EP2032688A1 - Dispositif de culture cellulaire, procédé de fabrication du dispositif, et procédé de culture cellulaire - Google Patents

Dispositif de culture cellulaire, procédé de fabrication du dispositif, et procédé de culture cellulaire

Info

Publication number
EP2032688A1
EP2032688A1 EP07764371A EP07764371A EP2032688A1 EP 2032688 A1 EP2032688 A1 EP 2032688A1 EP 07764371 A EP07764371 A EP 07764371A EP 07764371 A EP07764371 A EP 07764371A EP 2032688 A1 EP2032688 A1 EP 2032688A1
Authority
EP
European Patent Office
Prior art keywords
elastomer
cell culture
cells
resin
cross
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP07764371A
Other languages
German (de)
English (en)
Inventor
Bernd Hoffmann
Bodo Borm
Rudolf Merkel
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Forschungszentrum Juelich GmbH
Original Assignee
Forschungszentrum Juelich GmbH
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Forschungszentrum Juelich GmbH filed Critical Forschungszentrum Juelich GmbH
Publication of EP2032688A1 publication Critical patent/EP2032688A1/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0068—General culture methods using substrates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/20—Material Coatings
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
    • C12M25/02—Membranes; Filters
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/30—Synthetic polymers

Definitions

  • 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.
  • 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.
  • the object of the invention is to provide a cell culture device which enables cell culture under in vivo similar conditions.
  • 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.
  • 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.
  • the elastomer advantageously allows quasi a culture of the cells under in situ or in vivo similar conditions.
  • 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.
  • 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.
  • the cells optionally adhering to the elastomer and the progress of the cell culture can be well observed.
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • the maximum layer thickness of device and elastomer to be penetrated optimally for most objectives of light microscopes should not exceed about 250 ⁇ m.
  • 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.
  • 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.
  • ground substance is used synonymously with the main constituent in the polymerized elastomer.
  • 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 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.
  • 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.
  • 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.
  • the macromolecular structure of the matrix of polymerizable end group silicone resins causes a reduction in the rate of diffusion of the reactive species, e.g. As the vinyl groups and the concentration thereof.
  • the reactive species e.g. As the vinyl groups and the concentration thereof.
  • 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.
  • 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.
  • 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.
  • ABCR vinyl-terminated polydimethylsiloxane
  • HMS 301 as an example of a methylhydrosiloxane-dimethylsiloxane copolymer for a cross-linking agent
  • 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.
  • DMS-T21 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.
  • DMS-T22 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.
  • 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.
  • 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 ( ⁇ , ⁇ - (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.
  • n-hexyl methacrylates ethylene glycol dimethacrylates
  • n-decyl methacrylates 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.
  • 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.
  • An unstructured surface of an elastomer can be applied very quickly to the surface of a suitable cell culture device by a suitable method.
  • industrial serial production of cell culture devices according to the invention from the known cell culture devices is advantageously possible.
  • 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.
  • the base substance and optionally copolymer (cross-linking agent) it is possible to reproducibly produce layer thicknesses of about 100 nanometers to a few centimeters.
  • 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.
  • 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.
  • the device can also be held on a turntable by means of a vacuum.
  • the elastomer 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.
  • 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 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • biomolecules such as 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.
  • An inventive cell culture method provides cells z. B. from a preculture applied to an optionally modified elastomer of the device and to cultivate.
  • 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.
  • 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.
  • the necessary boundary conditions for the culture and for the elastomer must be adapted.
  • auxiliaries such as vitamins, the temperature or the shape of the culture vessel may be mentioned as boundary conditions.
  • the differentiation of the cell can be specifically controlled via the Young modulus of the elastomer during the cell culture process.
  • 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.
  • 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.
  • 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 Naturallichtaufhahme.
  • 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 Naturallichtaufhahme.
  • FIG. 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).
  • 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 ⁇ m 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.
  • the elastomer was coated with a 2.5 ⁇ g / 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 ⁇ m 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.
  • 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.
  • FIG. 2a 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
  • layer 1 the liquid arranged on the PDMS.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Chemical & Material Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Biomedical Technology (AREA)
  • Genetics & Genomics (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Sustainable Development (AREA)
  • Cell Biology (AREA)
  • Clinical Laboratory Science (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention concerne un dispositif de culture cellulaire caractérisé par une surface en élastomère non structuré. Grâce à l'élasticité de son milieu, il permet de cultiver des cellules dans des conditions proches de la nature. L'invention concerne en outre un procédé de fabrication d'un dispositif selon l'invention, ainsi qu'un procédé de culture cellulaire utilisant ce dispositif.
EP07764371A 2006-06-24 2007-06-14 Dispositif de culture cellulaire, procédé de fabrication du dispositif, et procédé de culture cellulaire Withdrawn EP2032688A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE102006029051A DE102006029051A1 (de) 2006-06-24 2006-06-24 Zellkulturvorrichtung, Verfahren zur Herstellung der Vorrichtung und Zellkulturverfahren
PCT/DE2007/001054 WO2007147389A1 (fr) 2006-06-24 2007-06-14 Dispositif de culture cellulaire, procédé de fabrication du dispositif, et procédé de culture cellulaire

Publications (1)

Publication Number Publication Date
EP2032688A1 true EP2032688A1 (fr) 2009-03-11

Family

ID=38578623

Family Applications (1)

Application Number Title Priority Date Filing Date
EP07764371A Withdrawn EP2032688A1 (fr) 2006-06-24 2007-06-14 Dispositif de culture cellulaire, procédé de fabrication du dispositif, et procédé de culture cellulaire

Country Status (5)

Country Link
US (1) US20090186411A1 (fr)
EP (1) EP2032688A1 (fr)
JP (1) JP2009540805A (fr)
DE (1) DE102006029051A1 (fr)
WO (1) WO2007147389A1 (fr)

Families Citing this family (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE102009049454A1 (de) * 2009-10-14 2011-04-21 Forschungszentrum Jülich GmbH Vorrichtung zur Untersuchung von Zellen mit einem Elastomer sowie Verwendung der Vorrichtung
US20130029422A1 (en) * 2011-07-26 2013-01-31 Vasiliy Nikolaevich Goral Composite Substrate for 3D Cell Culture
EP2594632A1 (fr) 2011-11-18 2013-05-22 Miltenyi Biotec GmbH Procédé et dispositif de modification cellulaire
JP2015107110A (ja) * 2013-10-22 2015-06-11 株式会社メニコン 軟質培養容器
EP3277796B1 (fr) * 2015-03-31 2020-04-22 Kuhner Shaker Gmbh Matrices de libération pour la libération contrôlée de matériaux dans un milieu environnant
JP6724321B2 (ja) * 2015-09-15 2020-07-15 Tdk株式会社 積層電子部品
EP3431582A1 (fr) * 2017-07-18 2019-01-23 Koninklijke Philips N.V. Matériaux de culture de cellules
EP3798300A1 (fr) * 2019-09-24 2021-03-31 Koninklijke Philips N.V. Matériaux de culture de cellules

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991019783A1 (fr) * 1990-06-15 1991-12-26 E.I. Du Pont De Nemours And Company Surfaces en polymere elastomere servant de support a des cellules de mammiferes et procedes de preparation associes
US7517453B2 (en) * 2003-03-01 2009-04-14 The Trustees Of Boston University Microvascular network device
US20060240548A1 (en) * 2003-06-26 2006-10-26 Mordechai Deutsch Materials for constructing cell-chips, cell-chip covers, cell-chips coats, processed cell-chips and uses thereof
CN101189271A (zh) * 2004-02-13 2008-05-28 北卡罗来纳大学查珀尔希尔分校 制造微流体设备的功能材料和新型方法
WO2005087913A1 (fr) * 2004-03-11 2005-09-22 Nagoya Industrial Science Research Institute Dispositif de culture

Non-Patent Citations (10)

* Cited by examiner, † Cited by third party
Title
BROWN X Q ET AL: "Evaluation of polydimethylsiloxane scaffolds with physiologically-relevant elastic moduli: interplay of substrate mechanics and surface chemistry effects on vascular smooth muscle cell response", BIOMATERIALS, ELSEVIER SCIENCE PUBLISHERS BV., BARKING, GB, vol. 26, no. 16, 1 June 2005 (2005-06-01), pages 3123 - 3129, XP025280622, ISSN: 0142-9612, [retrieved on 20050601] *
BROWN X.Q.; OOKAWA K.; WONG J.Y.: "Evaluation of polydimethylsiloxane scaffolds with physiologically-relevant elastic moduli: interplay of substrate mechanics and surface chemistry effects on vascular smooth muscle cell response", BIOMATERIALS, vol. 26, no. 16, 1 June 2005 (2005-06-01), pages 3123 - 3129, XP025280622
GOFFIN J.M. ET AL: "Focal adhesion size controls tension-dependent recruitment of alpha-smooth muscle actin to stress fibers", THE JOURNAL OF CELL BIOLOGY, vol. 172, no. 2, 16 January 2006 (2006-01-16), pages 259 - 268, XP003024831
GOFFIN JEROME M ET AL: "Focal adhesion size controls tension-dependent recruitment of alpha-smooth muscle actin to stress fibers", JOURNAL OF CELL BIOLOGY, vol. 172, no. 2, January 2006 (2006-01-01), pages 259 - 268, ISSN: 0021-9525 *
LEE J N ET AL: "Compatibility of mammalian cells on surfaces of poly(dimethylsiloxane)", LANGMUIR 20041221 AMERICAN CHEMICAL SOCIETY US, vol. 20, no. 26, 21 December 2004 (2004-12-21), pages 11684 - 11691 *
LEE J.N. ET AL: "Compatibility of Mammalian Cells on Surfaces of Poly(dimethylsiloxane)", LANGMUIR, vol. 20, no. 26, 2004, pages 11684 - 11691, XP003025765
LEE J.N. ET AL: "Solvent Compatibility of Poly(dimethylsiloxane)- BAsed Microfluidic devices", ANAL. CHEM., vol. 75, 2003, pages 6544 - 6554, XP003025764
LIAO H ANDERSSON A-S SUTHERLAND D PETRONIS S KASEMO B THOMSEN P: "Response of rat osteoblast-like cells to microstructured model surfaces in vitro", BIOMATERIALS, ELSEVIER SCIENCE PUBLISHERS BV., BARKING, GB, vol. 24, no. 4, 1 February 2003 (2003-02-01), pages 649 - 654, XP004393190, ISSN: 0142-9612 *
LIAO H. ET AL: "Response of rat osteoblast-like cells to microstructured model surfaces in vitro", BIOMATERIALS, vol. 24, no. 4, 1 February 2003 (2003-02-01), pages 649 - 654, XP004393190
See also references of WO2007147389A1 *

Also Published As

Publication number Publication date
JP2009540805A (ja) 2009-11-26
DE102006029051A1 (de) 2007-12-27
WO2007147389A1 (fr) 2007-12-27
US20090186411A1 (en) 2009-07-23

Similar Documents

Publication Publication Date Title
EP2032688A1 (fr) Dispositif de culture cellulaire, procédé de fabrication du dispositif, et procédé de culture cellulaire
Park et al. Increased poly (dimethylsiloxane) stiffness improves viability and morphology of mouse fibroblast cells
DE3855631T2 (de) Biokompatible polyorganosiloxan-zusammensetzung für zellkultur-vorrichtung
Cimetta et al. Production of arrays of cardiac and skeletal muscle myofibers by micropatterning techniques on a soft substrate
KR20100074164A (ko) 컴플라이언트 표면의 멀티웰 배양 플레이트
CN104704067A (zh) 光滑的自润滑聚合物表面
EP1880764A1 (fr) Porte échantillons destiné à l'analyse de la croissance cellulaire
US20130029422A1 (en) Composite Substrate for 3D Cell Culture
US20140141503A1 (en) Cell culture substrate having uniform surface coating
Pedron et al. Microfluidic approaches for the fabrication of gradient crosslinked networks based on poly (ethylene glycol) and hyperbranched polymers for manipulation of cell interactions
DE102009002577B4 (de) Zellkulturträger für die Kultivierung von humanen Stamm- und Vorläuferzellen sowie ein Verfahren zur Kultivierung
CN107119012A (zh) 一种制备纳米粗糙化pdms基底的新方法
WO2010075933A1 (fr) Substrats permettant de sélectionner et d'influencer spécifiquement le fonctionnement de cellules
Cerca et al. Comparative evaluation of coagulase-negative staphylococci (CoNS) adherence to acrylic by a static method and a parallel-plate flow dynamic method
Zhang et al. Anti-bio adhesive behavior and mechanism of polystyrene microspheres enhanced PEG-based hydrogels
Ahmed et al. Prolonged morphometric study of barnacles grown on soft substrata of hydrogels and elastomers
EP2492012B1 (fr) Dispositif de recouvrement pour un porte-échantillons
JP2010200679A (ja) 細胞培養容器、細胞培養方法、および細胞評価方法
DE102010022675B4 (de) Verfahren zur Herstellung einer Hydrogel-Mikrostruktur
Leclerc et al. In vitro cyto-biocompatibility study of thin-film transistors substrates using an organotypic culture method
EP2665810B1 (fr) Procédé pour l'immobilisation et la préparation de structures de multicomposants fonctionnelles de matrice extracellulaire
Pedron et al. Combinatorial approach for fabrication of coatings to control bacterial adhesion
JP2023178108A (ja) 細胞培養基材
CN111918958B (zh) 培养基材、培养基材的制造方法、干细胞的培养方法及培养装置
JP7219891B2 (ja) 細胞培養用積層体、医療器具および医療器具の使用方法

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20081218

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IS IT LI LT LU LV MC MT NL PL PT RO SE SI SK TR

AX Request for extension of the european patent

Extension state: AL BA HR MK RS

TPAC Observations filed by third parties

Free format text: ORIGINAL CODE: EPIDOSNTIPA

17Q First examination report despatched

Effective date: 20090512

TPAC Observations filed by third parties

Free format text: ORIGINAL CODE: EPIDOSNTIPA

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION HAS BEEN WITHDRAWN

18W Application withdrawn

Effective date: 20100612