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WO2022001631A1 - Dispositif de préparation de grappes de cellules, procédé de construction correspondant et application associée - Google Patents

Dispositif de préparation de grappes de cellules, procédé de construction correspondant et application associée Download PDF

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Publication number
WO2022001631A1
WO2022001631A1 PCT/CN2021/099718 CN2021099718W WO2022001631A1 WO 2022001631 A1 WO2022001631 A1 WO 2022001631A1 CN 2021099718 W CN2021099718 W CN 2021099718W WO 2022001631 A1 WO2022001631 A1 WO 2022001631A1
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cell
microcavity
substrate
template
cells
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Chinese (zh)
Inventor
姚睿
苏奕君
梁少君
薛胜楠
申函宁
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Tsinghua University
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Tsinghua University
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    • 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
    • C12M23/02—Form or structure of the vessel
    • C12M23/12—Well or multiwell plates
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J3/00—Processes of treating or compounding macromolecular substances
    • C08J3/24—Crosslinking, e.g. vulcanising, of macromolecules
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J5/00—Manufacture of articles or shaped materials containing macromolecular substances
    • C08J5/18—Manufacture of films or sheets
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L83/00—Compositions of macromolecular compounds obtained by reactions forming in the main chain of the macromolecule a linkage containing silicon with or without sulfur, nitrogen, oxygen or carbon only; Compositions of derivatives of such polymers
    • C08L83/04—Polysiloxanes
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L89/00—Compositions of proteins; Compositions of derivatives thereof
    • 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
    • C12M3/00—Tissue, human, animal or plant cell, or virus culture apparatus
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J2383/00—Characterised by the use of macromolecular compounds obtained by reactions forming in the main chain of the macromolecule a linkage containing silicon with or without sulfur, nitrogen, oxygen, or carbon only; Derivatives of such polymers
    • C08J2383/04—Polysiloxanes
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J2389/00—Characterised by the use of proteins; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J2483/00—Characterised by the use of macromolecular compounds obtained by reactions forming in the main chain of the macromolecule a linkage containing silicon with or without sulfur, nitrogen, oxygen, or carbon only; Derivatives of such polymers
    • C08J2483/04—Polysiloxanes
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J2489/00—Characterised by the use of proteins; Derivatives thereof

Definitions

  • the present application relates to the technical fields of biotechnology and tissue engineering, and in particular, to a device for preparing cell clusters, a construction method and applications thereof.
  • the traditional cell culture method is flat culture.
  • the monolayer cell culture obtained by flat culture is not the same as the physiological structure of cells in vivo, which will affect the survival and function of cells.
  • a quasi-3D environment can be constructed.
  • 3D cell culture can recreate cell-cell, cell-matrix interactions and reduce the differences between in vitro culture and native tissues.
  • cells such as embryonic stem cells, mesenchymal stem cells, and various tumor cells have the characteristic of clumps.
  • pancreatic ⁇ cells need to form cell clusters to maintain their functions, and the construction of cell clusters is beneficial to cell culture and differentiation.
  • organoids have also increased rapidly recently, and organoids often require co-culture of multiple cells to ensure their structural development and functional expression.
  • the downstream applications of cell clusters include drug detection, in vivo transplantation, etc. These downstream applications generally require 10 8 to 10 9 cells to obtain reliable results, so a large number of cell clusters are prepared in the early stage. In view of the wide application of cell clusters, large-scale preparation of cell clusters is very necessary.
  • Clusters are critical. Another important factor is the stiffness of the substrate. Different cell types have different requirements for material stiffness, so a suitable culture substrate is required when preparing cell clusters.
  • the research on cell clusters generally involves laying a substrate in a conventional multi-well culture dish and planting a certain number of cells to form clusters by self-assembly of cells.
  • this method can provide a soft bottom that is beneficial for cell aggregation and culture, it is limited in quantity and size, and it is difficult to obtain a large number of uniform cell clusters.
  • the introduction of microcavity arrays with controllable dimensions can solve this problem and obtain cell clusters of uniform size and considerable number.
  • the bottom of the multi-well culture dish is equipped with a microcavity array, which can prepare a large number of uniform cell clusters.
  • the plastic substrate is too rigid, and the size and shape of the micropores cannot meet the custom requirements, and cannot be reused. Costs and fees are exorbitant.
  • microcavity arrays for culturing cell clusters.
  • a common method is to prepare polydimethylsiloxane stamps by a template method, and then use the stamps to prepare microcavity arrays for culturing cells.
  • Most of the hydrogel substrates prepared by this method are hydrogel substrates such as PEG and agar, which have a limited range of mechanical properties and cannot be reused.
  • the method is to obtain a micro-pattern template with a vertical wall by photolithography, and then prepare a micro-cavity by a template method. This method can only prepare microcavities with vertical walls, and it is difficult to obtain non-vertical walls. The generation of cell aggregates largely depends on the intercellular force.
  • the purpose of the present application is to provide a device for preparing cell clusters and a construction method and application thereof.
  • the present application provides a device for preparing cell clusters, the device comprising a cell culture plate and a substrate fixed in a microwell of the cell culture plate and having a microcavity array.
  • Each microcavity constitutes a space for cell cluster growth (roughly determines the size and space of cell cluster growth); the shape of the microcavity can be selected from cylindrical, square, rectangular, triangular, diamond, Any regular or irregular shape such as hexagonal prism, inverted cone, etc., especially the non-vertical wall surface such as inverted cone is the priority; the upper surface area of the microcavity is 0.04 ⁇ 1mm 2 , the depth of the microcavity is 100 ⁇ 500 ⁇ m, and the substrate The number of microcavities arranged on the top is about 1000 to 5000 (depending on the size of the microcavity and the overall size of the template). Arrays can contain microcavities of different shapes, sizes, and arrangements.
  • FIG. 1 The schematic diagram of the device provided in this application is shown in FIG. 1 .
  • FIG. 2 The schematic diagram of the microcavity is shown in Figure 2.
  • the material used to make the substrate can be selected from polypropylene, polystyrene, polyacrylamide, polylactic acid, polyhydroxy acid, polylactic acid alkyd copolymer, polydimethylsiloxane (PDMS), polyanhydride, polyacid Ester, polyamide, polyamino acid, polyacetal, polycyanoacrylate, polyurethane, polypyrrole, polyester, polymethacrylate, polyethylene, polycarbonate, polyethylene oxide, silk fibroin , silk fibroin derivatives, chitosan, gelatin, gelatin derivatives, alginate, agar, matrigel, collagen, collagen derivatives, hyaluronic acid, hyaluronic acid derivatives, cellulose, cellulose-derived materials, At least one of proteoglycan, proteoglycan derivatives, glycoproteins, glycoprotein derived materials, laminin, fibronectin and fibrin, etc., preferably a mixture of polydimethylsiloxane and silk fibroin.
  • the cell culture plate is a commercial multi-well culture plate, preferably a 6-well plate, a 12-well plate or a 24-well plate.
  • the elastic modulus of the substrate is 0.1 MPa to 10 MPa, and the elastic modulus can be adjusted according to the composition, concentration and curing characteristics of the microcavity substrate material.
  • the pH of the substrate is 3-12.
  • the thickness of the substrate is 100 ⁇ m ⁇ 2 cm.
  • extracellular matrix components that are conducive to the adhesion and growth of cell clusters can be added as needed, such as collagen, matrigel, proteoglycan, glycoprotein, hyaluronic acid, laminar connection protein or fibronectin, etc.
  • the mass percentage concentration of the extracellular matrix material is 0.1%-80%, preferably 1%-25%.
  • the device is reusable, and can be cleaned, sterilized and used again after one use, cell clusters are collected, and the micro-pattern accuracy and sterility can be maintained.
  • the present application provides a method for constructing a device for preparing cell clusters, including: firstly preparing a micro-pattern template with adjustable size and customizable shape, and then obtaining a micro-pattern template with a micro-cavity array by a two-step template method. base. details as follows:
  • stamps For the liquid or semi-solid stamp material, the stamp material is covered on the template by using the template method, and then peeled off from the template after curing and forming, to obtain the over-molded stamp.
  • the mold micro-pattern and the stamp material (such as polymethyl methacrylate) are laminated, pressurized, heated, and then mechanically or chemically peeled off to obtain a stamped stamp.
  • the base material described in step 3) can be selected from polypropylene, polystyrene, polyacrylamide, polylactic acid, polyhydroxy acid, polylactic acid alkyd copolymer, polydimethylsiloxane (PDMS), polyanhydride , Polyester, Polyamide, Polyamino acid, Polyacetal, Polycyanoacrylate, Polyurethane, Polypyrrole, Polyester, Polymethacrylate, Polyethylene, Polycarbonate, Polyethylene oxide, Silk Fibroin, Silk Fibroin Derivatives, Chitosan, Gelatin, Gelatin Derivatives, Alginate, Agar, Matrigel, Collagen, Collagen Derivatives, Hyaluronic Acid, Hyaluronic Acid Derivatives, Cellulose, Cellulose At least one of derived materials, proteoglycans, proteoglycan derivatives, glycoproteins, glycoprotein derived materials, laminin, fibronectin and fibrin, etc., preferably a mixture of polydimethylsiloxane and silk
  • the shape of the microcavity on the template described in step 1) can be selected from any regular or irregular shape such as cylinder, square column, rectangular column, triangular column, diamond column, hexagonal column, inverted cone, etc., especially
  • the non-vertical wall surface such as inverted cone is the priority; the upper surface area of the microcavity is 0.04-1 mm 2 , the depth of the micro-cavity is 100-500 ⁇ m, and the number of micro-cavities arranged on the template is 1000-5000.
  • the material used for making the template in step 1) can be selected from silicon, aluminum, iron, tin, glass, polymethyl methacrylate, polydimethylsiloxane, polycaprolactone, polytrimethylene carbonate, At least one of polytetrafluoroethylene, polyethylene oxide, polyethylene vinyl acetate, polydioxanone, polyether ether ketone, and the like.
  • the seal material described in step 2) is a degradable material or a non-degradable material
  • the degradable material can be selected from gelatin, gelatin derivatives, agar, agarose, At least one of F-127, polyvinyl alcohol, polyethylene glycol, etc.
  • the non-degradable material can be selected from polymethyl methacrylate, epoxy resin, phenolic resin, polyvinyl chloride resin, unsaturated polyester At least one of resin, gypsum, silica gel (silica gel matrix), and the like.
  • the mass percentage concentration of the seal material in step 2) is 0.1%-80%, preferably 1%-25%.
  • the concentration of the cross-linking agent in step 3) is 0.1 mM to 10 M, preferably 1 mM to 100 mM.
  • the cross-linking agent can be selected from hydrogen-containing silicone oil, silane coupling agent, divalent cation, genipin, glutaraldehyde, adipic acid dihydrazide, epichlorohydrin, carbodiimide, thrombin and the like. At least one of derivatives, etc., preferably hydrogen-containing silicone oil.
  • the base material and the cross-linking agent in step 3) are mixed in a volume ratio of 1000:1 to 1:1000, preferably 10:1 to 1:10.
  • the conditions for curing in step 3) are: a temperature of 10-100° C. and/or a light intensity of 0.5-1000 lx.
  • the method for constructing the device for preparing cell clusters includes:
  • a polymethyl methacrylate template with a columnar microcavity array is fabricated by photolithography
  • A3. Mix PDMS and silk fibroin with a cross-linking agent, and after defoaming treatment (vacuum to remove air bubbles), cover it on the stamp of the flip, overnight at room temperature, and solidify to form a mixed substrate of PDMS and silk fibroin;
  • the stamp is melted by heating and removed from the mixed substrate of PDMS and silk fibroin to obtain a substrate with a microcavity array;
  • step A3 the mass ratio of PDMS, silk fibroin and cross-linking agent is 5-10:0.6-60:1, and the cross-linking agent is hydrogen-containing silicone oil.
  • the method for constructing the device for preparing cell clusters comprises:
  • a silicon wafer template with an inverted tapered microcavity array is fabricated by a wet etching process
  • the micropattern surface of the silicon wafer template is attached to the polymethyl methacrylate (PMMA) sheet, heated and pressurized to obtain a microcavity structure on the PMMA, and the silicon wafer template and PMMA are mechanically peeled off to obtain Non-degradable replica stamp;
  • PMMA polymethyl methacrylate
  • step B3 the mass ratio of PDMS, silk fibroin and cross-linking agent is 5-10:0.6-60:1, and the cross-linking agent is hydrogen-containing silicone oil.
  • the present application provides the above device, or the application of the device prepared according to the above method in cell cluster culture.
  • This device is suitable for:
  • Stem cell culture and induced differentiation such as embryonic stem cells and mesenchymal stem cells are cultured in clusters and further induced to differentiate, etc.;
  • Multi-cell co-culture such as endothelial cells, fibroblasts and hepatocytes, pancreatic islet cells co-culture, tissue fragments and mesenchymal stem cells co-culture in clusters, etc.
  • the cells suitable for culturing in this device can be selected from one or more of the following cells: embryonic stem cells, pluripotent stem cells, induced pluripotent stem cells, stem cells derived from various organs, progenitor cells derived from various organs, Mesenchymal stem cells, cells derived from various stem cells, fibroblasts derived from various organs, epithelial cells derived from various organs, epidermal cells derived from various organs, endothelial cells derived from various organs, and various organ sources muscle cells, amniotic cells, cone cells, nerve cells, blood cells, red blood cells, white blood cells, platelets, vascular cells, phagocytes, immune cells, lymphocytes, eosinophils, basophils, plasma cells, mast cells , antigen presenting cells, cells of the mononuclear phagocyte system, melanocytes, chondrocytes, bone-derived cells, smooth muscle cells, skeletal muscle cells, cardiomyocytes, secretory cells, adipocyte
  • the cells are particularly preferably stem cells, more preferably embryonic stem cells or mesenchymal stem cells.
  • This device can collect clusters by simple operation.
  • hydrophobic materials can be selected to prepare microcavity array substrates, or the substrate materials can be treated with surfactants to weaken the adhesion of cells to the substrates.
  • the detachment and collection of cell clusters can be completed by pipetting, which is convenient for subsequent operations.
  • the device can ensure the stable growth of cell clusters through a suitable substrate, conduct long-term culture and downstream differentiation studies of cell clusters, and be further applied to cell therapy, high-throughput model construction, organoid construction, drug screening, tissue regeneration and In vitro artificial systems, etc.
  • the device provided in this application includes a substrate with a microcavity array and a commercial multi-well culture plate; each microcavity forms a space for the growth of cell clusters, and can also have non-vertical walls, and the properties of cell clusters obtained through shape and volume control.
  • the device provided by the present application can prepare cell clusters conveniently and on a large scale, and can be used repeatedly; the elastic modulus of the substrate can be adjusted to adapt to the conditions required by the co-culture of various cells and various cells.
  • the cell clusters prepared by this device have high activity, uniform size, complete shape, controllable performance and good biological performance, and can be used for cell therapy, high-throughput model construction, multi-cell co-culture, stem cell differentiation, organoid construction, drug Screening, tissue regeneration and in vitro artificial systems.
  • the large-scale cell cluster preparation device has the characteristics of reusability, non-vertical wall surface, rapid large-scale preparation, regulated physicochemical properties, and high-throughput culture.
  • the large-scale cell cluster preparation device in this application is reusable.
  • the large-scale cell cluster preparation device in the present application can be reused after cleaning and sterilization, and can maintain the precision of the micropattern and the sterile environment required for cultivation.
  • the device may have non-vertical walls.
  • the device can be prepared by using a non-vertical wall template obtained by etching technology, which makes up for the shortage of only vertical wall microcavity prepared by photolithography technology or one-step template method.
  • the device can be obtained by a rapid, controllable and large-scale preparation method.
  • the device prepares the micro-cavity substrate by a two-step template method, avoids the reversal of the male and female molds of the micro-cavity template, and can be applied to complex molds and can also be prepared and obtained on a large scale.
  • the device can adjust its physicochemical properties as required. By regulating the physical and chemical factors such as cell cluster size, substrate material properties, mechanical strength, etc., the homogeneous growth, long-term culture and downstream research of cell clusters can be realized. It can be used for the culture and differentiation of different cell types and different cell sizes. And function maintenance, the detachment and collection of cell clusters can also be completed.
  • the device can realize high-throughput preparation of cell clusters.
  • the large-scale cell cluster preparation device in this application can realize the shape and size of various microcavities on the same template and the same substrate by means of computer-aided design and micro-patterning technology, so as to realize different cell cluster sizes under the same culture conditions.
  • the comparison with distribution is of great significance for flux studies and screening.
  • FIG. 1 is a schematic diagram of the apparatus for preparing cell clusters and the culture of cell clusters in the present application.
  • FIG. 2 is a schematic diagram of different microcavity shapes of the apparatus for preparing cell clusters of the present application. Wherein, 1) to 4) are different microcavity shapes.
  • FIG. 3 is a schematic diagram of the template method process during the preparation of the device of the present application.
  • FIG. 4 shows the differentiation of adipose stem cells in the cell cluster preparation device according to the preferred embodiment of the present application.
  • A is the light microscope image of adipose stem cells
  • B is the light microscope image of islet-like cells after differentiation
  • FIG. 5 shows the co-culture of islet-like cells and endothelial cells in the cell cluster preparation device according to the preferred embodiment of the present application.
  • FIG. 6 is the difference in culturing cells between the PDMS and silk fibroin mixed substrate and the pure PDMS substrate in the comparative example of the present application.
  • the present application provides a device and method for large-scale preparation of cell clusters.
  • micron-scale cell/co-culture cell clusters can be obtained, and the prepared cells/co-culture cell clusters have high activity and large size. It has the characteristics of uniformity, complete shape, controllable performance and good biological performance.
  • the device is reusable, and a suitable substrate can be used to ensure the stable growth of cell clusters for long-term culture and downstream differentiation studies.
  • the detachment and collection of cell clusters can also be completed as required for subsequent work.
  • tens of thousands of cells/co-cultured cell clusters with high activity, uniform size, complete shape, controllable performance and good biological performance can be obtained at one time through a microcavity structure with adjustable size and a substrate with suitable stiffness. It meets various requirements for downstream applications such as cell therapy, high-throughput model construction, multi-cell co-culture, stem cell differentiation, organoid construction, drug screening, tissue regeneration, and in vitro artificial systems.
  • the schematic diagram of the device provided in this application is shown in FIG. 1 .
  • the cell cluster preparation device provided in the present application is mainly composed of a substrate with a microcavity array.
  • Each microcavity of the microcavity array substrate provided by the present application is a space for cell growth, and its shape and volume affect the properties and physiological functions of the cell clusters.
  • the shape can be cylindrical, square, rectangular, triangular, diamond, hexagonal, inverted cone and irregular shape, etc., especially the non-vertical wall surface such as inverted cone is the priority,
  • the upper surface area of the microcavity is 0.04-1 mm 2
  • the depth of the microcavity is 100-500 ⁇ m, which roughly determines the size and space of the cell cluster growth.
  • the schematic diagram of the microcavity can be seen in Figure 2.
  • microcavity array contained about 1000 to 5000 microcavities, depending on the size of the microcavity and the overall size of the template.
  • the array pattern and size can be the same or different, and the arrangement can be neat and uniform, or customized.
  • Substrate arrays can contain microcavities of different shapes, sizes, and arrangements.
  • the cell cluster preparation device provided in this application can be used with commercial conventional multi-well culture plates (eg, 6-well plate, 12-well plate, 24-well plate, etc.).
  • the elastic modulus of the microcavity substrate of the device is 0.1 MPa to 10 MPa, and the elastic modulus can be adjusted by the composition, concentration and curing characteristics of the microcavity substrate material.
  • the device is reusable, and can be cleaned, sterilized and used again after one use, cell clusters are collected, and the micro-pattern accuracy and sterility can be maintained.
  • the present application provides a preparation method of the above cell cluster preparation device.
  • the device preparation method includes the following steps:
  • stamps For the liquid or semi-solid stamp material, the stamp material is covered on the template by the template method, and then peeled off from the template after curing to obtain a re-molded stamp.
  • stamp material such as polymethyl methacrylate
  • thermoplastic solid materials using the hot pressing method, the mold micro-pattern and the stamp material (such as polymethyl methacrylate) are laminated, pressurized, heated, and then mechanically or chemically peeled off to obtain a stamped stamp.
  • a variety of cells can be successively planted on the microcavity substrate to realize the cultivation of multi-cell co-culture cell clusters with controllable size and biological properties.
  • the microcavity template is a self-defined, reusable template with a microcavity array.
  • the microcavity template can be selected from a commercialized product, such as the multi-well culture plate Agreewell TM 400 (Stemcell) with a 400 ⁇ m microwell array at the bottom, or a custom template can be selected, using a microchannel well-known in the art. Patterning methods such as dry etching, wet etching, laser cutting engraving, micro-pattern mold forming, etc. are custom-made in combination with computer-aided design.
  • the material of the microcavity template can be silicon, aluminum, iron, tin, glass, polymethyl methacrylate, polydimethylsiloxane, polycaprolactone, polytrimethylene carbonate, polytetrafluoroethylene, poly Ethylene oxide, polyethylene vinyl acetate, polydioxanone, polyether ether ketone, etc.
  • the microcavity template can controllably adjust the shape, size and quantity of the microcavity on the microcavity template, and the shape can be cylindrical, square column, rectangular column, triangular column, diamond column, hexagonal column, inverted cone and no Regular shape, etc., especially the non-vertical wall surface such as inverted cone is the priority, the upper surface area of the microcavity is 0.04 ⁇ 1mm 2 , the depth of the microcavity is 100 ⁇ 500 ⁇ m, and the number is about 1000 ⁇ 5000, depending on the size of the microcavity and the template Overall size is determined.
  • the shape and size of the microcavity template need not be uniform.
  • the overall size and shape of the micro-chamber template generally depends on the commercial conventional multi-well culture plate used. It is generally circular, with a diameter equal to or slightly smaller than the pore size of the multi-well culture plate. The thickness does not determine the final substrate thickness. It ranges from 100 ⁇ m to 2 cm.
  • the mold-turning stamp is a degradable or non-degradable material used for intermediate mold-turning, obtained by a template method or a hot-pressing method, and its pattern is the male mold of the template microcavity (female mold), which is used for two-step mold-turning. , rubbing onto the substrate to prepare the microcavity substrate.
  • the shapes of the stamps can be cylindrical, square, rectangular, triangular, diamond, hexagonal, inverted cone and irregular, etc. Most of them are protruding structures, and the upper surface area of the structure is 0.04 ⁇ 1 mm 2 , the height is 100 to 500 ⁇ m, the number is about 1000 to 5000, and the shape and size are not necessarily uniform.
  • the degradable materials used for the stamp can be gelatin, gelatin derivatives, agar, agarose, Sacrificial materials such as F-127, polyvinyl alcohol, polyethylene glycol, etc., are convenient to be removed by sacrificial methods such as heating and water solubility after the subsequent preparation of the substrate.
  • the non-degradable material used for the stamping can be at least one of polymethyl methacrylate, epoxy resin, phenolic resin, polyvinyl chloride resin, unsaturated polyester resin, gypsum, silica gel, and the like.
  • the mass percentage concentration of the stamp material is 0.1% to 80%, preferably 1% to 25%.
  • the microcavity substrate is the substrate for culturing cell clusters.
  • Available materials for microcavity substrates are polypropylene, polystyrene, polyacrylamide, polylactic acid, polyhydroxy acid, polylactic acid alkyd, polydimethylsiloxane, polyanhydride, polyester, polyamide, Polyamino acid, polyacetal, polycyanoacrylate, polyurethane, polypyrrole, polyester, polymethacrylate, polyethylene, polycarbonate, polyethylene oxide, silk fibroin, silk fibroin derivative Compounds, Chitosan, Gelatin, Gelatin Derivatives, Alginate, Agar, Matrigel, Collagen, Collagen Derivatives, Hyaluronic Acid, Hyaluronic Acid Derivatives, Cellulose, Cellulose-Derived Materials, Proteoglycans, Proteoglycans At least one of derivatives, glycoproteins, glycoprotein-derived materials, laminin, fibronectin, and fibrin, etc
  • the mass percentage concentration of the base material may be 0.1% to 80%, preferably 1% to 25%.
  • the forming and fixing of the micro-cavity substrate is a two-step overmold using the overmold stamp. Cover the unmolded and fixed base material on the stamp, and ensure that the base material is closely attached to the stamp by vacuum defoaming, standing, etc., and then use a cross-linking agent to cure according to the characteristics of the base material and stamp material. , thermal curing, light curing, UV curing, pH adjustment curing and other methods to complete the forming and fixing of the base material, and obtain a microcavity substrate equivalent to a microcavity template.
  • extracellular matrix components that are conducive to the adhesion and growth of cell clusters such as collagen, matrigel, proteoglycan, glycoprotein, hyaluronic acid, laminin, can be added to the microcavity substrate as needed. or fibronectin.
  • the mass percentage concentration of the extracellular matrix material is 0.1%-80%, preferably 1%-25%.
  • the elastic modulus of the microcavity substrate is 0.1 MPa to 100 MPa, which is adjusted by the composition, concentration and curing characteristics of the substrate material.
  • the composition and concentration of the base material can be selected according to the characteristics of the cultured cell clusters.
  • the ratio of curing agent can be adjusted for materials cured by cross-linking agent.
  • the curing time and curing temperature can be adjusted for thermally cured materials. In general, the greater the proportion of curing agent, the higher the curing temperature, the longer the curing time, and the greater the light intensity, the greater the elastic modulus of the obtained substrate.
  • the cross-linking agent used can be hydrogen-containing silicone oil, silane coupling agent, divalent cation, genipin, glutaraldehyde, adipic acid dihydrazide, epichlorohydrin, carbodiimide, thrombin and its derivatives At least one of such, preferably hydrogen-containing silicone oil.
  • the concentration of the cross-linking agent used is 0.1 mM to 10 M, preferably 1 mM to 100 mM.
  • the base material and the crosslinking agent solution are mixed in a volume ratio of 1000:1 to 1:1000, preferably 10:1 to 1:10.
  • the thermal curing temperature of the base material is 10-100°C.
  • the light intensity of the base material is 0.5-1000 lx.
  • the pH value of the base material is 3-12.
  • the biodegradable mold stamp After the microcavity substrate is molded and fixed, the biodegradable mold stamp needs to be sacrificed and removed according to its material properties.
  • the stamp material is temperature-sensitive, such as gelatin, gelatin derivatives, agar, agarose and other high-temperature dissolving materials can be removed by heating, such as Low-temperature dissolving materials such as F-127 can be removed by freezing, and water-soluble materials such as polyvinyl alcohol and polyethylene glycol can be removed by water-dissolving.
  • the non-degradable mold stamp can be mechanically peeled off after the microcavity substrate is formed and fixed.
  • the substrate with microcavity array is obtained by two-step overmolding, and has the same microcavity array as the above-mentioned microcavity template, and the shape can be cylindrical, square column, rectangular column, triangular column, diamond column, six Prismatic, inverted cone and irregular shapes, etc., especially the non-vertical wall such as inverted cone is the priority, the upper surface area of the microcavity is 0.04 ⁇ 1mm 2 , the depth of the microcavity is 100 ⁇ 500 ⁇ m, and the number is 1000 ⁇ 5000 Left and right, shape and size need not be uniform.
  • the microcavity substrate has a certain array of microcavities.
  • the shape and volume of each microcavity constitute the physical space for the growth of cell clusters. Together with the number of cells to be planted, the size of the cell clusters is determined, which in turn affects the functional expression of the cell clusters.
  • the substrate with the microcavity array is adhered to the conventional cell culture plate to form a whole, and the construction methods include but are not limited to the following two:
  • microcavity substrate After designing and preparing the microcavity substrate of the required size and shape, cut it into an appropriate shape, and embed it in a commercial conventional multi-well culture plate. Using the pressure and the adhesion of the microcavity substrate material, the microcavity substrate is connected to the multi-well culture dish. The surface is connected to form a whole, which can be used immediately after sterilization or stored for later use.
  • micro-pattern base material and the mold-turning stamp on a commercial conventional multi-hole culture plate at the same time, and remove the mold-turning stamp after the micro-pattern base material is formed, to obtain an integrated micro-pattern base-multi-purpose culture plate. After sterilization treatment Can be used immediately or saved for later use.
  • Polymethyl methacrylate was purchased from Taobao e-commerce Baibang acrylic processing store, with an average molecular weight of about 2 million Daltons.
  • Polydimethylsiloxane (PDMS) was purchased from Dow Corning Company, item number 7450507, which is a two-component silicone rubber with a viscosity of 5500cps (25°C) before mixing and a viscosity of 3900cps (25°C) after mixing.
  • the basic components are mainly dimethyl silicone oil and platinum-based catalyst.
  • the curing agent is mainly hydrogen-containing silicone oil.
  • Silk fibroin was purchased from Sigma-Aldrich Company, item number 5154, with an average molecular weight of 100KDa.
  • Example 1 The method of making a cell cluster preparation device with a degradable stamp
  • the microcavity template is a silicon wafer with an inverted tapered microcavity array prepared by wet etching, and a circular template with an overall diameter of 3 cm is prepared.
  • the template is an inverted tapered array containing 1500 microcavities. The distance is 200 ⁇ m, the microcavity is an inverted cone with a square upper surface, the upper surface area is 0.25 mm 2 , and the depth is 500 ⁇ m.
  • the imprinted stamp is made of agar material. Agar solution with a concentration of 5% was used, dissolved at high temperature and high pressure, and poured into the micro-cavity template while still hot, with 2ml of agar per pour, and vacuum was used to remove air bubbles before the agar solution solidified. After the agar has cooled and solidified, carefully remove the agar stamp. The preparation of 12 stamps is done here.
  • the basic components of PDMS (the main components are dimethyl silicone oil and platinum-based catalyst) and the curing agent (the main components are hydrogen-containing silicone oil) in the Sylgard 184 silicone elastomer kit (Dow Corning Company, product number 7450507) Mix well, add 100 mg of silk fibroin per gram of PDMS, stir well, and perform vacuum defoaming treatment.
  • the prepared 12 agar stamps were placed in a 60 mm diameter petri dish with the micropatterned surface facing up, and the PDMS-silk fibroin mixture was cast on the stamp surface overnight at room temperature.
  • the sacrificial agar was heated to obtain a substrate containing 12 microcavity arrays.
  • a hole puncher take out the part containing the microcavity array in the PDMS and silk fibroin mixture according to the pore size of a conventional 12-well petri dish, and cut it to an appropriate thickness as needed.
  • the resulting microcavity substrates are uniform in size and contain identical microcavity arrays.
  • the microcavity base was embedded in a 12-well petri dish, and the microcavity base was connected with the surface of the 12-well petri dish by using the pressure and the adhesion of PDMS itself.
  • the PDMS was subjected to high temperature and high pressure sterilization treatment, and the hydrophobic state was maintained after treatment.
  • the conditions of autoclaving are: 105°C, 0.15MPa for 30min.
  • the morphology, structure and properties of the prepared device are as follows:
  • the bottom of a conventional 12-well petri dish is a microcavity base composed of PDMS and silk fibroin, the whole base is a circle with a diameter of 3cm, the base surface is a microcavity array, the shape of the microcavity is an inverted square pyramid, and the upper surface area is 0.25mm 2 .
  • the depth is 500 ⁇ m
  • the array density is 214 micropores/cm 2
  • the distance between adjacent microcavities is 200 ⁇ m
  • the substrate elastic modulus is 5 MPa
  • the pH value is 7.4.
  • Example 2 The method of making a cell cluster preparation device with a non-degradable stamp
  • the microcavity template is a silicon wafer with an inverted tapered microcavity array prepared by wet etching, and a circular template with an overall diameter of 3 cm is prepared.
  • the template is an inverted tapered array containing 1500 microcavities. The distance is 200 ⁇ m, the microcavity is an inverted cone with a square upper surface, the upper surface area is 0.25 mm 2 , and the depth is 500 ⁇ m.
  • the non-degradable overmolded stamp is made of polymethyl methacrylate (PMMA).
  • PMMA polymethyl methacrylate
  • the micropatterned surface of the silicon wafer template is bonded to a polymethyl methacrylate (PMMA) sheet, heated to 130 °C, pressurized to 1 MPa, held for 30 s, and annealed to obtain a microcavity structure on the PMMA.
  • the silicon wafer template and PMMA were mechanically peeled off to obtain a non-degradable overmolded stamp.
  • the preparation of 12 stamps is completed here, and this stamp can be reused.
  • the basic components of PDMS in Sylgard 184 silicone elastomer kit (the main components are dimethyl silicone oil and platinum catalyst) and the curing agent (the main components are hydrogen-containing silicone oil) in the Sylgard 184 silicone elastomer kit are thoroughly mixed in a mass ratio of 5:1, and then add to each gram of PDMS. 100 mg of silk fibroin, fully stirred, and vacuum defoamed.
  • the prepared 12 PMMA stamps were placed in a 60mm diameter petri dish with the micropatterned surface facing up, and the PDMS-silk fibroin mixture was cast on the stamp surface overnight at room temperature.
  • the PMMA stamp and the PDMS-silk fibroin substrate were mechanically peeled off to obtain a substrate containing 12 microcavity arrays.
  • Using a hole puncher take out the part containing the microcavity array in the PDMS and silk fibroin mixture according to the pore size of a conventional 12-well petri dish, and cut it to an appropriate thickness as needed.
  • the resulting microcavity substrates are uniform in size and contain identical microcavity arrays.
  • the microcavity base was embedded in a 12-well petri dish, and the microcavity base was connected with the surface of the 12-well petri dish by using the pressure and the adhesion of PDMS itself.
  • the device is subjected to high temperature and high pressure sterilization treatment, and the hydrophobic state is maintained after treatment.
  • the conditions of autoclaving are: 105°C, 0.15MPa for 30min. Autoclaving does not destroy the spatial structure of the substrate.
  • the stamp is rinsed with alcohol and dried for later use.
  • the morphology, structure and properties of the prepared device are as follows:
  • the bottom of a conventional 12-well petri dish is a microcavity base composed of PDMS and silk fibroin, the whole base is a circle with a diameter of 3cm, the base surface is a microcavity array, the shape of the microcavity is an inverted square pyramid, and the upper surface area is 0.25mm 2 .
  • the depth is 500 ⁇ m
  • the array density is 214 micropores/cm 2
  • the distance between adjacent microcavities is 200 ⁇ m
  • the substrate elastic modulus is 5 MPa
  • the pH value is 7.4.
  • Example 3 Using a cell cluster preparation device to induce adipose stem cells to differentiate into islet cell clusters
  • DMEM medium and DMEM/F-12 medium were mixed in a 2:1 volume ratio, Nicotinamide (5mM), Activin A (4nM), Exendin-4 (20nM), Pentagastrin were added (20nM), hepatocyte growth factor (200pM), B-27 supplement (1%), N-2 supplement (1%), double antibody (1%).
  • This application improves the existing adipose stem cell-islet cell differentiation solution, increases the concentration of four types of growth factors that promote pancreatic differentiation, Activin A, Exendin-4, Pentagastrin, and hepatocyte growth factor, and reduces B -27 supplement and N-2 supplement, two types of medium components that maintain stem cell stemness, improve the efficiency of adipose-derived stem cells to differentiate into pancreatic cells.
  • T75 adipose stem cells ATCC
  • A is the morphological light microscope image of adipose stem cells
  • B is the morphological light microscope image of adipose stem cells after 6 days of differentiation in the cell cluster preparation device. Clusters with clear edges, uniform size and complete shape;
  • Harvest cells collect the supernatant from each well in the device; add 1 ml of PBS to each well by pipetting once, collect the liquid, and repeat the process 1 more time. Transfer to a 50ml centrifuge tube, centrifuge at 1000rpm for 3min, discard the supernatant, remove most of the single cells, and place it in a petri dish.
  • Immunofluorescence staining Cell clusters were washed with phosphate buffered saline (PBS); fixed with 4% paraformaldehyde for 30 minutes at room temperature, and then washed once with PBS for 5 minutes; with 0.3% Triton-X (Sigma, X100) on ice Permeabilized for 20 minutes, then washed once with PBS for 5 minutes; blocked with 5% bovine serum albumin (Multicell, 800-096-EG) for 1 hour, washed once with PBS for 5 minutes; The primary antibody, Pdx1 (Abeam, ab47383), was incubated at 4°C overnight.
  • PBS phosphate buffered saline
  • Pdx1 Abeam, ab47383
  • the microcavity array is used for cell culture, which is a quasi-3D environment.
  • the culture period is shorter, and cell clusters can be formed after 2 days; the clustering efficiency is higher, close to 100%, almost no single cells are produced; the differentiation efficiency is higher, nearly 80% of cells express islet-related proteins; cell clusters are obtained efficiently and conveniently, avoiding operations such as digestion, and reducing the loosening and apoptosis of cell clusters; the size of cell clusters is consistent It has good properties and can guarantee the biological properties of cell clusters.
  • adipose stem cells have strong adherence ability and are difficult to be cultured in suspension, and the device of the present application can be used to build a quasi-3D environment; the clustering efficiency is higher, almost no single cells are produced; the differentiation efficiency is higher; cell clusters The clusters are more compact and the contact between cells is more sufficient; the size of the cell clusters is consistent, which ensures the biological properties of the cell clusters.
  • Example 4 Co-cultivation of islet cells and endothelial cells using a cell cluster preparation device
  • adipose stem cells obtained by differentiation of adipose stem cells, and the differentiation method is similar to that of Example 3.
  • the adipose stem cells were cultured in DMEM medium supplemented with 10% FBS and 1% double antibody, and differentiated into islet-like cells from the adipose stem cell-islet-like cell differentiation medium.
  • DMEM medium and DMEM/F-12 medium were mixed at a ratio of 2:1, Nicotinamide (5mM), Activin A (4nM), Exendin-4 (20nM), Pentagastrin (20nM) were added ), hepatocyte growth factor (200pM), B-27 supplement (1%), N-2 supplement (1%), double antibody (1%).
  • Endothelial cells (PUMC-HUVEC-T1, Peking Union Cell Resource Center) were cultured in DMEM medium supplemented with 10% FBS and 1% double antibody (penicillin-streptomycin).
  • Co-culture medium Islet cell differentiation medium and endothelial cell fluid were mixed in a volume ratio of 1:1.
  • Figure 5 shows the co-culture of islet cells and endothelial cells in the cell cluster preparation device of Example 2.
  • A is the morphological light microscope image of islet-like cells and endothelial cells co-cultured. It can be seen that loose cells gradually aggregate to form tight clusters with clear edges and uniform size of cell clusters.
  • immunofluorescence staining was used to detect the expression of islet ⁇ -cell-specific marker proteins (such as Pdx1) and endothelial cell-specific marker proteins (such as CD31).
  • Harvest cells collect the supernatant from each well in the device; add 1 ml of PBS to each well by pipetting once, collect the liquid, and repeat the process 1 more time. Transfer to a 50ml centrifuge tube, centrifuge at 1000rpm for 3min, discard the supernatant, remove most of the single cells, and place it in a petri dish.
  • Immunofluorescence staining Cell clusters were washed with phosphate buffered saline (PBS); fixed with 4% paraformaldehyde for 30 minutes at room temperature, and then washed once with PBS for 5 minutes; with 0.3% Triton-X (Sigma, X100) on ice Permeabilized for 20 minutes, then washed once with PBS for 5 minutes; blocked with 5% bovine serum albumin (Multicell, 800-096-EG) for 1 hour, washed once with PBS for 5 minutes; Primary antibodies, Pdx1 (Abeam, ab47383) and CD31 (Abeam, ab24590), were incubated overnight at 4°C.
  • PBS phosphate buffered saline
  • CD31 Abeam, ab24590
  • Figure 5 shows the co-culture of islet cells and endothelial cells in the cell cluster preparation device of Example 2.
  • B is the immunofluorescence staining of the co-cultured cells
  • the islet cells in the cell cluster are Pdx1 positive
  • the endothelial cells are CD31 positive, which proves that the islet cells and endothelial cells can maintain the expression of differentiation function in the cell cluster preparation .
  • the microcavity array is used for cell culture, which is a quasi-3D environment.
  • the culture period is shorter, and cell clusters can be formed after 2 days; the clustering efficiency is higher, close to 100%, almost no single cells are produced; the differentiation efficiency is higher, nearly 80% of cells express islet-related proteins; the contact between different cells can be strengthened to achieve the purpose of co-culture; cell clusters are obtained efficiently and conveniently, avoiding operations such as digestion, Reduce the loosening and apoptosis of cell clusters; the size of cell clusters is consistent, and the biological properties of cell clusters can be guaranteed.
  • adipose stem cells have strong adherence ability and are difficult to be cultured in suspension, and the device of the present application can be used to build a quasi-3D environment; the clustering efficiency is higher, almost no single cells are produced; the differentiation efficiency is higher; cell clusters The clusters are more compact and the contact between cells is more sufficient; the size of the cell clusters is consistent, which ensures the biological properties of the cell clusters.
  • Example 2 a mixture of PDMS and silk fibroin was used as the microcavity substrate.
  • PDMS is a common material that can be used as an elastic substrate for cell culture and other fields.
  • This application uses the mixture of PDMS and silk fibroin as the base material, and has achieved remarkable technical effects:
  • Silk fibroin has good biocompatibility and can be used as a cell culture medium. After mixing with PDMS as a substrate, it can regulate cell growth. Its nano-scale porous structure enhances oxygen exchange and penetration, which is more conducive to rapid cell growth. proliferation.
  • Silk fibroin has hydrophobicity. As a mixed substrate, it is conducive to the shedding of cells from the substrate and facilitates the collection of cells without damage. Pure PDMS is used as the base material to prepare the device of the present application, and the recovery rate of cell clusters is 85%. The mixed system of PDMS and silk fibroin is used as the base material to prepare the device of the present application, and the recovery rate of cell clusters can reach 94%.

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Abstract

La présente invention concerne un dispositif de préparation de grappes de cellules, un procédé de construction correspondant et une application associée. Le dispositif comprend une plaque de culture cellulaire, et un substrat fixé dans un puits de la plaque de culture cellulaire et comportant un réseau de microcavités. Chaque microcavité forme un espace pour la croissance de la grappe de cellules, et peut également avoir une surface de paroi non verticale. Les propriétés des grappes de cellules sont obtenues par la régulation de la forme et du volume. Le procédé de préparation associé comprend les étapes suivantes : préparation d'un gabarit de microcavité à taille ajustable, puis obtention d'un substrat comportant un réseau de microcavités au moyen d'un retournement en deux étapes. Le dispositif selon la présente invention peut être utilisé pour préparer des grappes de cellules à grande échelle de manière commode, et peut être utilisé de manière répétée. Le module élastique du substrat peut être ajusté pour satisfaire aux exigences de co-culture de divers types de cellules et de multiples types cellulaires. Les grappes de cellules préparées à l'aide de ce dispositif présentent une activité élevée, une taille uniforme, une forme complète, des performances maîtrisables et de bonnes propriétés biologiques. Elles peuvent être utilisées pour la thérapie cellulaire, la construction de modèles à haut rendement, la co-culture multi-cellulaire, la différenciation des cellules souches, la construction d'organoïdes, le criblage de médicaments, la régénération des tissus et les systèmes artificiels in vitro.
PCT/CN2021/099718 2020-06-29 2021-06-11 Dispositif de préparation de grappes de cellules, procédé de construction correspondant et application associée Ceased WO2022001631A1 (fr)

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CN115491285A (zh) * 2022-09-23 2022-12-20 哈尔滨工业大学 可替换的类器官芯片、柔性制造类器官设备及方法
CN115491285B (zh) * 2022-09-23 2024-05-24 哈尔滨工业大学 可替换的类器官芯片、柔性制造类器官设备及方法
CN115895884A (zh) * 2022-11-30 2023-04-04 苏州大学 一种用于细胞三维培养的超疏水孔板，多器官微流控芯片及其应用

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