EP2032688A1

EP2032688A1 - Cell culture apparatus, method for producing the apparatus and cell culture method

Info

Publication number: EP2032688A1
Application number: EP07764371A
Authority: EP
Inventors: Bernd Hoffmann; Bodo Borm; Rudolf Merkel
Original assignee: Forschungszentrum Juelich GmbH
Current assignee: Forschungszentrum Juelich GmbH
Priority date: 2006-06-24
Filing date: 2007-06-14
Publication date: 2009-03-11
Also published as: DE102006029051A1; WO2007147389A1; JP2009540805A; US20090186411A1

Abstract

The invention relates to a cell culture apparatus for cells, characterized by a surface composed of an unstructured elastomer. It is thus possible for cells to be cultured under close to natural conditions in relation to their environmental elasticity. A method for producing an apparatus according to the invention is disclosed, as is a cell culture method using such an apparatus.

Description

Cell culture device, method of making the device, and cell culture methods

The invention relates to a cell culture device, a method for producing the device and a cell culture method with such a device.

Known from the prior art are cell culture vessels and devices made of plastics, such as polypropylene, polystyrene or glass. These vessels or devices are available in a variety of designs and have been routinely used for many years, for example in the form of cell culture bottles, test tubes, multiwell plates, petri dishes and so on.

Polypropylene and polystyrene are thermoplastics. The substances are physiologically harmless and also approved for food packaging without restrictions.

The types of glass used for the cell culture are, for example, soda-lime glass or borosilicate glass. The glasses are popular for laboratory use due to their optical properties and excellent resistance to chemicals and high temperatures. These surfaces allow the culture of cells, without which further chemical modifications of the surface would be mandatory.

Unfortunately, however, the glasses and plastics mentioned do not permit long-term examination and culture of the cells under in vivo-like conditions.

Despite this significant disadvantage, they are routinely used in many scientifically and economically relevant issues, such as drug screening or cancer research.

From Discher et al. (Discher D.E., Janmey P., Yu-Li Wang (2005), Tissue cells feel and respond to the stiffness of their substrates, Science, 310, 1139-1143), it is known that tissue cells can respond to the stiffness of their substrates.

Disadvantageous are also the substrates known therefrom for long-term cell cultures and unsuitable for storage or storage. The object of the invention is to provide a cell culture device which enables cell culture under in vivo similar conditions.

The object is achieved by a device according to the main claim and a method for their preparation according to the independent claim. Advantageous embodiments will be apparent from the claims referring back to each of them.

The cell culture device according to the invention is characterized by an unstructured surface of an elastomer. The cell culture device thus has a flat surface made of an elastomer.

Within the scope of the invention, it has been recognized that the elastic property of a cell culture surface, in physical terms in the form of Young's modulus, is of crucial importance in the culture of cells under in vivo-like conditions. The Young modulus is also known as modulus of elasticity or modulus of elasticity.

Elastomers have a reproducibly settable Young modulus, depending on the nature of the starting materials and on the production process.

Elastomers are advantageously much more suitable for culture of cells than the hard surface cell culture vessels used in the prior art. It depends less on the chemical composition of the elastomer, but rather on the physical property, expressed in the form of Young's modulus.

It was recognized that the elastic surface in terms of softness and elasticity can be described as near-natural. The elastomer advantageously allows quasi a culture of the cells under in situ or in vivo similar conditions.

In the context of the invention, it was further recognized that the materials commonly used according to the prior art, such as polypropylene or polystyrene, have a much too high hardness with an E-modulus of about 10 ⁹ Pa. Such materials are not to be described as near-natural in terms of elasticity. The same applies to soda lime glass (E modulus 7.3 * 10 ¹⁰ Pa, Poisson number: 0.22), which is also frequently used, and to borosilicate glass (E modulus 6.3 * 10 ¹⁰ Pa, Poisson number: 0.2). In the context of the invention, it was also recognized that the culture conditions and surfaces of cell culture devices used hitherto in terms of their stiffness and hardness are in contrast to the natural environment of cells. In particular, the cells of multicellular cells are in contact with much softer and more elastic surfaces in contact, as is specified with the glass or plastic types used in the laboratory.

The surface of the device according to the invention has a layer or a film of unstructured elastomer for receiving the cells. With this surface, the cells are brought into contact and adhere for culture either directly or through a culture-promoting layer.

Such an elastomer layer or film accordingly has a flat unstructured surface. The layer or the film has no (micro) structures whatsoever.

In the context of the invention it was recognized that structures such. As trenches or island-like surveys, be recognized by many cell types and the cells so adversely to an uninfluenced development and proliferation and / or spread by cell division prevent. By a flat surface of an elastomer, therefore, the natural development and proliferation of the cells is advantageously supported.

Advantageously, the area of the unstructured elastomer is approximately at least one cm ² . This is about the same as the surface of a cover glass.

The concept of the invention in its broadest embodiment encompasses a cell culture device with an unstructured elastomer as the surface for the cells, wherein each elastomer can be used as the surface of the cell culture device which is chemically stable and biocompatible for the cells used. The elastomer used should advantageously also be reproducible in terms of elasticity.

The unstructured elastomer covers the part of the surface of the cell culture device which is intended to receive the cells in the form of a layer which z. B. is designed as a thin film. The elastomer thus forms the background of the cells. The elastomer is transparent in one embodiment of the invention.

As a result, the cells optionally adhering to the elastomer and the progress of the cell culture can be well observed.

Since the material of the cell culture vessel as such, that is without the elastomer, is generally also transparent, it is advantageously possible to closely observe the cells through the outer wall of the device and through the elastomer surface on the inner wall of the device.

In a further advantageous embodiment of the invention, the surface of the device made of the elastomer has a refractive index in a range from about 1.3 to 1.5 and in particular a refractive index of 1.4.

The devices according to the invention together with elastomer are particularly suitable for receiving adherent cells, which include over 95% of all known animal cell types.

The Young modulus of the elastomer in the device according to the invention can be between 1

MPa below 1 kPa, depending on the problem to be set.

An elasticity of the elastomer down to 500 Pa is reproducibly adjustable.

The device according to the invention advantageously has an elastomer with a Poisson ratio or a Poisson's ratio in a range from about 0.3 to 0.5. A Poisson ratio of 0.5 characterizes the elastomer as incompressible or volume constant. It is mathematically and physically accurately detectable.

In a particularly advantageous embodiment of the invention, the surface of the device comprises an elastomer which is not based on water.

The elastomer on the surface of the device according to the invention then advantageously remains permanently and has a correspondingly high shell life. It therefore does not shrink due to evaporation of the water, such. As the known and in this context disadvantageous agar or acrylic surfaces. It is particularly advantageous if the shell life of the elastomer is at least one year. By shell life is meant the time window in which the device can be stored after its manufacture without altering the elastomer until it is used.

The elastomer in the device according to the invention may advantageously be sterilized. By sterilization by z. B. gamma or UV irradiation, the elastomer is not affected.

The layer thickness of the elastomer in the cell culture device may be between about one hundred nanometers to several centimeters.

The layer thickness of the elastomer as well as the thickness of the transparent device should advantageously be adapted to each other and selected so that the cells can be examined by means of a microscope.

Then the maximum layer thickness of device and elastomer to be penetrated optimally for most objectives of light microscopes should not exceed about 250 μm.

As elastomers, a variety of different chemical substances from different groups are available and can be used according to the invention as the surface of the cell culture device. By way of example only and not by way of limitation, elastomers from the groups of siloxane resins, butadiene resins and acrylate resins are listed. These resins have slightly hydrophobic properties and are basically suitable for the cell culture method according to the invention.

The resins regularly have reactive groups, which form a bond with each other and thereby can lead to crosslinking of the elastomer.

In the following, the term ground substance is used synonymously with the main constituent in the polymerized elastomer.

In the case of siloxane resins and butadiene resins, the reactive groups are present in more or less long unreactive chain segments. The reactive groups of the siloxane resins, like the reactive groups of the butadiene resins, can bond with each other without that a copolymer would have to be used. Copolymers additionally crosslink the reactive groups of the siloxane and butadiene resins.

Acrylate resins have only reactive groups, which are crosslinked alone or with the addition of copolymers.

The basic substance of the elastomer is thus composed of monomeric, oligomeric or polymeric constituents which are polymerized by crosslinking to superordinate structures.

Crosslinking of the elastomer constituents of the matrix and cross-linking agent (copolymer) involves various techniques. The term cross-linker and copolymer is used synonymously below.

The crosslinking is for example by suitable copolymers, by UV light, ozone or z. B. controlled by platinum catalysts at elevated temperatures. High temperatures usually increase the reaction rate during crosslinking.

Catalyst driven polymerizations are fast and efficient and are e.g. For example, for SiI xanharze with a platinum catalyst, eg. B. by means of platinum Divinyltetramethyldisoloxan, (SP 6830.0 the company. ABCR "a better choice for chemical reagents") performed.

Resins are available and can be used according to the invention, the basic constituents of which crosslink by themselves in air at room temperature without addition of copolymer.

The longer the selected chain length of the base substance (unreactive chain segment) of the elastomer according to the invention, the higher the viscosity, that is, the lower the corresponding elasticity of the elastomer after addition of a suitable cross-linker.

Conversely, with decreasing chain length of the elastomer, the elasticity increases after crosslinking of the elastomer. In terms of elasticity, each elastomer can be accurately and reproducibly adjusted by suitably selecting factors such as the ground substance chain length, the mixture ratio between the base substance and cross-linking agent (copolymer), or the addition of thinners.

Before polymerization, the macromolecular structure of the matrix of polymerizable end group silicone resins, as compared to a monomeric resin, causes a reduction in the rate of diffusion of the reactive species, e.g. As the vinyl groups and the concentration thereof. These disadvantages can be eliminated by lowering the viscosity, for example via reactive thinners, and by adding crosslinkers.

Reactive thinners have a dual function. In addition to lowering the viscosity of the starting product, they also increase the number of reactive groups per unit volume of resin.

Elastomers from the group of butadiene resins are suitable for a crosslinking reaction analogous to the vulcanization of rubber with sulfur. They are resins in which the main chains of the oligomeric constituents are crosslinked as the basic substance via reactive sites distributed throughout the chain by monomeric units of a crosslinker. These oligomer chains can be linked together directly without the addition of a cross-linking agent. However, the high viscosity of these oligomers can be reduced by reactive diluents and crosslinkers.

Elastomers, which are formed from the group of acrylate resins, are preferably based on monomers as the basic substance of the resin component. The resin consists of freely moving reactive groups. Thereby, a low viscosity with good diffusion and high concentration of reactive groups can be achieved.

For elastomers of siloxane resins, too, the mixing ratio of the base substance to a crosslinker of very high concentrations of cross-linking agent for the production of low elasticities or high Young Moduli (> 1 MPa) up to mixing ratios with low concentration of cross-linking agent for producing high elasticities. Elastomers with particularly good cell culture properties are mixtures of a vinyl-substituted siloxane as the basic substance and a memylhydrosiloxane-dimethylsiloxane copolymer as cross-linking agent. After crosslinking, the PDMS can be used as a surface for the cell culture.

Other siloxanes, such as hydroxysiloxane with chlorosilane are crosslinked directly with each other or else with the addition of a copolymer and are also used in the context of the invention as a support for cell culture.

Particularly advantageous PDMS elastomers according to the invention are formed from a matrix with oligomers having a molecular weight of 100 to 200,000.

A particularly suitable PDMS elastomer is made from the sylgard 184 kit (Dow Corning). This kit comprises as a basic substance vinyl-terminated siloxane resin and methylhydrosiloxane-dimethylsiloxane copolymer cross-linking agent in separate units, which are mixed together and polymerized according to the desired elasticity in suitable mixing ratios.

DMS-V31 as the basic substance of a vinyl-terminated polydimethylsiloxane (ABCR) and HMS 301 as an example of a methylhydrosiloxane-dimethylsiloxane copolymer for a cross-linking agent (ABCR) are also outstandingly suitable for forming elastomers for cell culture.

The PDMS basic constituents, ie the vinyl-terminated siloxane DMS-V31 and the methylhydrosiloxane-dimethylsiloxane copolymer as cross-linking agent (HMS-301) are both in a liquid state at room temperature. The polymerization is preferably carried out in the presence of a platinum catalyst at preferably ~ 60 ° C.

As a thinner for PDMS, DMS-T21 (ABCR) is exemplified as a polydimethylsiloxane. This is not reactive because there are no methylhydrosiloxane groups. They have a viscosity of 100 cSt. It can be mixed with up to 30% by volume in not cross-linked PDMS. As diluents for PDMS, by way of example also DMS-T22 (ABCR) is mentioned as a polydimethylsiloxane; this is not reactive because there are no methylhydrosiloxane groups. They have a viscosity of 200 cSt. They can be mixed into not yet cross-linked PDMS with up to 30 percent by volume.

The PDMS elastomers and the elastomers based on butadiene and acrylate resin can be reproducibly applied uniformly thick to the surface of the cell culture device in a simple and inexpensive manner.

A person skilled in the art will use a suitable reference for the reproducible production of elastomers for this purpose.

In this regard, reference is made to the following references and home pages, the content of which is hereby incorporated by reference in this application, in particular with regard to the elastomers, crosslinkers and thinners specified there:

■ W. Noil, Chemistry and Technology of Silicones, Academic Press, New York (1968).

T. C. Kendrick, B. Parbhoo, J.W. White, "Siloxane Polymers and Copolymers," in The Chemistry of Organic Silicon Compounds Pt2 (edited by S. Patai and Z. Rappoport), 21, p. 1289-1361, John Wiley, Chichester (1989).

■ SJ. Clarson, J.A. Semlyen, Siloxane Polymers, Prentice Hall, New Jersey (1993).

^■ JW White, RC Treadgold, "Organofunctional siloxane," in siloxanes polymer (edited by SJ. Clarson and YES Semlyen), 4, pl93-215, Prentice Hall, New Jersey (1993).

^■ W. Gardiner, JW White, "Specialty Silicones as Building Blocks for Organic Polymer Modification," in High Value polymer (edited by AH Fawcett), Royal Society of Chemistry, Cambridge (1990).

^■ M. Brook, Silicon in Organic, Organometallic and Polymer Chemistry, John Wiley and Sons, New York (2000).

^■ Dow Corning, Homepage, Silanes selection guide

^■ ABCR, Homepage, Siloxane product list ■ M. Heger, Development of a Stereolithography Resin for Elastomeric Products, Ph.D. thesis, Darmstadt, Germany (2001).

For siloxane resins of a base substance and a cross-linking agent, depending on the position of the reactive groups, ie terminal position or within the chain, all known siloxane groups can be used both as a basic substance or as cross-linking agent.

By way of example only and not by way of limitation, some important elastomers of various groups are summarized below for cell culture devices according to the invention:

Elastomers from the group of vinyl-functionalized siloxanes are cross-linkable with themselves by means of peroxides and by adjusting the temperature. However, they are also cross-linkable with hydride-functionalized siloxanes by suitable choice of platinum catalysts.

Elastomers from the group of the hydride-functionalized siloxanes are cross-linked with vinyl-functionalized siloxanes optionally with platinum catalysts. However, they are also cross-linkable with silanol-functionalized siloxanes by metal salt catalysts.

Elastomers from the group of silanol-functionalized siloxanes are cross-linkable with hydride-functionalized siloxanes by metal salt catalysts. But they are also cross-linkable with themselves by room temperature vulcanizations. They are also cross-linkable with amino-functionalized siloxanes _v

Elastomers from the groups of the amino-functionalized siloxanes, the epoxy-functionalized siloxanes and the carbinol-functionalized siloxanes are likewise suitable siloxanes in the context of the invention, that is to say they can be used as a substrate for cells in cell culture devices.

Elastomers from the group of methacrylate / acrylate-functionalized siloxanes are cross-linkable with themselves by radical formers, including UV light. Elastomers from the group of mercapto-functionalized siloxanes are also cross-linkable with themselves but also with vinyl-functionalized siloxanes by radical formers, including UV light.

Elastomers from the group of chorine / dimethylamine-functionalized siloxanes (α, ω- (methacryloxypiopropylethylsilyl) polydimethylsiloxanes, methacryloxypropyldimethylchlorosilanes) are cross-linked with hydride-functionalized siloxanes by hydrolyzing the chlorosilane bond.

Suitable diluents for the elastomers are, by way of example and not limitation, polydimethylsiloxanes without reactive groups and medium to low chain lengths, or siloxanes having reactive groups, but which do not participate in the reaction in the cross-linking reaction used.

Butadiene resins as a basic substance in the context of the invention are exemplary and not limiting nature of carboxy-terminated oligobutadienes.

Butadiene resins as crosslinkers or thinners are z. Hexanediol diacrylates, cyclohexyl methacrylates, methyl acrylates, ethylene glycol dimethacrylates, polyethylene glycol) monomethacrylates, linseed oil, styrenes, stearyl methacrylates, 2-hydroxyethyl acrylates, copolymers of n-butyl acrylates and t-butyl acrylates, n-hexyl methacrylates, 2-dimethylaminoethyl methacrylates, n- Decylmethacrylate.

An acrylate resin as a basic substance may, for. B. be 2-hydroxypropyl acrylates.

Acrylate resins as crosslinkers or thinners are z. For example, n-hexyl methacrylates, ethylene glycol dimethacrylates, n-decyl methacrylates.

Elastomers, copolymers and thinners which fall under one of the groups mentioned and / or are to be taken from the incorporated references, are expressly covered by the spirit of the invention. A variety of slightly different elastomers can be formed from a single combination of base and cross-linker alone, e.g. From the sylgard 184 kit. For this purpose, the Young's modulus in the abovementioned range can be varied, for example, by changing the mixing ratio between the basic substance and the cross-linking agent.

Of course, this also applies to the other elastomers mentioned above and cited in the literature. Such minor changes are also expressly covered by the spirit of the invention.

An unstructured surface of an elastomer can be applied very quickly to the surface of a suitable cell culture device by a suitable method. As a result, industrial serial production of cell culture devices according to the invention from the known cell culture devices is advantageously possible.

A uniform, the cell culture device covering layer of the elastomer is virtually arbitrarily adjustable by a person skilled in the art, provided that he has a suitable method, such. B. applies a spin coating or a casting process.

With the mixtures of base substance and optionally copolymer (cross-linking agent) to be used, it is possible to reproducibly produce layer thicknesses of about 100 nanometers to a few centimeters. For this purpose, the base substance and optionally a cross-linking agent and / or catalyst are mixed and evenly distributed in or on the cell culture device, so that the surface which is provided for receiving the cell, is formed from the unstructured elastomer.

The crosslinking to the elastomer takes place regularly after the application of the elastomer to the cell culture device.

To produce highly elastic surfaces for cell culture, it is advantageous to use a method by which an unstructured, flat elastomer film can be reproducibly produced on the surface of the device in the industrial sense. As a result, the series production of the inventive cell culture devices is made possible. Rotary processes are particularly advantageous for the production of very thin and uniform layers of the elastomer.

In doing so, a small amount of uncrosslinked liquid elastomer is placed on the surface of the device and distributed by centrifugal force through a suitable rotating device.

In the case of elastomers comprising a cross-linking agent, this is added in advance to the basic substance and mixed with this, optionally a catalyst is added.

The device together with liquid elastomer is clamped, for example, in a spin-coater. Alternatively, the device can also be held on a turntable by means of a vacuum.

By rotation at z. B. 1000 rpm per minute, the elastomer is distributed as a very uniform unstructured film on the bottom of the cell culture vessel. The layer formed then serves to accommodate the cells in the cell culture process.

After the optionally catalyst-driven curing, at least one elastomer from the group of siloxane resins retains its elastic properties over a virtually unlimited period of time and without any additional effort.

The resulting layer thickness is dependent on the viscosity and the amount of the still uncrosslinked elastomer, the rotational speed and the acceleration and process duration during spin coating.

A person skilled in the art is readily able to reproducibly produce various layer thicknesses of elastomers with the given process parameters.

It can, for. For example, a small amount of the uncrosslinked elastomer may be regularly applied and distributed to the surface of a desired device at about 1000 revolutions per minute by means of a spin coater. An alternative to the rotation process is to cast the elastomer without subsequent rotation or coating, by means of which suitable layer thicknesses are applied to surfaces by means of a suitable apparatus or a rolling process in which the elastomer is rolled onto surfaces by means of a roll or a rolling process or a combination of these various methods a possible alternative to the rotation process.

In particular, petri dishes, microscope slides or coverslips with a corresponding surface can be produced from a surface facing out of the cell culture from one of the elastomers mentioned. In addition, however, multiwell plates, cell culture bottles and other forms commonly used in cell culture according to the prior art are to be uniformly coated with an elastomer.

The idea of the invention can be applied to any hitherto known cell culture vessel or to any cell culture device.

A person skilled in the art can therefore coat any cell culture device which can be removed from the relevant laboratory and specialist catalogs with a surface made of an elastomer. It is thus particularly advantageous to provide a new class of cell culture vessels and devices.

A washing step of the cross-linked elastomer with isopropanol, especially pure isopropanol, increases the transparent properties of the elastomer.

This is very important for an unstructured flat surface. As a rule, a washing step in the alcohol of about 3 hours is sufficient for this purpose.

Particularly at higher elasticities of the elastomer of the device according to the invention with Young Moduli <50 kPa, a washing step with isopropanol causes non-cross-linked constituents to diffuse out of agglomerated bubbles of the layer. The washing step thus improves the observability of the cells.

It is conceivable to use another substance or alcohol for this purpose.

In any case, the surface of the cell culture device with the elastomer should look for better observability of the cells, preferably like that of glass. The elastomer of the cell culture device can be activated in a very particularly advantageous embodiment of the invention by physisorption of biomolecules.

By physisorption, special adhesion proteins are applied to the surface of the device. Physiosorption generally refers to a passive surface coating in which biomolecules settle out of an aqueous solution onto the underlying surface and build up a more or less uniform layer. This defines defined adhesion conditions for cells, which allow the analysis of very specific questions. By way of example, the study of biochemical aspects of cell-substrate interactions under defined conditions may be mentioned.

For this purpose, biomolecules such. For example, fibronectin, laminin, collagen, tenascin or hyaluronic acid are first dissolved in buffer. The liquid together with biomolecule is applied to the already crosslinked elastomer. The biomolecule deposits passively on the elastomer as a thin layer. The residual liquid is then removed again.

The biomolecule for any elastomer is selected essentially on the basis of the question or the cells.

This has the particularly advantageous effect of at least partially abolishing the hydrophobic property of the elastomer while at the same time allowing specific interactions under in vivo conditions between the protein as biomolecule and the cells. This makes the elastomers particularly well suited for cell culture processes.

However, it is also conceivable to carry out the cell culture method according to the invention in a device according to the invention without such changes, since some cell types also manage without elastomers altered by physisorption.

The variety of possible combinations with respect to the parameters type of elastomer, elasticity and type of physisorption open, as indicated in the exemplary embodiment, with respect to the differentiation of cells after addition of therapeutics a variety of Possibilities to deal with specific questions which could not be dealt with by the previous culture methods.

It is conceivable to arrange separate webs as a structure for complex problems in the elastomer in order to detect the reaction of the cells to chemicals or therapeutics. It is possible in this way to analyze chemotaxis in the broadest sense as well as the division behavior under these conditions.

It is conceivable to arrange more complex sequences of layers, for. B. by stamping biomolecules by means of micro- or nanocontact stamp after previous passivation of the intermediate areas.

An inventive cell culture method provides cells z. B. from a preculture applied to an optionally modified elastomer of the device and to cultivate.

The term cell culture is broad in terms of the cells used. It includes prokaryotic and eukaryotic cells as well as multicellular structures. In particular for cells of eukaryotic origin, the devices according to the invention are advantageous, since these cells generally adhere.

The term cell culture includes, inter alia, the analysis of cells in drug discovery under natural elasticity. The differentiation, the division rates and / or the migration behavior of the cells on the elastomer are of particular interest.

Depending on the type of cell used, the necessary boundary conditions for the culture and for the elastomer must be adapted.

By way of example, the selection of a suitable synthetic, liquid nutrient medium, auxiliaries such as vitamins, the temperature or the shape of the culture vessel may be mentioned as boundary conditions.

In a very particularly advantageous embodiment of the invention, the differentiation of the cell can be specifically controlled via the Young modulus of the elastomer during the cell culture process. Thus, fibroblasts which have been applied to the elastomer are advantageously differentiable depending on the Young's modulus of the elastomer into various cell types such as cardiac fibroblasts or myofibroblasts. This in turn is of interest in drug analysis and the provision of new therapeutics.

In the context of the invention, it has been recognized that the number of cell divisions per unit of time can also be controlled as a measure of the division rate of cells via the Young modulus of the elastomer surface of the cell culture vessel. Depending on the type of cell used, it increases on soft surfaces by up to 40% compared to devices according to the prior art.

In addition, the invention will be described with reference to exemplary embodiments and the accompanying figures.

Show it:

Fig. 1: Schematic representation of the type of crosslinking of some resins (prior art) for cell culture devices according to the invention.

Fig. 2: Adherent fibroblasts after six days of culture in a Petri dish made of polypropylene (prior art) with a centrally arranged cover glass as a cell culture soil. a) Antibody labeling of the cytoskeleton. b) to a) corresponding Durchlichtaufhahme.

Fig. 3: Adherent fibroblasts after six days of culture in a Petri dish made of polypropylene with a centrally arranged cover glass and an elastic surface of PDMS arranged thereon with a Young modulus of 38 kPa as a cell culture soil. a) Antibody labeling of the cytoskeleton. b) to a) corresponding Durchlichtaufhahme.

4 shows a confocal created side view of a cell culture device according to FIG. 3.

Fig. 1 shows schematically the type of crosslinking of three types of resin, which can serve according to the invention as an unstructured substrate for cells (prior art). For the arrangement of FIG. 3, fibroblasts were isolated and plated at a concentration of about 5,000 cells per cm ² on a PDMS surface made from a sylgard 184 kit.

The kit comprises vinyl-terminated siloxane as the basic substance and methylhydrosiloxane-dimethylsiloxane as copolymer and admixed Pt catalyst.

The mixture of siloxane and copolymer was prepared in a mixing ratio of 50: 1 to ground substance to crosslinker, mixed and degassed in Eksikator.

A cover glass measuring 25 x 75 mm and a thickness of -100 μm was mounted on the turntable of a spin coater of type Delta 10T from Süss Microtech and coated with 3 ml of the not yet crosslinked PDMS. The rotation process was carried out for 30 seconds at 1000 revolutions per minute.

The underside of the coverslip was then cleaned of excess PDMS with n-heptane. The curing took place in dust-protected containers at 60 ° C overnight, with an elasticity of about 25 kPa is achieved.

After crosslinking, the elastomer was coated with a 2.5 μg / cm ² fibronectin as an additional layer favoring adhesion of the cells. Fibronectin ensures adhesion of cells under near-natural and defined conditions. For this purpose, a corresponding small amount was applied and withdrawn after 20 minutes, the residual liquid again.

The elastomer was washed in 2-propanol with gentle shaking for 15 hours to wash out excess silicone oil which was not cross-linked. This causes in the specified PDMS Porymer with a Young's modulus of -25 kPa about a 40% volume reduction while improving the transparency and a post-curing to about 38 kPa. The layer thickness of the elastomer produced after this step was about 40 μm at a Poisson ratio of about 0.5. The coverslip was stuck over the elastomer from below to a centrally located hole in the bottom of a Petri dish. The connection between the PDMS surface and the cell culture vessel was made directly via the adhesive properties of the cross-linked PDMS already on the surface.

Alternatively, it is possible to increase the adhesion by using an additionally used cell-compatible adhesive.

As a comparative sample, an identical coverslip without such an elastomer was glued to an otherwise identical Petri dish.

Embryonic myocardial fibroblasts were isolated and seeded as single cells on the elastomer (Figure 3) or directly on the glass surface of the coverslip (Figure 2).

The cells according to FIG. 3, like the cells in FIG. 2, were incubated for six days at 37 ^° C. in a suitable nutrient solution. Subsequently, the cells were fixed with formaldehyde and labeled with an antibody against smouth muscle actin and incubated. Smouth muscle actin is a marker for the natural function of force generation of cells. The corresponding fixing and labeling protocols can easily be found by a person skilled in the art from the corresponding specialist literature.

The comparison between Fig. 2a) and Fig. 3a) shows that after 6 days of culture, only the fibroblasts on PDMS (Fig. 3a) had formed a distinct, dense and parallel oriented cytoskeleton. On the background of the comparison sample (FIG. 2 a) formed by the cover glass, however, this structure, which is absolutely essential for the function, of the parallel orientation of the cytoskeleton was not formed.

The corresponding transmitted light images show that cell counts on the PDMS surface (FIG. 3 b) compared with the glass surface (FIG. 2 b) increased by about 40%. This proves the increased cell division rate on PDMS towards glass and indicates a significantly lower cell stress when using an elastic surface. The experiment shows that the morphology of the cells and thus their structure and function are positively influenced by the choice of an elastomer as substrate. The same applies to the cell number.

The resulting layer thickness of the elastomer 3 is about 40 microns, as shown in Fig. 4. Layer 4 shows the adjacent air, layer 2 represents the cover glass and layer 1 the liquid arranged on the PDMS.

Claims

Patent claims

1. cell culture device with an unstructured surface for receiving cells, characterized in that the surface is formed of an elastomer.

2. Cell culture device according to claim 1, characterized by a transparent elastomer.

3. Device according to one of the preceding claims, characterized by a refractive index of the elastomer in a range of about 1.3 to 1.5, in particular 1.4.

4. Device according to one of the preceding claims, characterized by an elastomer having a Young modulus of 1 MPa to 500 Pa.

5. Device according to one of the preceding claims, characterized in that the surface of the elastomer has a layer promoting the adhesion and culture of the cells.

6. Device according to the preceding claim 5, characterized by a favoring layer of fibronectin, laminin, collagen, tenascin or hyuronic acid.

7. Device according to one of the preceding claims, characterized by an elastomer having a Poisson's ratio in a range of about 0.3 to 0.5.

8. Device according to one of the preceding claims, characterized by an elastomer which is not based on water.

9. Device according to one of the preceding claims, characterized by an elastomer of a base material of a siloxane resin, a butadiene resin or an acrylate resin.

10. Device according to the preceding claim, characterized by a vinyl-terminated siloxane resin.

11. Device according to one of the preceding claims, characterized by an elastomer comprising a methylhydrosiloxane-dimethylsiloxane copolymer as a cross-linking agent.

12. Device according to one of the preceding claims, characterized by a Petri dish, a slide, a coverslip, a multiwell plate, a cell culture flask, a sample carrier for the investigation of chemical and / or biological samples in general, in particular a sample carrier with webs and channels in the sense of a microfluidic Systems or a cell culture chamber.

13. Device according to one of the preceding claims, characterized in that it comprises a material suitable for in situ hybridizations and / or antibody labeling of the cells.

14. A method for producing a cell culture device, characterized in that a provided for receiving cells surface of the device is coated with an unstructured surface made of an elastomer.

15. The method of claim 14, wherein an elastomer of a siloxane resin, butadiene resin or acrylate resin is selected.

16. The method according to claim 15, characterized by

Choice of a vinyl-terminated siloxane resin.

17. The method according to claim 15, characterized by

Choice of a chloride-terminated siloxane resin.

18. The method according to any one of claims 14 to 17, wherein the resin is mixed with a copolymer as a cross-linking agent and optionally with a catalyst prior to coating the surface of the device.

19. The method according to any one of claims 14 to 18, wherein a transparent elastomer is formed.

20. The method according to any one of the preceding claims 14 to 19, characterized by

Execution of a rotation process for coating the device with the elastomer.

21. The method according to any one of claims 14 to 19, characterized by

Execution of a coating process, rolling process or casting process.

22. The method according to any one of the preceding claims 14 to 21, characterized by selecting a temperature of about 60 ° to 120 ° Celsius during the cross-linking of the elastomer.

23. The method according to any one of the preceding claims 14 to 22, characterized by a polymerization of the elastomer under UV light.

24. The method according to any one of the preceding claims 14 to 23, wherein the polymerized elastomer is washed with isopropanol.

25. The method according to any one of the preceding claims 14 to 24, characterized by a sterilization of the device after curing of the elastomer.

26. The method according to any one of the preceding claims 14 to 25, wherein a Young modulus of the elastomer of 1 MPa to 500 Pa is prepared.

27. cell culture method, characterized in that

Cells are cultivated in a cell culture device on an unstructured elastomer.

A cell culture method according to claim 27, wherein cells are cultured on a siloxane resin, butadiene resin or acrylate resin.

29. The cell culture method according to claim 27 or 28, wherein the differentiation, and / or the rate of division and / or the migration behavior of the cells is controlled by the Young's modulus of the elastomer during the cultivation.

Cell culture method according to one of the preceding claims 27 to 29, wherein cells are cultured with the addition of therapeutics on the planar elastomer.

31. Cell culture method according to one of the preceding claims 27 to 30, are cultivated in the fibroblasts on the elastomer.

A cell culture method according to any one of the preceding claims 27 to 31, wherein a kit comprising PDMS is used as the elastomer.

33. The cell culture method according to any one of claims 27 to 32, characterized by a sylgard 184 kit.

34. The cell culture method according to any of the preceding claims 27 to 33, wherein cells are cultured on an elastomer having a Young's modulus of 1 MPa to 500 Pa.

35. The cell culture method according to any one of the preceding claims 27 to 34, wherein cells are cultured on a transparent elastomer.

36. Cell culture method according to one of the preceding claims 27 to 35, characterized in that the cells are cultured on an elastomer having a layer promoting the adhesion and culture of the cells, for. On a layer comprising fibronectin, laminin, collagen, tenascin or hyaluronic acid.