that is suitable for cultivating cells including handling such cell.

Examples of devices for cultivating cells include cell culture flasks, cell culture dishes, cell culture plates (e.g. multiwell or microwell plates), cell culture bags, and reactors (e.g.

bioreactors).

Further examples of devices for cultivating cells which are suitable for handling cell culture containing liquids include tubes, pipettes and pipette tips, and syringes.

Preferably, the device for cultivating cells is selected from flasks, cell culture dishes, cell culture plates, cell culture bags, bioreactors, tubes, pipettes, and syringes. In particular, the device for cultivating cells is selected from flasks, cell culture dishes, and cell culture plates.

The devices for cultivating cells can be lab scale devices, semi industrial size devices or industrial size devices. Typical industrial sizes of reactors, such as bioreactors are for instance 3 L, 50-200 L or 1000-2000 L. Typical sizes of a well in a cell culture plate is from 0.001 to 10 ml. A biosensor is usually an analytical device designed to detect an analyte. The biosensors normally comprise at least one bio-recognition element, a biotransducer component and in many cases an electronic processing system. In many cases the biosensors also comprise tubes or pipes that are designed to transport fluids comprising the analyte towards and away from the biorecognition element as well as a housing to protect the biosensor. An analyte may mean a substance that shall be detected in an analytical process. Normally bioreceptors are biomolecules from organisms or receptors modeled after biological systems to interact with the analyte of interest. Typical bioreceptors include proteins, antibody/antigen, enzymes, nucleic acids/DNA, sugars, carbohydrates, cells or cellular structures, or biomimetic materials. In a preferred embodiment, bioreceptors are proteins such as antigens and antibodies, peptides, DNA, RNA, sugars and carbohydrates.

The biocompatible device comprises on its surface an adsorbed layer of the polymer P. The whole surface or parts of the surface comprises the adsorbed layer of the polymer P.

Preferably, at least the part of the surface which usually comes into contact with biological material (e.g. cells) under normal operating conditions comprises the adsorbed layer of the polymer P. The layer of the polymer P is adsorbed to the surface, which usually means that there are no covalent chemical bonds between the polymer and the surface. The term “adsorbed” usually refers to physisorption, and usually not to chemisorption.

Preferably, the surface is free of other adsorbed polymers beside the polymer P. Preferably, the surface is free of other layers (e.g. adsorbed, or covalently bound layers) beside the adsorbed layer of the polymer P.

The adsorbed layer may comprise at least one polymer P, such as one, two or three different polymers P. Preferably, the adsorbed layer consists of the polymer P. In one form the adsorbed layer is free of other polymers beside the polymer P. In another form the adsorbed layer is free of biological compounds, pharmaceuticals or biologically active compounds.

The surface of biocompatible device can be made of any biocompatible material, e.g. material on which cells can be grown, be it with the cells being attached to the surface or not. Preferably, the surface is at least partly made of glass, quartz, silicon, metals, metal oxides or organic polymers. In another preferred form, the surface is at least partly made of organic polymers, such as polyolefines, preferably polystyrene, polyethylene, polypropylene, fluorinated polyolefines, or partially fluorinated polyolefines. In another preferred form, the surface is at least partly made of organic polymers, such as polyolefines (e.g. polyethylene, polypropylene), polystyrene, polycarbonate, polysulfones (e.g. polyethersulfones), fluorinated polyolefines, or partially fluorinated polyolefines, where polystyrene is preferred.

Suitable organic polymers for the surface include

- polycarbonate,

- polystyrene,

- hydrophilized polystyrene,

- polyamide,

- poly(methyl methacrylate),

- polyesters,

- polysulfones (like polyethersulfones),

- polyvinylchloride,

- polyvinylidene chloride,

- fluorinated or partially fluorinated polyolefins (like fluorinated polyethylene or polypropylene),

- polyolefines [such as polyethylene (like low density polyethylene, ultralow density

polyethylene, linear low density polyethylene, high density polyethylene, high molecular weight polyethylene, ultrahigh molecular weight polyethylene), polypropylene (like oriented polypropylene, biaxially oriented polypropylene), cyclic olefin polymers (COP, like polynorbornene) or cyclic olefin copolymers (COC, like copolymers of ethylene and norbornene)].

Preferably, the surface is at least partly made of

polyolefines,

polystyrene,

fluorinated or partially fluorinated polyolefines.

In another preferred form, the surface is at least partly made of

polyolefines,

polycarbonate,

polysulfones,

polystyrene,

fluorinated or partially fluorinated polyolefines.

In another preferred form the surface is at least partly made of

polyethylene,

polypropylene,

- COC, - COP,

polycarbonate,

polystyrene, or

fluorinated or partially fluorinated polyolefines

In one especially preferred embodiment, the surface is at least partly made of polystyrene.

The term“surfaces is at least partly made of a material” usually means that at least 50 %, preferably at least 80%, and in particular at least 95% of the surface is made of the material.

The surface usually refers to that part of the surface of the biocompatible device which in general comes into contact with biological material (e.g. cells) under normal operating conditions. In a form the at least 50 %, preferably at least 80%, and in particular at least 95% of the surface is made of the organic polymer, such as polyolefines (preferably polyethylene or polypropylene), polycarbonate, polystyrene, fluorinated or partially fluorinated polyolefines, cyclic olefin polymers, or cyclic olefin copolymers. In another form the at least 50 %, preferably at least 80%, and in particular at least 95% of the surface is made polystyrene.

The polymer P is a copolymer of at least one monomer M and at least one macromonomer (e.g. the ester E). In the context of this application, this shall mean that polymer P comprises these monomers in polymerized form. Preferably, the polymer P consists of the monomer M and the macromonomer, such as the ester E. In another form the polymer P is free of other monomers beside the monomer M and the macromonomer, such as the ester E.

The polymer P normally comprises the monomer M and the macromonomer (e.g. the ester E) in a molar ratio from 0.05:1 to 50:1 , preferably from 0.2:1 to 15:1 , more preferably from 0.3:1 to 10:1 and especially preferably from 0.5:1 to 4:1. In another form the polymer P comprises the monomer M and the macromonomer (e.g. the ester E) in a molar ratio from 1 : 3 to 3 : 1 , preferably from 1 : 2 to 2 : 1 , and in particular from 1 : 1.5 to 1.5 : 1.

In a preferred form the macromonomer is the ester E. The ester E is the ester of methacrylic acid and polyethylene oxide, and/or of acrylic acid and polyethylene oxide. In a preferred embodiment the polyethylene oxide is esterified on one end with (meth)acrylic acid and has been functionalized on the other end, for example by pro forma etherification with an alkyl group like methyl, ethyl, propyl or butyl, preferably methyl. The latter are normally obtained by alkoxylation of alcohols like methanol. In a preferred form the ester E is an ester of (meth)acrylic acid and polyethylene glycol mono C₁-C₄ alkyl ether, in particular an ester of (meth)acrylic acid and polyethylene glycol mono methyl ether. In a less preferred embodiment said polyethylene oxide is esterified on one end with (meth)acrylic acid and bears a hydroxy group on the other end.

Polyethylene oxide in this context shall mean a polyalkylene oxide that consists essentially of oxyethylene units and optionally a terminal alkyl ether group. In particular, polyethylene oxide comprises less than 10 mol% of oxyalkylene units different from oxyethylene. Preferably, polyethylene oxide as used in this context comprises less than 5 mol %, more preferably less than 1 mol % of oxyalkylene units different from oxyethylene. In an especially preferred embodiment polyethylene oxide as used herein consists of oxyethylene units and a terminal alkyl ether group. Polyethylene oxide is in many cases prepared by ring opening polymerization of ethylene oxide using alcohols like methanol, ethanol, n/iso-propanol or n/sec/tert-butanol as a starter.

In one form the macromonomer is a polyethylene glycol (meth)acrylamide, which may be selected from mono-N or di-N substituted (meth)acrylamide, where mono-N substituted are preferred. The polyethylene glycol may be selected from the aforementioned polyethylene oxide.

Preferably, macromonomer (e.g. the ester E) has an average molar mass Mn of 300 to

10.000 g/mol, more preferably 500 to 10,000 and even more preferably 800 to 10,000 g/mol, especially preferably 1 ,000 to 10,000 g/mol and particularly preferably 1500 to 10,000 g/mol.

In another embodiment, the macromonomer (e.g. the ester E) has an average molar mass Mn of 300 to 8,000 g/mol, more preferably 300 to 5,000 and even more preferably 300 to

3,000 g/mol and especially preferably 300 to 2000 g/mol.

In especially preferred embodiments, macromonomer (e.g. the ester E) has an average molar mass Mn of 500 to 8000 g / mol, 1000 to 5000 g/mol, 800 to 3000 g/mol, 1000 to 3000 g/mol,

800 to 2500 g/mol or 1500 to 2000 g/mol.

The monomer M is selected from alkyl (meth)acrylate, aryloxyalkyl (meth)acrylate, alkyl (meth)acrylamide, or aryl (meth)acrylamide. Mixtures of momoner M are also possible.

Preferably, monomer M is C1-C18 alkyl (meth)acrylate or phenoxyethyl (meth)acrylate. In one form momoner M is C1-C18 alkyl (meth)acrylate, preferably C1-C6 alkyl (meth)acrylate. In another form monomer M is phenoxyethyl (meth)acrylate. Suitable alkyl (meth)acrylates are C1-C18 alkyl (meth)acrylate, preferably C1-C12 alkyl (meth)- acrylate, in particular C1-C6 alkyl (meth)acrylate. The alkyl unit of the alkyl (meth)acrylate may be linear, branched or cyclic, preferably linear or branched. In a preferred form alkyl (methacry late is methyl acrylate, ethyl acrylate, propyl acrylate, butyl acrylate, butyl methacrylate, or methyl methacrylate. In particular the momoner M is selected from butyl acrylate and butyl methacrylate. In another form the monomer M is selected from n-butyl (meth)acrylate, preferably n-butyl methacrylate.

Suitable aryloxyalkyl (meth)acrylates are phenyloxyalkyl (meth) acrylates, such as

phenoxyethyl acrylate and phenoxyethyl methacrylate, wherein phenoxyethyl acrylate is preferred.

Suitable alkyl (meth)acrylamides are N-C₁-C₂₂ alkyl (meth)acrylamides, preferably N-C₄-C₂₀ (meth)alkyl acrylamides, such as N-tert-butyl acrylamide, N-hexadecyl acrylamide, or N-octa- decyl acrylamide.

Suitable aryl (meth)acrylamides are N-aryl (meth)acrylamides, preferably N-phenyl acrylamide.

The polymer P has usually a number average molar mass Mn of 2,000 to 100,000 g/mol, preferably of 3,000 to 80,000, and in particular of 10,000 to 40,000. The polymer P has usually a weight average molar mass Mw of 5,000 to 200,000 g/mol, preferably of 7,000 to 150,000, and in particular of 20,000 to 80,000. All values for the average molar mass Mn or Mw given in this application may be determined by gel permeation chromatography (GPC), e.g. using the method as described in the experimental section of this application.

The polymer P is preferably a statistical copolymer in which monomer M and macromonomer (e.g. the ester E) are distributed statistically.

The polymer P is normally prepared by radical polymerization of the monomer M and the macromonomer (e.g. the ester E), e.g. by solution polymerization or emulsion polymerization.

Preferably the polymer P is prepared by solution polymerization.“Solution polymerization” means that all starting materials are at least partly dissolved in the same solvent and that the polymerization reaction takes place in homogenous phase, without additional surfactants having to be present. In one preferred embodiment, the monomer M and the macromonomer (e.g. the ester E) are dissolved in suitable solvents like alcohols like methanol, ethanol, 1 -propanol, 2-propanol, butanol or mixtures thereof and are then polymerized. Preferably, such solvents for the solution polymerization comprise at least 50 % by weight, preferably 70% and more preferably 80 % by weight of alcohols like methanol, ethanol, 1-propanol, 2-propanol, butanol or mixtures thereof. Preferably, such solvents for the solution polymerization comprise 20 % by weight or less, preferably 10 % by weight or less of water.

The radical polymerization can be initiated by oxidative radical starters like organic peroxides (e.g. tert-butyl-2,2-dimethylpropaneperoxoate, sodium persulfate, potassium persulfate, metachloroperbenzoic acid) or azo starters like azo-bisisobutyrodinitrile or 2,2'-Azobis(2-methyl- butyronitrile).

The biocompatible device comprises on its surface an adsorbed layer of a polymer P, where the adsorbed layer may be a self-assembled monolayer of the polymer P.

A“self-assembled monolayer” means typically a molecular assembly formed spontaneously on a surface by adsorption. A self-assembled monolayer forms usually spontaneously on such surfaces without any further process step being required. Self-assembled monolayers can for example be characterized by atomic force microscopy (AFM) or X-ray photoelectron

spectroscopy (XPS) or in situ methods such as quartz crystal microbalance or surface plasmon resonance spectroscopy.

The self-assembled monolayer of the polymer P normally has a thickness that correlates with the size of the individual molecules adsorbed to that surface, e.g. it is normally smaller than 100 nm, such as 1 to 50 nm, preferably 1 to 20 nm, and in particular 1 to 10 nm.

In a form the biocompatible device comprises on its surface an adsorbed layer of a polymer P which is a copolymer of at least one macromonomer selected from an ester E of (meth)acrylic acid and polyethylene oxide, and at least one monomer M selected from alkyl (meth)acrylate or aryloxyalkyl (meth)acrylate, where the biocompatible device is a device for cultivating cells (e.g. selected from cell culture flasks, cell culture dishes, cell culture plates, cell culture bags, reactors, tubes, pipettes, pipette tips, and syringes), and where the surface is at least partly made of polyolefins, polystyrene, fluorinated or partially fluorinated polyolefines. In a form the biocompatible device comprises on its surface an adsorbed layer of a polymer P which is a copolymer of at least one macromonomer selected from an ester E of (meth)acrylic acid and polyethylene oxide, and at least one monomer M selected from butyl (meth)acrylate (e.g. n-butyl methacrylate), where the biocompatible device is a device for cultivating cells (e.g. selected from cell culture flasks, cell culture dishes, cell culture plates, cell culture bags, reactors, tubes, pipettes, pipette tips, and syringes), and where the surface is at least partly made of polyolefins, polystyrene, fluorinated or partially fluorinated polyolefines.

In another form the biocompatible device comprises on its surface an adsorbed layer of a polymer P which is a copolymer of at least one macromonomer selected from an ester E of (meth)acrylic acid and polyethylene oxide, and at least one monomer M selected from C₁-C₁₈ alkyl (meth)acrylate or phenoxyethyl (meth)acrylate, where the biocompatible device is a device for cultivating cells (e.g. selected from cell culture flasks, cell culture dishes, cell culture plates, cell culture bags, reactors, tubes, pipettes, pipette tips, and syringes), and where the surface is at least partly made of polyolefins or polystyrene.

In another form the biocompatible device comprises on its surface an adsorbed layer of a polymer P which is a copolymer of at least one macromonomer selected from an ester E of (meth)acrylic acid and polyethylene oxide, and at least one monomer M selected from butyl (meth)acrylate (e.g. n-butyl methacrylate), where the biocompatible device is a device for cultivating cells (e.g. selected from cell culture flasks, cell culture dishes, cell culture plates, cell culture bags, reactors, tubes, pipettes, pipette tips, and syringes), and where the surface is at least partly made of polyolefins or polystyrene.

In another form the biocompatible device comprises on its surface an adsorbed layer of a polymer P which is a copolymer of at least one macromonomer selected from an ester E of (meth)acrylic acid and polyethylene oxide, and at least one monomer M selected from C1-C18 alkyl (meth)acrylate or phenoxyethyl (meth)acrylate, where the polymer P comprises the monomer M and the macromonomer in a molar ratio from 0.3:1 to 10:1 , where the biocompatible device is a device for cultivating cells (e.g. selected from cell culture flasks, cell culture dishes, cell culture plates, cell culture bags, reactors, tubes, pipettes, pipette tips, and syringes), and where the surface is at least partly made of polystyrene.

In another form the biocompatible device comprises on its surface an adsorbed layer of a polymer P which is a copolymer of at least one macromonomer selected from an ester E of (meth)acrylic acid and polyethylene oxide, and at least one monomer M selected from butyl (meth)acrylate (e.g. n-butyl methacrylate), where the polymer P comprises the monomer M and the macromonomer in a molar ratio from 0.3:1 to 10:1 , where the biocompatible device is a device for cultivating cells (e.g. selected from cell culture flasks, cell culture dishes, cell culture plates, cell culture bags, reactors, tubes, pipettes, pipette tips, and syringes), and where the surface is at least partly made of polystyrene.

In a form the biocompatible device comprises on its surface an adsorbed layer of a polymer P which is a copolymer of at least one macromonomer selected from an ester E of (meth)acrylic acid and polyethylene oxide, and at least one monomer M selected from alkyl (meth)acrylate or aryloxyalkyl (meth)acrylate, where the adsorbed layer may be a self-assembled monolayer of the polymer P,

where the biocompatible device is a device for cultivating cells (e.g. selected from cell culture flasks, cell culture dishes, cell culture plates, cell culture bags, reactors, tubes, pipettes, pipette tips, and syringes), and where the surface is at least partly made of polyolefins, polystyrene, fluorinated or partially fluorinated polyolefines.

where the biocompatible device is a device for cultivating cells (e.g. selected from cell culture flasks, cell culture dishes, cell culture plates, cell culture bags, reactors, tubes, pipettes, pipette tips, and syringes), and where the surface is at least partly made of polyolefins, polycarboantes, polysulfones, polystyrene, fluorinated or partially fluorinated polyolefines.

In a form the biocompatible device comprises on its surface an adsorbed layer of a polymer P which is a copolymer of at least one macromonomer selected from an ester E of (meth)acrylic acid and polyethylene oxide, and at least one monomer M selected from butyl (meth)acrylate (e.g. n-butyl methacrylate), where the adsorbed layer may be a self-assembled monolayer of the polymer P,

In a form the biocompatible device comprises on its surface an adsorbed layer of a polymer P which is a copolymer of at least one macromonomer selected from an ester E of (meth)acrylic acid and polyethylene oxide, and at least one monomer M selected from butyl (meth)acrylate (e.g. n-butyl methacrylate), where the adsorbed layer may be a self-assembled monolayer of the polymer P, where the biocompatible device is a device for cultivating cells (e.g. selected from cell culture flasks, cell culture dishes, cell culture plates, cell culture bags, reactors, tubes, pipettes, pipette tips, and syringes), and where the surface is at least partly made of polyolefins, polycarbonates, polysulfones, polystyrene, fluorinated or partially fluorinated polyolefines.

In another form the biocompatible device comprises on its surface an adsorbed layer of a polymer P which is a copolymer of at least one macromonomer selected from an ester E of (meth)acrylic acid and polyethylene oxide, and at least one monomer M selected from C₁-C₁₈ alkyl (meth)acrylate or phenoxyethyl (meth)acrylate, where the adsorbed layer may be a self-assembled monolayer of the polymer P,

where the biocompatible device is a device for cultivating cells (e.g. selected from cell culture flasks, cell culture dishes, cell culture plates, cell culture bags, reactors, tubes, pipettes, pipette tips, and syringes), and where the surface is at least partly made of polyolefins or polystyrene.

In another form the biocompatible device comprises on its surface an adsorbed layer of a polymer P which is a copolymer of at least one macromonomer selected from an ester E of (meth)acrylic acid and polyethylene oxide, and at least one monomer M selected from butyl alkyl (meth)acrylate (e.g. n-butyl methacrylate), where the adsorbed layer may be a self-assembled monolayer of the polymer P,

The invention also relates to a process for making the biocompatible device comprising the following steps:

A) providing a biocompatible device, and B) applying to the surface of the biocompatible device a solution S of the polymer in a solvent L.

In another form the invention relates to a process for making the biocompatible device which comprises on its surface an adsorbed layer of a polymer P which is a copolymer of

at least one macromonomer selected from an ester E of (meth)acrylic acid and polyethylene oxide or a polyethylene glycol (meth)acrylamide, and

at least one monomer M selected from alkyl (meth)acrylate, aryloxyalkyl (meth)acrylate, alkyl (meth)acrylamide or aryl (meth)acrylamide,

comprising the following steps:

A) providing a biocompatible device, and

solvent L.

at least one monomer M selected from butyl (meth)acrylate (e.g. n-butyl methacrylate), comprising the following steps:

A) providing a biocompatible device, and

solvent L.

Typically, the biocompatible device is obtainable by the process for making the biocompatible device.

The solution S normally comprises 0.001 to 10 % by weight of polymer P based on the solution S, preferably 0.05 to 5 % by weight and even more preferably 0.05 to 0.3 % by weight.

Suitable solvents L are water or alcohols like methanol, ethanol, n/iso-propanol or n/sec/iso/tert- butanol. Preferably, the solvent L comprises water and alcohol. Suitable alcohols are Ci to C₁₂ alcohols, preferably Ci to Cs alcohols, and in particular Ci to C6 alcohols. In another form the solvent L comprises an alcohol, such as methanol, ethanol, n/iso-propanol or n/sec/iso/tert- butanol, preferably ethanol or n/iso-propanol. In another form the solvent L is a mixture of water and an alcohol, preferably a mixture of water and ethanol or a mixture of water and isopropanol. Preferably, the solution S is an aqueous solution.“Aqueous” in this context shall mean that said polymer P is dissolved in a solvent or solvent mixture that comprises at least 50 % by weight, preferably at least 70 % by weight, more preferably at least 90 % by weight and particularly preferably at least 99 % by weight of water. In a preferred embodiment, the solvent L in which said at least one polymer P is dissolved is water. The aqueous solution S is usually a clear solution without any turbidity. In another embodiment, the aqueous solution S comprises polymer P at least partly in dissolved state but shows turbidity. Preferably, solution S is an aqueous solution comprising at least 50 % of water.

In another preferred embodiment, solution S comprises at least 50% by weight, preferably at least 70 % by weight, more preferably at least 90 % by weight and particularly preferably at least 99 % by weight of at least one alcohol like methanol, ethanol, n/iso-propanol or

n/sec/iso/tert-butanol.

In another preferred embodiment, the solvent L comprises at least 50% by weight, preferably at least 55 % by weight, and in particular at least 60 % by weight of at least one alcohol like methanol, ethanol, n/iso-propanol or n/sec/iso/tert-butanol.

In another preferred form, the solvent L comprises ethanol and water, and the ethanol content may be at least 50 wt% (e.g. at least 60 wt%), preferably from 50 to 99 wt%, or 50 to 90 wt%, or 55 to 80 wt%, or 55 to 70 wt%, or 58 to 66 wt%. In another preferred form, the solvent L

comprises ethanol and water, and the ethanol content may be at least 50 vol% (e.g. at least 60 vol%), preferably from 50 to 99 vol%, or 55 to 90 vol%, or 60 to 80 vol%, or 65 to 75 vol%.

In another preferred form, the solvent L comprises isopropanol and water, and the isopropanol content may be at least 50 wt% (e.g. at least 60 wt%), preferably from 50 to 99 wt%, or 50 to 90 wt%, or 55 to 85 wt%, or 60 to 85 wt%. In another preferred form, the solvent L comprises isopropanol and water, and the isopropanol content may be at least 50 vol% (e.g. at least 70 vol%), preferably from 50 to 99 vol%, or 55 to 95 vol%, or 60 to 90 vol%, or 75 to 85 vol%.

In another preferred form the solvent L comprises ethanol and water, and the ethanol content may be at least 50 wt%, or the solvent L comprises isopropanol and water, and the isopropanol content may be at least 50 wt%.

In another preferred form the solvent L comprises ethanol and water, and the ethanol content may be at least 50 vol%, or the solvent L comprises isopropanol and water, and the isopropanol content may be at least 50 vol%. In another form the solution S comprises 0.001 to 10 % by weight of polymer P based on the solution S (preferably 0.05 to 5 % by weight and even more preferably 0.05 to 0.3 % by weight), and a) the solvent L comprises ethanol and water, and the ethanol content may be at least 50 wt%, or b) the solvent L comprises isopropanol and water, and the isopropanol content may be at least 50 wt%.

In one form the process for making the biocompatible device comprises the following steps:

A) providing the biocompatible device, and B) applying to the surface of the biocompatible device a solution S of the polymer in a solvent L, where the solvent L comprises at least 50% by weight of at least one alcohol like methanol, ethanol, n/iso-propanol or n/sec/iso/tert-butanol.

A) providing the biocompatible device, and B) applying to the surface of the biocompatible device a solution S of the polymer in a solvent L, where the solvent L comprises ethanol and water, and the ethanol content may be at least 50 wt%, or the solvent L comprises isopropanol and water, and the isopropanol content may be at least 50 wt%.

In another form the process for making the biocompatible device comprises the following steps: A) providing the biocompatible device, and B) applying to the surface of the biocompatible device a solution S of the polymer in a solvent L, where the solution S comprises 0.001 to 10 % by weight of polymer P based on the solution S (preferably 0.05 to 5 % by weight and even more preferably 0.05 to 0.3 % by weight), and a) the solvent L comprises ethanol and water, and the ethanol content may be at least 50 wt%, or b) the solvent L comprises isopropanol and water, and the isopropanol content may be at least 50 wt%.

Typically, the biocompatible device in step A) corresponds to the biocompatible device before it was treated by step B).

Optionally, the process may further comprise the following step:

C1) removing the supernatant solution S; or

C2) removing the solvent L.

In one form the process may further comprise the following step:

C1) removing the supernatant solution S.

Upon the application of solution S to the surface, the polymer P will normally self-organize to form a layer, in many cases a self-assembled monolayer, of polymer P on the surface. In many cases it will be sufficient to apply solution S to surface O and wait for a short period of time, for example 1 minute to 1 day, preferably 5 minutes to 4 hours. Next, the supernatant solution S can be removed, for example mechanically (for example by wiping or using a pipette) or by exchanging solution S by water or an (aqueous) solution that does not comprise of polymer P.

The step C1) of removing the supernatant solution S may comprise exchanging solution S for a solution or for a pure solvent or solvent mixture that does not contain polymer P (for example a cell culture medium or water or a buffer solution).

The step C1) usually results in the formation of self-adsorbed monolayer of the polymer P on the surface.

In another form the process may further comprise the following step:

C2) removing the solvent L.

The removing of the solvent L may be achieved by drying or evaporation of the solvent L, e.g. at ambient or elevated temperature, or at ambient or reduced pressure. Preferably, the removing of the solvent L is achieved by drying at ambient temperature and ambient pressure.

Step C2) may be considered as a forced deposition wherein polymer P is applied from a solution and subsequently the solution is not withdrawn as a whole, but only the solvent L is removed, for example by evaporation, leaving the formerly dissolved polymer P deposited on the surface. A forced deposition can be applied by filling wells of a plate with the solution of polymer P followed by drying, alternatively, a forced deposition can be applied by dip coating, spin-coating, spraying, draw-down bar application, and other methods.

The thickness and the amount per area of the adsorbed layer usually depends on the process for making the biocompatible device, especially if step C1) or C2) were applied. In general, the adsorbed layer may have a thickness of 1 nm to 10 pm. In general, the polymer P is adsorbed on the surface in an amount of at least 50 ng/cm², preferably at least 100 ng/cm², which may be determined by quartz crystal microbalance.

When the biocompatible device was obtained by the process comprising step C1) of removing the supernatant solution S, then the adsorbed layer may have a thickness of 1 nm to 100 nm, preferably 1 to 20 nm, and in particular 1 to 10 nm. When the biocompatible device was obtained by the process comprising step C1) of removing the supernatant solution S, then the polymer P is adsorbed in an amount of 50 to 5000 ng/cm², preferably 100 to 3000 ng/cm², and in particular 200 to 1000 ng/cm² on the surface.

When the biocompatible device was obtained by the process comprising step C2) of removing the solvent L, then the adsorbed layer may have a thickness of 0.01 pm to 100 pm, preferably 0.1 to 20 pm, and in particular 0.5 to 10 pm.

When the biocompatible device was obtained by the process comprising step C2) of removing the solvent L, then the polymer P is adsorbed in an amount of 0.5 to 5000 pg/cm², preferably 5 to 500 pg/cm², and in particular 30 to 300 pg/cm² on the surface.

The process for making the biocompatible device may be achieved in situ when cultivating cells in the supernatant medium above the surface of the biocompatible device. The process for making the biocompatible device may comprise the following steps:

A) providing the biocompatible device (e.g. a device for cultivating cells),

solvent L, where the solution S is a cell culture medium, and

C) cultivating cells in the supernatant medium above the surface of the biocompatible device.

The cell culture medium may comprise 0.001 to 30 % by weight, preferably 0.01 to 5 % by weight and even more preferably 0.05 to 1 % by weight of the polymer P based on the medium. It is assumed that through this process adsorbed layer of the polymer P is prepared in situ that allows for efficient cultivation of such cell. Suitable solvent L are described above.

Any kind of cell culture medium may be used, wherein aqueous cell culture mediums with the solvent L comprising water are preferred. The cell culture medium may contain cells, e.g. those which are cultivated in step C). The supernatant medium of step C) usually corresponds to the solution S which is a cell culture medium. The supernatant medium of step C) usually comprises the polymer P and the solvent L (preferably water) applied in step B).

Between the step B) of applying the solution S and the step C) of cultivating the cells is preferably no removing of the solution S or the solvent L. Usually, the amount of the solution S in the biocompatible device in step C) is at least the same as in step B). Usually, the solution S is identical in step B) and in step C). Usually, the supernatant medium in step C) is identical to the solution S of step B). The present invention also relates to a process for cultivating cells, comprising the following steps: a) providing the biocompatible device which is a device for cultivating cells, and

b) cultivating cells in the supernatant medium above the surface of the biocompatible device.

Optionally, the biocompatible device is subjected to a sterilization prior to cultivating cells in step b), e.g. by exposing the biocompatible device to gaseous ethylene oxide, electron-beam, x- ray or gamma-irradiation. In case the biocompatible device is subjected to sterilization, the biocompatible device is obtainable by the process comprising the step C2) of removing the solvent L.

The cells may be adherent cells or non-adherent cells, where adherent cells are preferred. Suitable cells are any cells derived from multicellular organisms, preferably cells derived from plant, animal or human.

Preferred cells are

stem cells,

progenitor cells,

differentiated cells,

cancer cells, and

cancer cell derived cell lines.

More preferred cells are stem cells and cancer cells.

Examples for stem cells are induced pluripotent stem cells, adult stem cells or embryonic stem cells. Examples for adult stem cells are hematopoietic stem cells, mammalian stem cells, intestinal stem cells, mesenchymal stem cells, endothelial stem cells, neural stem cells, olfactory adult stem cells, neural crest stem cells, or testicular cells, where mesenchymal stem cells and hematopoietic stem cells are preferred.

Examples for differentiated cells are blood cells (e.g. human blood cells) and cells derived from human or animal organs (e.g. organs like mammalian gland, colon, liver, kidney, pancreas, prostate, lung, stomach or brain).

Examples for cancer cells are human cancer cells (e.g. breast cancer cells or prostate cancer cells). In a form the process for cultivating cells comprises the following steps: a) providing the biocompatible device which is a device for cultivating cells, and

b) cultivating cells in the supernatant medium above the surface of the biocompatible device, where the biocompatible device comprises on its surface an adsorbed layer of a polymer P which is a copolymer of at least one macromonomer selected from an ester E of (meth)acrylic acid and polyethylene oxide, and at least one monomer M selected from alkyl (meth)acrylate or aryloxyalkyl (meth)acrylate, and where the surface is at least partly made of polyolefins, polystyrene, fluorinated or partially fluorinated polyolefines.

In a form the process for cultivating cells comprises the following steps: c) providing the biocompatible device which is a device for cultivating cells, and

d) cultivating cells in the supernatant medium above the surface of the biocompatible device, where the biocompatible device comprises on its surface an adsorbed layer of a polymer P which is a copolymer of at least one macromonomer selected from an ester E of (meth)acrylic acid and polyethylene oxide, and at least one monomer M selected from alkyl (meth)acrylate or aryloxyalkyl (meth)acrylate, and where the surface is at least partly made of polyolefins, polycarbonate, polysulfones, polystyrene, fluorinated or partially fluorinated polyolefines.

In a form the process for cultivating cells comprises the following steps: a) providing the biocompatible device which is a device for cultivating cells, and

b) cultivating cells in the supernatant medium above the surface of the biocompatible device, where the biocompatible device comprises on its surface an adsorbed layer of a polymer P which is a copolymer of at least one macromonomer selected from an ester E of (meth)acrylic acid and polyethylene oxide, and at least one monomer M selected from butyl (meth)acrylate (e.g. n-butyl methacrylate), and where the surface is at least partly made of polyolefins, polystyrene, fluorinated or partially fluorinated polyolefines.

d) cultivating cells in the supernatant medium above the surface of the biocompatible device, where the biocompatible device comprises on its surface an adsorbed layer of a polymer P which is a copolymer of at least one macromonomer selected from an ester E of (meth)acrylic acid and polyethylene oxide, and at least one monomer M selected from butyl (meth)acrylate (e.g. n-butyl methacrylate), and where the surface is at least partly made of polyolefins, polycarbonate, polysulfones, polystyrene, fluorinated or partially fluorinated polyolefines.

In another form the process for cultivating cells comprises the following steps: a) providing the biocompatible device which is a device for cultivating cells, and

b) cultivating cells in the supernatant medium above the surface of the biocompatible device, where the biocompatible device comprises on its surface an adsorbed layer of a polymer P which is a copolymer of at least one macromonomer selected from an ester E of (meth)acrylic acid and polyethylene oxide, and at least one monomer M selected from C₁-C₁₈ alkyl

(meth)acrylate or phenoxyethyl (meth)acrylate, and where the surface is at least partly made of polyolefins or polystyrene.

b) cultivating cells in the supernatant medium above the surface of the biocompatible device, where the biocompatible device comprises on its surface an adsorbed layer of a polymer P which is a copolymer of at least one macromonomer selected from an ester E of (meth)acrylic acid and polyethylene oxide, and at least one monomer M selected from butyl (meth)acrylate (e.g. n-butyl methacrylate), and where the surface is at least partly made of polyolefins or polystyrene.

b) cultivating cells in the supernatant medium above the surface of the biocompatible device, where the biocompatible device comprises on its surface an adsorbed layer of a polymer P which is a copolymer of at least one macromonomer selected from an ester E of (meth)acrylic acid and polyethylene oxide, and at least one monomer M selected from C1-C18 alkyl

(meth)acrylate or phenoxyethyl (meth)acrylate, where the polymer P comprises the monomer M and the macromonomer in a molar ratio from 0.3:1 to 10:1 , and where the surface is at least partly made of polystyrene.

b) cultivating cells in the supernatant medium above the surface of the biocompatible device, where the biocompatible device comprises on its surface an adsorbed layer of a polymer P which is a copolymer of at least one macromonomer selected from an ester E of (meth)acrylic acid and polyethylene oxide, and at least one monomer M selected from butyl (meth)acrylate (e.g. n-butyl methacrylate), where the polymer P comprises the monomer M and the macromonomer in a molar ratio from 0.3:1 to 10:1 , and where the surface is at least partly made of polystyrene.

b) cultivating cells in the supernatant medium above the surface of the biocompatible device, where the biocompatible device comprises on its surface an adsorbed layer of a polymer P which is a copolymer of at least one macromonomer selected from an ester E of (meth)acrylic acid and polyethylene oxide, and at least one monomer M selected from alkyl (meth)acrylate or aryloxyalkyl (meth)acrylate, where the adsorbed layer may be a self-assembled monolayer of the polymer P, and

where the surface is at least partly made of polyolefins, polystyrene, fluorinated or partially fluorinated polyolefines.

b) cultivating cells in the supernatant medium above the surface of the biocompatible device, where the biocompatible device comprises on its surface an adsorbed layer of a polymer P which is a copolymer of at least one macromonomer selected from an ester E of (meth)acrylic acid and polyethylene oxide, and at least one monomer M selected from butyl (meth)acrylate (e.g. n-butyl methacrylate), where the adsorbed layer may be a self-assembled monolayer of the polymer P, and

where the surface is at least partly made of polyolefins, polycarbonate, polysulfones, polystyrene, fluorinated or partially fluorinated polyolefines. In another form the process for cultivating cells comprises the following steps: a) providing the biocompatible device which is a device for cultivating cells, and

(meth)acrylate or phenoxyethyl (meth)acrylate, where the adsorbed layer may be a self-assembled monolayer of the polymer P, and

where the surface is at least partly made of polyolefins or polystyrene.

The invention has various advantages: The biocompatible devices are suitable for cultivating cell cultures, especially for the cultivation of normally adherent cells as non-adherent cell cultures, where they show improved anti-adhesive behavior. Avoiding cell adhesion in this device improves three-dimensional growth of cells, which resembles more closely the natural growth of cells by increasing cell to cell interaction and resembling natural drug, oxygen and nutrient gradients. They are easy and economical to make, they allow for the growing of cell cultures with a high circularity (meaning a round cell morphology; the circularity is a parameter defining how spherical an object is; it is calculated by 4*PI* cell area/square of cell perimeter), they allow for the growing of cells with a very low degree of adhesion to the surface, they allow for the growing of adherent cells with a round spheroid morphology, they allow for the growing of cells that are essentially free floating in the medium and can be easily harvested, or they allow for the cultivation of embryonic stem cells or of pluripotent cells while avoiding a premature specialization of such cells. Culturing in biocompatible devices allows the generation of three-dimensional cell aggregates of consistent size and shape. The usage of a single polymer layer, in contrast to thick surface coatings, allows for a surface modification which is uniform and does not influence the geometry of the biocompatible device.

Examples

Molecular weights were determined by Size Exclusion Chromatography using a mixed bed scouting column for water soluble linear polymers at 35°C. The eluent used was 0.01 M phosphate buffer at pH=7.4 containing 0.01 M sodium azide. The polymer used as 1.5 mg/ml_ concentrated solution in the eluent. Before injection all samples were filtered through a 0.2 pm filter. The calibration was carried out with narrow poly(ethylene glycol) samples having molecular weights between 106 and 1.378.000 g/mol.

Example 1 : Preparation of MMA : PEGMA550 3:1

382 parts by weight of isopropanol, 10.5 parts by weight of PEGMA550 (methoxy polyethylene glycol methacrylic ester, Mn of 550 g/mol) and 5 parts by weight of MMA (methyl methacrylate) were mixed under nitrogen and heated to 75°C. 0.21 parts by weight of tert-butyl-2,2-dimethyl- propaneperoxoate dissolved in 10 parts by weight of isopropanol were added. After 5 min 1.89 parts by weight of tert-butyl-2,2-dimethylpropaneperoxoate dissolved in 90 parts by weight of isopropanol were added within 3.5 hours and 94.5 parts by weight of PEGMA550 and 45 parts by weight of MMA dissolved in 108 parts by weight of isopropanol were added within 3 hours. Afterwards, the reaction was kept for 2 hours at 75°C. Afterwards, 1.05 parts by weight of tert- butyl-2,2-dimethylpropaneperoxoate dissolved in 10 parts by weight of isopropanol were added and the reaction mixture kept at 75°C for 30 minutes. Then, again, 1.05 parts by weight of tert- butyl-2,2-dimethylpropaneperoxoate dissolved in 10 parts by weight of isopropanol were added and the total reaction mixture was kept at 75°C for 2.5 hours. Finally, the reaction mixture was subjected to purification by water steam distillation.

Number average molecular weight Mn = 3910 g/mol, weight average molcular weight Mw = 7420 g/mol.

Example 2: Preparation of MMA : PEGMA2000 3:1

330 parts by weight of isopropanol, 34.7 parts by weight of PEGMA2000 (methoxy polyethylene glycol methacrylic ester, Mn of 2000 g/mol) 50wt% solution in water and 2.51 parts by weight of MMA were mixed under nitrogen and heated to 75°C. 0.27 parts by weight of tert-butyl-2,2-di- methylpropaneperoxoate dissolved in 4 parts by weight of isopropanol were added. After 5 min, 2.43 parts by weight of tert-butyl-2,2-dimethylpropaneperoxoate dissolved in 36 parts by weight of isopropanol were added within 3.5 hours and 312 parts by weight of PEGMA2000 and 22.6 parts by weight of MMA dissolved in 270 parts by weight of isopropanol were added within 3 hours. Afterwards, the reaction was kept for 2 hours at 75°C. Finally, the reaction mixture was subjected to purification by water steam distillation.

Number average molecular weight Mn = 14680 g/mol; weight average molecular weight Mw = 22550 g/mol.

Example 3: Preparation of POEA : PEGMA2000 3:1

352 parts by weight of isopropanol, 25 parts by weight of PEGMA2000 50wt% solution in water and 3.47 parts by weight of phenoxyethyl acrylate POEA were mixed under nitrogen and heated to 75°C. 0.22 parts by weight of tert-butyl-2,2-dimethylpropaneperoxoate dissolved in 5 parts by weight of isopropanol were added. After 5 min, 1.98 parts by weight of tert-butyl-2,2-dimethyl- propaneperoxoate dissolved in 45 parts by weight of isopropanol were added within 3.5 hours and 225 parts by weight of PEGMA2000 and 31.2 parts by weight of phenoxyethyl acrylate dissolved in 108 parts by weight of isopropanol were added within 3 hours. Afterwards, the reaction was kept for 2 hours at 75°C. Afterwards, 1.1 parts by weight of tert-butyl-2,2-di- methylpropaneperoxoate dissolved in 5 parts by weight of isopropanol were added and the reaction mixture kept at 75°C for 30 minutes. Then, again, 1.1 parts by weight of tert-butyl-2,2- dimethylpropaneperoxoate dissolved in 5 parts by weight of isopropanol were added and the total reaction mixture was kept at 75°C for 10.5 hours. Finally, the reaction mixture was subjected to purification by water steam distillation.

Number average molecular weight Mn = 16860 g/mol; weight average molecular weight Mw = 26700 g/mol.

Example 4: Preparation of n-BMA : PEGMA2000 3 : 1

225 parts by weight of isopropanol, 18.9 parts by weight of PEGMA2000 50wt% solution in water and 1.94 parts by weight of n-BMA (n-butyl methacrylate) were mixed under nitrogen and heated to 80°C. 0.15 parts by weight of tert-butyl-2,2-dimethylpropaneperoxoate dissolved in 5 parts by weight of isopropanol were added. After 5 min, 1.35 parts by weight of tert-butyl-2,2-di- methylpropaneperoxoate dissolved in 45 parts by weight of isopropanol were added within 4 hours and 170.1 parts by weight of PEGMA2000 dissolved in 45 parts by weight of isopropanol were added within 3 hours and 17.46 parts by weight of n-BMA dissolved in 45 parts by weight of isopropanol were added within 2 hours. Afterwards, the reaction was kept for 2 hours at 80°C. Afterwards, 0.75 parts by weight of tert-butyl-2,2-dimethylpropaneperoxoate dissolved in 5 parts by weight of isopropanol were added and the reaction mixture kept at 80°C for 30 minutes. Then, again, 0.75 parts by weight of tert-butyl-2,2-dimethylpropaneperoxoate dissolved in 5 parts by weight of isopropanol were added and the total reaction mixture was kept at 80°C for 2.5 hours. Finally, the reaction mixture was subjected to purification by water steam distillation.

Number average molecular weight Mn = 22980 g/mol; weight average molecular weight Mw = 43210 g/mol.

Example 5: Coating of model surfaces and anti-adhesive evaluation

Method:

The amounts of polymer P or milk absorbed on the surface was determined by Quartz-Crystal Microbalance (QCM), which measures the resonance frequency of a freely oscillating quartz crystal after excitation. QCM measurements were performed using standard flow-through methods with a flow rate of 50 pL/min at 23°C. A shift in resonance frequency scales inversely proportionally with mass changes at the quartz surface. The amounts were calculated from the shift of the 7^th overtone of the resonance frequency according to the method of Sauerbrey with a mass sensitivity of ~10 ng/cm² within the measurement series.

Surface Material:

Surfaces made of organic polymers were generated on the QCM sensor quartz surface by coating it with a thickness of 10 to 500 nm (determined through the weight difference of the QCM sensor) of the organic polymer. The thickness of the organic polymer coating does not affect the results of the QCM measurement as long as it is within the specified range. For generating a surface made of polystyrene (PS) on the QCM sensor, the dip-coating method was used. For dip-coating, the sensor was briefly immersed into a 1 wt% solution of the respective organic polymer in /V-Methyl-pyrrolidone and subsequently dried at 200°C using a heat gun. A heat gun was applied to facilitate the removal of the high boiling solvent NMP.

Adsorption of polymer P and of milk:

The experiments comprised the following steps and used an aqueous HEPES (4-(2- Hydroxyethyl)-1 -piperazine ethanesulfonic acid) buffer (10 mmol/L) with pH 7 (“buffer”):

1) buffer until a stable baseline was achieved;

2) 2 h 0.1 wt% polymer P solution in buffer;

3) 2 h buffer;

4) 0.5 h 0.1 wt% milk powder in buffer;

5) 0.5 h buffer.

Adsorption of the polymer P layer on polystyrene surfaces was carried out by equilibrating the coated quartz sensor surface with 0.1 wt% polymer P solution until a monolayer was formed (step 2) above). Afterwards, the sensor surface was rinsed with buffer until a stable mass reading was obtained (step 3) above).

Milk fouling was monitored during exposure of the samples to 0.1 wt% solutions of milk powder for 0.5 h. The final mass change was recorded after another 0.5 h of rinsing with buffer (steps 4) and 5) above). The results (based on double determination) are given in Table 1.

Table 1 :

Example 6: Evaluation of reduced cell adherence in cell culture

The adherence and spreading behavior was evaluated using a fibroblast cell line, namely 3T3 cells clone A31 (ECACC; Lot No. 03L010, passage 82). Cells were maintained in Dulbecco’s Minimal Essential Medium supplemented with Newborn Bovine Calf Serum (10 v/v%), 4 mM Glutamine and 1 % Penicillin/Streptomycin in T75 flasks with 20 mL medium at 37°C in a humidified atmosphere containing 5% CO2 to a confluence of 80-90 %. For the analysis of adherence on different polystyrene surfaces, semi-confluent 3T3 cells from passage 6-15 were trypsinized using Trypsin/EDTA (0,05%/0,02%) in PBS, incubated at 37°C and 5% CO2 and seeded at approximately 30.000 cells/mL using 1 ml per well of polystyrene-based 24 multiwell plates.

The adsorbed layer of polymer P on the polystyrene plates was made as follows: Solid polymer P powder was dissolved in water at a final concentration of 0.1 % and passed through a filter for sterilization. Sterile solution was pipetted into wells (2 ml per well) and incubated at room temperature for 3 h. After incubation the polymer solution was removed using a standard pipette. Afterwards wells were washed twice, by adding and removing 2 ml of sterile water into each well of the 24 well plate, followed by cell seeding as described previously. The adherence was characterized using an automated light microscopy (IncuCyte System, Sartorius) with a 10x objective, phase contrast and photo documentation taken 16 pictures per well every 8 hours. Quantitative analysis of cell adherence was performed by means of automatic microscopy and image analysis using the corresponding software of the automated microscope (IncuCyte S3 2018B, confluence mask). The percentage of adherent cells was determined after 4 days of culture via surface analysis within the well. Cells that do not adhere to the surface of the plate, accumulate into cell aggregates. In contrast, adherent cells spread on the surface of the cell culture plate and can be quantified via a confluence analysis, which determines the surface covered by cells. The results were summarized in Table 2.

Table 2:

Example 7: Evaluation of reduced cell adherence by spheroid formation

To further analyze the adherence and cell repellent properties the formation of spheroid structures was studied. Typically, cell spheroids will only form if cells are grown on a fully cell and protein repellent surface. Again, a fibroblast cell line was used, namely 3T3 cells clone A31 (ECACC; Lot No. 03L010). Cells were maintained in Dulbecco’s Minimal Essential Medium supplemented with Newborn Bovine Calf Serum (10 v/v%), 4 mM Glutamine and 1%

Penicillin/Streptomycin in T75 flasks with 20 mL medium at 37°C in a humidified atmosphere containing 5% CO2 to a confluence of 80-90 %. For the analysis of adherence on different polystyrene surfaces, semi-confluent 3T3 cells from passage were trypsinized using

Trypsin/EDTA (0,05%/0,02%) in PBS, incubated at 37°C and 5% CO2 and seeded at approximately 30.000 cells/mL using 150 pi ml per well of polystyrene 96 multiwell plates.

The adsorbed layer of polymer P on a 96 well polystyrene plates with a U-bottom shape (Nunc 262162) was made as follows: Solid polymer nBMA-PEGMA2000 (Example 4) powder was dissolved in either (A) water or (B) in water/ethanol mixture (70 % ethanol (v/v)) at a final concentration of 1 % (w/v) and passed through a filter for sterilization. Sterile solution was pipetted into wells (150 mI per well) and incubated at room temperature for 1 h. After incubation the polymer solution was removed using a standard pipette. Afterwards wells were washed twice, by adding and removing 150 pi ml of sterile water into each well of the 96 well plate, followed by cell seeding as described previously.

The formation of cell spheroid was documented by light microscopy using an automated light microscopy (IncuCyte System, Sartorius) with a 10x objective, phase contrast and photo documentation (cf Fig. 1).

The percentage of singular spheroids were determined after 3 days of culture by counting.

Wells which have remaining non-repellent spots form either no or multiple spheroids, while wells with a fully non-adherent surface form singular spheroids. The results are summarized in Table 3.

Table 3:

Fig. 1 shows light microscopy images, where the plates were either coated from water (A) or from 70% ethanol (v/v) (B) or not coated (C). In 25% of the wells coated with nBMA- PEGMA2000 from water one singular spheroid formed. In 92 % of the wells coated with nBMA- PEGMA2000 in water/ethanol one singular spheroid formed. No cell aggregations formed in uncoated wells.

Example 8: Reduced protein binding on polymer coated plates

In addition to cell-based assay, the protein and cell repellent properties of nBMA-PEGMA2000 were studied using a biochemical assay to quantify protein attachment. In this case, the binding of an antibody-enzyme conjugate as model protein was used. Solid nBMA-PEGMA2000 (Example 4) was dissolved in either (A) water or (B) water/ethanol (70% ethanol (v/v)) at a final concentration of 1 % (w/v) and passed through a filter for sterilization. Sterile solution was pipetted into wells (150 mI per well of flat-bottom 96-well polystyrene plates) and incubated at room temperature for 10 sec., 60 sec, 300 sec, 600 sec or 3600 sec. After incubation the polymer solution was removed using a standard pipette and washed three times using 300 mI PBS buffer. After washing, 150 mI of antibody / alkaline phosphatase conjugate solution was dispensed into each well (Goat Anti Bovine IgG (101-035-003 Lot 13733, Jackson Immuno Research dilution 1 :2000 in PBS) and incubated for 1 h. After removing the antibody / alkaline phosphatase conjugate solution, plates were again washed three times with PBS buffer. To quantify the antibody / alkaline phosphatase conjugate, which was bound by the plate, the wells were incubated with 100 pi of substrate for the alkaline phosphatase (Tetramethylbenzidine mM in NaAcO pH 4,9 and 0.00022% H2O2) . After 5 min, the enzyme reaction was stopped using 100 mI of 2M H2SO4 per well. The amount of colored enzyme product, which correlates to the amount of enzyme bound to the surface, was quantified in a plate reader measuring light absorbance at 620 nm.

Fig. 2 shows the amount of surface coating depending on the incubation time t (x-axis) and for either polymer P dissolved in either (A) water or in (B) water/ethanol. The y-axis shows the OD A450 nm.

If polymer P was dissolved in water and incubated for 10 seconds on the polystyrene surface before washing, the surface could bind protein, shown by a strong alkaline phosphatase activity remaining on the plate. Longer incubation time of the polymer on the polystyrene surface for up to 1 h decreased alkaline phosphatase activity significantly, demonstrating reduced protein binding.

In contrast, if polymer P was dissolved in 70 % ethanol (v/v) to a concentration of 1 % (w/v), protein binding was fully prevented, even if the polymer solution was incubated with the plate for only 10 seconds. Hence, polymer P can adsorb to the surface of the plate and prevent protein binding when applied from aqueous solution. When applied from ethanolic solution the polymer adsorption process is even more efficient and leads to even more significant protein reduction.

Claims

1. A biocompatible device which comprises on its surface an adsorbed layer of a polymer P which is a copolymer of at least one macromonomer selected from an ester E of (meth)acrylic acid and

polyethylene oxide or a polyethylene glycol (meth)acrylamide, and at least one monomer M selected from alkyl (meth)acrylate, aryloxyalkyl (meth)acrylate, alkyl (meth)acrylamide or aryl (meth)acrylamide.

2. The biocompatible device according to claim 1 , where the biocompatible device is a

biosensor or a device for cultivating cells.

3. The biocompatible device according to claim 2, where the device for cultivating cells is selected from cell culture flasks, cell culture dishes, cell culture plates, cell culture bags, reactors, tubes, pipettes, pipette tips, and syringes.

4. The biocompatible device according to any of claims 1 to 3 where the macromonomer is the ester E of (meth)acrylic acid and polyethylene oxide.

5. The biocompatible device according to any of claims 1 to 4 where the monomer M is butyl (meth)acrylate.

6. The biocompatible device according to any of claims 1 to 5 where the polymer P comprises the monomer M and the macromonomer in a molar ratio from 0.3:1 to 10:1.

7. The biocompatible device according to any of claims 1 to 6, where the polymer P is

adsorbed on the surface in an amount of at least 50 ng/cm².

8. The biocompatible device according to any of claims 1 to 7, where the polymer P consists of the monomer M and macromonomer.

9. The biocompatible device according to any of claims 1 to 8, where the surface is free of other layers beside the adsorbed layer of the polymer P.

10. The biocompatible device according to any of claims 1 to 9, where the surface is at least partly made of polyolefins, polycarbonate, polystyrene, polysulfones, fluorinated or partially fluorinated polyolefines.

11. The biocompatible device according to any of claims 1 to 10, where there are no covalent chemical bonds between the polymer P and the surface.

12. A process for making a biocompatible device which comprises on its surface an adsorbed layer of a polymer P which is a copolymer of at least one macromonomer selected from an ester E of (meth)acrylic acid and

polyethylene oxide or an polyethylene glycol (meth)acrylamide, and at least one monomer M selected from alkyl (meth)acrylate, aryloxyalkyl (meth)acrylate, alkyl (meth)acrylamide or aryl (meth)acrylamide, comprising the following steps:

A) providing a biocompatible device, and

B) applying to the surface of the biocompatible device a solution S of the polymer P in a solvent L.

13. The process according to claim 13, further comprising the following step:

C1) removing the supernatant solution S; or

C2) removing the solvent L.

14. The process according to claims 13 or 14, where the solvent L comprises an alcohol, such as methanol, ethanol, n/iso-propanol or n/sec/iso/tert-butanol.

15. The process according to any of claims 13 to 15, where the solvent L comprises at least 50% by weight of methanol, ethanol, n/iso-propanol or n/sec/iso/tert-butanol.

16. A process for cultivating cells, comprising the following steps:

a) providing the biocompatible device which is a device for cultivating cells as defined in any of claims 1 to 12 or obtainable by the process as defined in any of claims 13 to 15, and