EP2356215A1 - Substrats permettant de sélectionner et d'influencer spécifiquement le fonctionnement de cellules - Google Patents

Substrats permettant de sélectionner et d'influencer spécifiquement le fonctionnement de cellules

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Publication number
EP2356215A1
EP2356215A1 EP09764742A EP09764742A EP2356215A1 EP 2356215 A1 EP2356215 A1 EP 2356215A1 EP 09764742 A EP09764742 A EP 09764742A EP 09764742 A EP09764742 A EP 09764742A EP 2356215 A1 EP2356215 A1 EP 2356215A1
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EP
European Patent Office
Prior art keywords
substrate
cells
cell
ligands
specific
Prior art date
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Application number
EP09764742A
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German (de)
English (en)
Inventor
Joachim P. Spatz
Nadine Perschmann
Ann-Kathrin Schmieder
Roberto Fiammengo
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.)
Max Planck Gesellschaft zur Foerderung der Wissenschaften eV
Original Assignee
Max Planck Gesellschaft zur Foerderung der Wissenschaften eV
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Application filed by Max Planck Gesellschaft zur Foerderung der Wissenschaften eV filed Critical Max Planck Gesellschaft zur Foerderung der Wissenschaften eV
Publication of EP2356215A1 publication Critical patent/EP2356215A1/fr
Withdrawn legal-status Critical Current

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    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/551—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being inorganic
    • G01N33/552—Glass or silica
    • 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
    • C12N2533/40—Polyhydroxyacids, e.g. polymers of glycolic or lactic acid (PGA, PLA, PLGA); Bioresorbable polymers
    • 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
    • C12N2533/50—Proteins
    • C12N2533/52—Fibronectin; Laminin
    • 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
    • C12N2533/50—Proteins
    • C12N2533/54—Collagen; Gelatin
    • 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
    • C12N2535/00—Supports or coatings for cell culture characterised by topography
    • C12N2535/10—Patterned coating

Definitions

  • the invention relates to methods and substrates for specifically influencing the function of cells by their adhesion to substrate surfaces with predetermined properties.
  • the invention also relates to examination facilities and
  • ECM extracellular matrix
  • Concepts for the artificial construction of a matrix for the regulation of cell cultures in vitro are very often based on polymeric and inorganic scaffolds.
  • the goal here is that the polymeric or inorganic scaffold give physical signals to the cell system for cell orientation, cell migration and cell spreading.
  • the pores of the scaffold give the cell system sufficient space to define the tissue structure after a certain culture period.
  • Traditional polymer systems include polytetrafluoroethylene, silicones or polyethylene.
  • inorganic frameworks for example, bioactive glass, ceramics or calcium phosphates can be used.
  • bioactive matrices in the field of implant technology.
  • the artificial matrices known in the prior art take into account only a part of the factors which under natural conditions determine the interaction between the extracellular matrix and different cell types.
  • the present invention is based on the surprising finding that not only are the chemical properties, e.g. Presence of zeolite ligands and / or signal molecules, but also the mechanical properties of a substrate, e.g. the hardness, strength or rigidity of the substrate, allowing mechanical stimulation of cells through the substrate, and the geometric properties, e.g. the spatial arrangement of cell attachment sites of a substrate not only have a significant impact on cell adhesion (Discher et al., (2005) Science 320: 1139-1143; Arnold et al., (2004) Chemphyschem 5: 383-388) also exert a direct and specific influence on certain cell functions of adherent cells in many cases.
  • the chemical properties e.g. Presence of zeolite ligands and / or signal molecules
  • mechanical properties of a substrate e.g. the hardness, strength or rigidity of the substrate, allowing mechanical stimulation of cells through the substrate
  • geometric properties e.g. the spatial arrangement of cell attachment sites of a substrate not
  • the present invention provides, in one aspect, substrates for binding cells according to claim 1, the substrates having different surface areas, each representing at least one condition affecting cell adhesion and / or cell function.
  • substrates for binding cells according to claim 1 the substrates having different surface areas, each representing at least one condition affecting cell adhesion and / or cell function.
  • such substrates are part of a biomaterial chip according to claim 19 or an examination device according to claim 21.
  • the invention also encompasses the use of the above substrates, chips and inspection devices in various, in particular medical, applications according to claims 25-34.
  • a method for selectively affecting protein synthesis of target cells on a substrate according to claim 36 which induces or affects the synthesis of desired proteins by the arrangement of zeolite ligands at predetermined distances on the substrate.
  • a method of selecting and / or identifying cells according to claim 41 wherein the specific response of cells residing on a substrate is recorded and evaluated for mechanical stimulation.
  • the conditions influencing cell adhesion and / or cell function are determined. Typically, at least determined by a geometric property and / or a mechanical property or a combi ⁇ nation of a geometric property and / or a mechanical property with a chemical property of the respective surface area.
  • a geometric property and / or a mechanical property or a combi ⁇ nation of a geometric property and / or a mechanical property with a chemical property of the respective surface area can also play an important role in influencing cell adhesion and / or cell function. Any combinations of further properties with the chemical, mechanical and geometric properties explained in more detail herein are therefore also encompassed by the present invention.
  • the chemical property of the substrate is first determined by the molecular structure of the inorganic or organic substrate.
  • the substrate may e.g. Glass, metal or a plastic.
  • On this base substrate e.g. Nano Design Schemee be provided with a predetermined distance from nanostructures. Such nanostructures are e.g. by depositing gold nanoclusters of desired size and spacings on a substrate.
  • the chemical property of a desired surface area or of several surface areas is preferably predetermined or decisively determined by the functionalization of the base substrate and / or the nanostructures with specific zeolite ligands, in particular extracellular matrix (ECM) molecules in natural tissues or fragments thereof.
  • ECM extracellular matrix
  • the cell ligands are selected from molecules that bind to cell adhesion receptors (CAM) of cells. More particularly, they are molecules that bind to cell adhesion receptors of the groups of cadherins, immunoglobulin superfamily (Ig-CAMS), selectins, and integrins, particularly integrins.
  • CAM cell adhesion receptors
  • Ig-CAMS immunoglobulin superfamily
  • the ligands are selected from fibronectin, laminin, fibrinogen, tenascin, VCAM-I, MadCAM-1, collagen, or a cell adhesion receptor, particularly integrins, specific binding fragment thereof or a cell adhesion receptor specific binding derivative thereof.
  • the geometric property of the substrate typically comprises the arrangement of cell sites, in particular functionalized with cell ligands, at predetermined intervals on the substrate.
  • the arrangement of the contact sites or cell ligands represents a nanostructure.
  • nanostructure refers to an array of nanometer-sized islands, hereinafter referred to as “nanostructure regions”, which may serve as contact sites and may be selectively occupied by other molecules, eg, cell ligands.
  • the size of the islands should not be greater than a countable amount of molecules that interact with the surface of the cells. Desirable is the size of an island, which allows only the interaction of a single molecule due to the size of the island.
  • Island diameter in the range of less than 100 nm, in particular those less than 20 nm, for example less than 10 nm, are of particular interest in this case and are preferably used.
  • the spacing of the islands must be in ranges between 1 and 1000 nanometers, especially 1-300 nm, e.g. 1 - 200 nm or preferably 1 - 100 nm, be flexible and adjustable to an accuracy of 1-2 nanometers.
  • This connection may involve a simple or multiple connection of a single cell. Multiple binding has the advantage in particular in the presence of currents or, more generally, applied external forces, of keeping the cells significantly more stable at the interface than would be possible by single bonds, and thus, if appropriate, facilitating their separation from mixed media.
  • micellar block copolymer nanolithography R. Glass, M. Moller, JP Spatz, Nanotechnology 2003, 14 (10), 1153-1160, Arnold, M. Cavalcanti-Adam, R. Glass, J. Blummel, W. Eck, M. Kantlehner, H. Kessler, and JP Sparrow. (2004) Chemphyzem. 5: 383-8, DE 199 52018.6; DE 19747813.1; DE 29747815.8; DE 19747816.6).
  • nanoclusters desired size and desired spacings are produced on a substrate.
  • These nanostructures are typically ordered quasi-hexagonally.
  • all materials are suitable as the substrate on which the desired nanostructures can be formed.
  • Some suitable but non-limiting examples are glass, silica and plastic surfaces, including hydrogels.
  • the nanostructure regions form gradients of at least one of the geometric (e.g., distances and sizes of the regions) and chemical properties (e.g., type of functionalization). These gradients can also be formed by a sequence of spatially separated regions (see Fig. 6).
  • the mechanical property comprises the hardness or rigidity of the substrate and / or its viscous, elastic or viscoelastic properties.
  • the hardness or stiffness expressed as a Young 's modulus, can be varied, for example, in a range from 0.1 kPa to 100 MPa.
  • the mechanical property may include mechanical stimulation of the cells through the substrate.
  • the base substrates used are, for example, elastic plastics such as polydimethylsiloxane (PDMS) or hydrogels, preferably based on PEO, particularly preferably polyethylene glycol diacrylate (PEGDA) hydrogels).
  • PDMS polydimethylsiloxane
  • PEGDA polyethylene glycol diacrylate hydrogels
  • PEGDA polyethylene glycol diacrylate hydrogels
  • These offer using PEGs of different molecular weights and using different mass percentages (concentrations) of a wide span with respect to the Young's modulus see (MPa to kPa areas). Nanostructures as described above can also be transferred to these base substrates (for example as described in DE 10 2004 043 908 A1).
  • These gold nanostructures which can have different particle density and thus varying particle spacings (in the form of a gradient or precise homogeneous regions) and can be selectively adjusted in this respect, serve as binding sites for various ligands and / or functional groups after transfer to the hydrogel there , such as thiol groups.
  • a surface (eg glass) of a base substrate is first activated / hydroxylated to link the PEG hydrogel and functionalized with a compound which, for example, contains an unsaturated function, eg allyltriethoxysilane (FIG , In this case, the unsaturated function serves to networking with the hydrogel during the polymerization process.
  • an unsaturated function eg allyltriethoxysilane
  • Nanostructuring of the hydrogel is made possible by a transfer process.
  • the nanostructure is first applied to a glass surface by means of a diblock copolymer micelle technique (eg similar to that described in DE 19747813.1; DE 29747815.8; DE 19747816.6) and then using a linker, for example a prophenol or N, N V - Bis (acryloyl) cystamine linker, transferred to the hydrogel.
  • a linker for example a prophenol or N, N V - Bis (acryloyl) cystamine linker
  • the representation of the entire substrate is now carried out by simultaneous transfer of the gold structure and attachment of the hydrogel during the polymerization process.
  • the respective surfaces function as a flow cell (FIG. 1b), whereby the polymer solution can be filled between both surfaces without bubbles.
  • the hydrogel is stored in water, resulting in a water absorption of the gel and a gentle detachment of the upper glass.
  • the PEG hydrogel can also be functionalized completely or partially with a carboxylic acid. This laterally controlled functionalization of the surface of the gel occurs through a transfer process.
  • a carboxylic acid preferably a long-chain and polyunsaturated carboxylic acid (fatty acid, eg linolenic acid) is first applied to a hydrophilic glass.
  • This process is associated with a high degree of lateral precision and enables functionalization in the form of a microstructure (FIG. 1c) which can also be transferred to the gel.
  • the unsaturated end of the acid is then cross-linked with the PEG hydrogel during the polymerization process, whereby the carboxylic acid is covalently bonded to the surface of the gel ( Figure Id).
  • FIG. 2 clearly shows in the left panel the exclusive growth of the cells on the carboxylic acid functionalized hydrogel side.
  • the unfunctionalized side has a passivating effect on cells.
  • the right panel shows the binding of the fluorescent dye Oregon Green 488 cadaverine by EDC / NHS activation of the carboxyl function.
  • a substrate which comprises at least 3 different surface areas with at least 3 different conditions influencing cell adhesion and / or cell function.
  • this substrate is characterized in that the at least 3 different conditions that influence the cell adhesion and / or cell function, the functional at least one surface area with certain cell ligands, the geometric arrangement of zeolite ligands in at least one surface area, the substrate hardness or substrate stiffness in at least one surface area. A combination of two or more properties in one or more of these surface areas is possible and generally preferred (FIG. 3).
  • the substrate has a three-dimensional structure.
  • a structure may e.g. a tube or microtube with a diameter of a few to several 100 microns.
  • Such tubes may be made of various plastics and hydrogels, e.g. based on polyalkylene glycol, and also nanostructures, e.g. prepared as described above.
  • the different surface regions of the substrate are spatially separated from one another by barriers.
  • the different surface areas may be in separate chambers of the substrate.
  • the present invention also includes supports comprising two or more of the substrates of the invention in a three-dimensional array.
  • supports comprising two or more of the substrates of the invention in a three-dimensional array.
  • two identical or different substrates which, for example, carry different zeolite ligands, can be set against each other.
  • the optimum distance can be achieved, for example, by means of a frame (eg made of Teflon). Ion) of certain thickness between the two substrates.
  • a medium exchange can be made by the weak pumping of new medium.
  • a preferred embodiment of the invention relates to a biomaterial chip which comprises at least one substrate according to the invention and is constructed from different separate chambers which represent different but specific conditions influencing cell adhesion and / or cell function as explained above and the completed cultivation of cells in each Chamber allow.
  • a biomaterial chip preferably comprises at least 16 chambers.
  • the subject matter of the present invention is also an examination device comprising a) a substrate, a carrier or a biomaterial chip as defined above, b) a sample holder in which the substrate or the carrier or biomaterial chip is arranged, c) a Measuring device for detecting at least one cell-specific analysis parameter and d) an evaluation device.
  • the measuring device comprises a direct or inverted optical microscope and preferably a device for digital image processing.
  • the cell-specific analysis parameter is, but is not limited to, the number of cells, the cell shape, or the presence of a label, especially fluorescent label, for, for example, adhesion molecules or certain specific proteins, eg, cell differentiation, or other specific molecules, eg, nucleic acids or membrane components.
  • a label especially fluorescent label, for, for example, adhesion molecules or certain specific proteins, eg, cell differentiation, or other specific molecules, eg, nucleic acids or membrane components.
  • Other suitable parameters will be for the Skilled skilled in the art, depending on the cells studied and the conditions of cultivation and be easily detectable by standard methods.
  • the above-described substrates, biomaterial chips, or assay devices may be used to identify suitable substrate conditions for a specific cell system or cell function.
  • this specific cell function is the synthesis of specific proteins.
  • the substrates, chips and assay devices according to the invention are particularly suitable for carrying out a screening with high numbers of samples and / or many different substrate conditions ("high-through-put-screening", HTS) by combining different ones for cell adhesion and / or Cell function of essential substrate parameters in one or more surface regions and their specific variation in other surface regions can be used to quickly and efficiently identify suitable or optimized substrate conditions for specific cell types or cell functions, as well as rapid selection and identification of specific cell types.
  • HTS high-through-put-screening
  • the investigated cells are not limited in cell type or type of species. Both prokaryotic and eukaryotic cells can be examined. Preferably, it is cells of a vertebrate, in particular a mammal, more preferably of humans. In a specific embodiment, the cells examined are stem cells or the differentiated cells derived therefrom.
  • the substrates, chips and inspection devices according to the invention have wide potential applications on many offer biology, biochemistry and medicine, including medical diagnostics. Special fields of application are eg investigations in immunology and allergology. A specific example of this is the identification of substrate conditions for triggering allergic reactions of T or mast cells.
  • Another possible application relates to the promotion or investigation of the selective cell colonization of boundary surfaces, in particular in cardiology or implant technology.
  • the result of this study may e.g. the identification of suitable or optimized matrix properties for implants in different body areas, e.g. Bone, ear, etc., be.
  • substrates, biomaterial chips, or assay devices can also be used to select or identify cells.
  • a related application is the identification of disease states characterized by the change in cell type, e.g. Cancer or malaria.
  • a further aspect of the present invention relates to a method for influencing the protein synthesis of target cells, comprising a) providing a substrate for binding cells to the surface of this substrate, the substrate having at least one surface region having zeolite ligands at predetermined intervals are arranged on the substrate, is functionalized; b) applying the target cells to the substrate; c) cultivating the target cells on the substrate, wherein the synthesis of desired proteins induced by the arrangement of Zellli- ganden at predetermined intervals on the substrate or influenced.
  • This method may further include additionally providing a particular mechanical property of the functionalized surface area that also affects cell function.
  • This mechanical property can be provided, for example, by predetermining a certain rigidity or hardness of the substrate or by providing mechanical stimulation of adhering cells.
  • this aspect relates to a method for the selection and / or identification of numbers which comprises: a) providing a substrate for binding cells to the surface of said substrate, wherein the substrate has at least one surface area providing mechanical stimulation to the surface Can exercise cells; b) applying the target cells to the substrate; c) mechanical stimulation of the target cells on the substrate; d) recording the response of the cells to the stimulation; e) Evaluation of the reaction of the cells and, if appropriate, comparison with reference values and thereby identification of cells of a specific cell type and / or a specific origin.
  • Fig. Ia shows schematically the functionalization of a base substrate (eg glass) with allyltriethoxyethane for attachment of a hydrogel
  • Fig. Ib shows schematically the attachment of the hydrogel and the transfer of a gold nanostructure to the hydrogel;
  • Fig. Ic shows the microstructuring of a hydrophilic glass with carboxylic acid for subsequent transfer to a hydrogel
  • Fig. Id shows schematically the functionalization of the tethered hydrogel by the carboxylic acid.
  • Fig. 2 shows various hydrogels bound to glass.
  • the right panel illustrates the transfer of the gold nanostructure by means of “electroless deposition", which increases the gold particles.
  • Fig. 3 is a schematic representation of a combination of three variable parameters on a substrate having different surface areas.
  • Figure 4 shows a comparison of the growth of cells on a carboxylic acid functionalized versus an unfunctionalized hydrogel.
  • Fig. 5 shows contact angle measurements of surfaces with and without carboxylic acid functionalization.
  • Fig. 6 shows schematically an embodiment of a substrate with several spatially separated surface areas, in which two parameters, namely the type of ligand (biomolecule 1-4) and the distances between the ligands are systematically varied.
  • Figure 7 shows the differential synthetic activity of 3T3 fibroblasts with respect to the protein fibronectin as a function of the structure of the substrate surface), as detected by gel electrophoresis of the corresponding mRNA.
  • 8 shows a bar graph of the different synthesis activity of mouse osteoblasts with respect to the protein Vinkulin as a function of the structure of the substrate surface.
  • KNNQKSEPLIGRKKT C-terminal Hepa ⁇ n- A 4 Pi binding domain
  • HSRNSI cell-binding domain
  • KNSFMALYLSKGRLVFALG LG4 module, La-Syndecan 2 minm-alpha 3
  • PPFLMLLKGSTR LG3 domain, laminin 5-A 3 ⁇ i alpha 3-chain
  • IAFQRN LG2 domain, laminin-alpha 1- Aeßi
  • HHLGGAKQAGDV (gamma chain) ⁇ ub ⁇ a
  • CAM Cell Adhesion Molecule
  • Other peptides cyclo (RGDfK) Oysss Q vss
  • a surface (glass) is first activated / hydroxylated and functionalized with allyltriethoxysilane (FIG. 1 a).
  • the unsaturated function serves for subsequent crosslinking with the hydrogel during the polymerization process.
  • the nanostructuring of the hydrogel is done by a transfer process.
  • the nanostructure is first applied to a glass surface by means of a diblock copolymer micelle technique, as already described, and then transferred to the hydrogel using a prophenol or N, IST-bis (acryloyl) cystamine linker.
  • the unsaturated end of the linker serves to form covalent bonds during the polymerization of the hydrogel.
  • the representation of the entire substrate is now carried out by simultaneous transfer of the gold structure and attachment of the hydrogel during the polymerization process.
  • the respective surfaces function as a flow cell (FIG. 1b), whereby the polymer solution can be filled between both surfaces without bubbles.
  • the hydrogel is stored in water, which leads to a water absorption of the gel and to a gentle detachment of the upper glass.
  • Such a PEG hydrogel can also be functionalized in whole or in part with a carboxylic acid. This laterally controlled functionalization of the surface of the gel occurs through a transfer process.
  • a long-chain and polyunsaturated carboxylic acid (fatty acid, eg linolenic acid) is first applied to a hydrophilic glass (FIG. 1c). The unsaturated end of the acid is then cross-linked with the PEG hydrogel during the polymerization process, whereby the carboxylic acid is covalently bonded to the surface of the gel ( Figure Id).
  • Figure 4 clearly shows the exclusive growth of cells on the carboxylic acid functionalized hydrogel side.
  • the unfunctionalized side has a passivating effect on cells.
  • the right panel shows the binding of the fluorescent dye Oregon Green 488 cadaverine by EDC / NHS activation of the carboxyl function.
  • 3T3 fibroblasts were applied to glass substrates containing an array of specific cell ligands, C- (-RGDfK-) -thiol, on gold nanostructures of different distances or a homogeneous surface.
  • These fibroblasts synthesize two different types of fibronectin, which differ in molecular weight.
  • both species are synthesized in almost the same amount.
  • the specification of a nanostructure and its variation can lead to a drastic preference for one or the other type of protein.
  • FIG. 7 shows the gel electrophoresis of the mRNAs which are responsible for the synthesis of the two different fibronectins. The results show a strongly different expression activity for both genes as a function of the distance between the nanostructural regions and thus the cell ligands (58 nm and 73 nm, respectively).
  • Fig. 8 shows a bar graph of the different synthetic activity of mouse osteoblasts with respect to the protein Vinkulin as a function of the structure of the substrate surface over a period of 24 h.

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  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention concerne un procédé et des substrats permettant de sélectionner et d'influencer spécifiquement le fonctionnement de cellules par leur adhérence à des surfaces de substrats ayant des propriétés prédéfinies. Ces substrats comportent différentes régions superficielles qui représentent une condition influençant l'adhérence de cellules et/ou le fonctionnement de cellules et ces conditions sont déterminées par une propriété géométrique et/ou une propriété mécanique ou une combinaison d'une propriété géométrique et/ou d'une propriété mécanique avec une propriété chimique de la région superficielle en question. L'invention concerne également des dispositifs d'analyse et des procédés d'analyse utilisant ces substrats pour identifier et sélectionner des types de cellules déterminés, pour identifier des conditions de substrat appropriées pour influencer un fonctionnement de cellules déterminé ou des types de cellules déterminés ou pour identifier des états maladifs qui sont caractérisés par une modification du type de cellules ou du fonctionnement des cellules.
EP09764742A 2008-12-08 2009-12-07 Substrats permettant de sélectionner et d'influencer spécifiquement le fonctionnement de cellules Withdrawn EP2356215A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE102008060991A DE102008060991A1 (de) 2008-12-08 2008-12-08 Subtrate zur Selektion und spezifischen Beeinflussung der Funktion von Zellen
PCT/EP2009/008723 WO2010075933A1 (fr) 2008-12-08 2009-12-07 Substrats permettant de sélectionner et d'influencer spécifiquement le fonctionnement de cellules

Publications (1)

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EP2356215A1 true EP2356215A1 (fr) 2011-08-17

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US (1) US20110275539A1 (fr)
EP (1) EP2356215A1 (fr)
CN (1) CN102317441A (fr)
DE (1) DE102008060991A1 (fr)
WO (1) WO2010075933A1 (fr)

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EP3431582A1 (fr) * 2017-07-18 2019-01-23 Koninklijke Philips N.V. Matériaux de culture de cellules
CN111699244B (zh) * 2017-12-22 2023-11-24 巴斯德研究所 基于水凝胶的器官芯片微流控装置
DE102018204203A1 (de) * 2018-03-20 2019-09-26 Carl Von Ossietzky Universität Oldenburg Verfahren zur Verbesserung und/oder Steuerung der Zelladhäsion und medizinisches Implantat, Zellkultursystem oder Zellassay mit verbesserter Zelladhäsion
EP3591401A1 (fr) 2018-07-06 2020-01-08 Euroimmun Medizinische Labordiagnostika AG Procédé de détection automatisée d'anticorps dans un échantillon biologique liquide à l'aide d'une puce à antigène et puce à antigène correspondante
EP3798300A1 (fr) * 2019-09-24 2021-03-31 Koninklijke Philips N.V. Matériaux de culture de cellules

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DE102008060991A1 (de) 2010-06-10
US20110275539A1 (en) 2011-11-10
CN102317441A (zh) 2012-01-11
WO2010075933A1 (fr) 2010-07-08

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