WO2013083073A1 - 细胞集落体外培养装置及其用途 - Google Patents
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Classifications
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0603—Embryonic cells ; Embryoid bodies
- C12N5/0606—Pluripotent embryonic cells, e.g. embryonic stem cells [ES]
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- C12M23/00—Constructional details, e.g. recesses, hinges
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- C12M23/10—Petri dish
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- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/34—Internal compartments or partitions
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- 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
- C12M25/14—Scaffolds; Matrices
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- C12N2502/00—Coculture with; Conditioned medium produced by
- C12N2502/13—Coculture with; Conditioned medium produced by connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
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- C12N2533/00—Supports or coatings for cell culture, characterised by material
- C12N2533/30—Synthetic polymers
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- C12N2533/00—Supports or coatings for cell culture, characterised by material
- C12N2533/50—Proteins
- C12N2533/54—Collagen; Gelatin
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- C12N2535/00—Supports or coatings for cell culture characterised by topography
- C12N2535/10—Patterned coating
Definitions
- the present invention relates to a cell colony in vitro culture apparatus and uses thereof. Specifically, the present invention relates to a cell colony in vitro culture apparatus and application thereof for regulating stem cell self-renewal and directed differentiation, a method for regulating stem cell self-renewal, a method for regulating directed differentiation, a method for forming a cell monolayer micropattern, and a cell for forming A method of multi-layer microclusters. Background technique
- Totipotent stem cell refers to stem cells with unlimited differentiation potential and can differentiate into all tissues and organs, including embryonic stem cells (ESC) and induced pluripotent stem cells (iPS).
- ESC embryonic stem cells
- iPS induced pluripotent stem cells
- Embryonic stem cells are isolated and cultured from early mammalian embryos and are characterized by developmental pluripotency, participation in the development of whole organisms, and the formation of various tissues and organs of the human body. Embryonic stem cells are obtained from the embryo of the gastrula stage, and further differentiated to form a specialized stem cell. Such as liver progenitor cells, bone marrow hematopoietic stem cells and the like. From a technical point of view, "all-round" embryonic stem cells are ideal for therapeutic cloning.
- iPS cells are common somatic cells after "initialization", but have similar functions as embryonic stem cells and can differentiate into various tissue cells. More importantly, it bypasses many ethical and legal obstacles that embryonic stem cell research has always faced, and its application prospects in the medical field are very broad.
- hESC human embryonic stem cells
- MEF mouse embryonic fibroblasts
- the extracellular matrix can also serve as a culture substrate to maintain self-renewal of hESC, such as one of matrigel, collagen, laminin, fibronectin, or a mixture thereof.
- the survival rate of hESC is low, so the classical method of subculture is to digest or chop its colonies into clusters containing about 100 cells.
- the key to operation is to keep the hESC in a state of cell clusters.
- this method is highly dependent on the quality and tactics of the operator and ultimately results in a random, heterogeneous, heterogeneous hESC colony of shape and size, but at the same time too large or too small cell clusters
- the size and cell composition of colonies regulate the differentiation-inducing factors and pluripotent maintenance factors, especially the Smadl signal directly related to colony size is the antagonism of embryonic stem cells derived from embryonic stem cells and embryonic stem cells.
- BMP2 bone morphogenetic protein-2
- GFF3 growth differentiation factor-3
- the spontaneous differentiation of embryonic stem cells can be rescued by regulating Smadl activation alone, suggesting that the size of hESC colonies can independently regulate stem cell differentiation and self-renewal, but can not regulate colony size under classical culture conditions, in random colony size, cell density and molecular size. Under the joint action of microenvironment, there must be uncontrollable spontaneous differentiation, which affects the proliferation and self-renewal of embryonic stem cells.
- the heterogeneity and size difference of pluripotent stem cell colonies also have an important influence on the differentiation fate of stem cells. Uncontrollable initial stem cell colonies usually lead to inconsistent or unstable differentiation results and differentiation efficiency, which is a common problem in the study of pluripotent stem cell differentiation. Through micropatteming technology, it is found that the smaller colonies differentiate into cardiomyocytes, and the trend of larger colonies to differentiate into neural cells is more obvious. The mechanical properties of pluripotent stem cell cultures are self-renewing. And the fate of differentiation also has an important impact. Studies have shown that when the elastic modulus of the substrate is about 0.6 kPa, it has a good maintenance effect on the self-renewal of mouse embryonic stem cells.
- This mechanical strength is similar to that in vivo; similarly, without the addition of chemical signal stimulation,
- the medium with higher mechanical strength has the effect of inducing differentiation of bone marrow mesenchymal stem cells into osteoblasts, and the mechanical strength is higher.
- Low substrate can induce stem cells to differentiate into adipocytes; more in-depth studies have shown that proper mechanical stress can inhibit the spontaneous differentiation of hESC and promote self-renewal, and can regulate hESC self-renewal when bidirectional cyclic stress and chemical signals work synergistically. And differentiation, is of great significance for research in the fields of stem cell differentiation and cell therapy.
- the most ideal culture platform for pluripotent stem cells should contain the following four factors under the premise of maintaining stem cell phenotype and normal karyotype: Controllable colony shape and size, physical and chemical properties adjustable medium bottom, single layer can be realized Adjustable and stable cell planting density has been achieved by various culture methods such as cell colonies and multi-layer cell clusters, and suitable subculture methods. Summary of the invention
- the present invention aims to solve at least one of the technical problems existing in the prior art.
- one of the objects of the present invention is to provide a culture substrate having a controllable colony shape and size, physical and chemical properties, a single layer of cell colonies, and a plurality of cell clusters, A cell colony in vitro culture device of a suitable passage method.
- the invention provides a cell colony in vitro culture device.
- the cell colony in vitro culture apparatus comprises: a micropatterned template having a micropattern cavity; a viscous hydrogel, the viscous hydrogel being formed in the micropatterning a lower surface of the template, wherein the micropatterned cavity defines a cell colony growth space.
- the inventors have surprisingly found that homogenized growth of cell colonies can be achieved using the cell colony in vitro culture apparatus of the present invention.
- the cell colony in vitro culture apparatus of the present invention integrates a micropatterned template and a viscous hydrogel, and can realize the shape and size of the cell colony, and the cell single layer or multilayer growth mode.
- the mechanical strength and chemical composition of the adherent substrate, growth factors and chemical signals, co-cultured cells and other factors are regulated, and the control means for each factor are independent of each other, and do not interfere with each other; cell proliferation and differentiation can be achieved.
- the in-situ culture is carried out to greatly enlarge the operation, and at the same time, it can reduce the treatment and damage of the cells; the preparation process can be easily carried out with the commercial culture, without changing the original operation steps and details of the cell culture and It can maintain a good aseptic environment; make full use of micro-patterning technology, and can easily construct a variety of cell co-culture environments at the same time, providing a convenient and practical means for high-throughput and high-content model construction and screening research.
- the cell colony in vitro culture apparatus of the present invention may further have the following additional technical features: According to an embodiment of the present invention, the cell colony in vitro culture apparatus of the present invention further comprises: a hard substrate, the hard The substrate is on the lower surface of the viscous hydrogel.
- the cell colony in vitro culture apparatus of the present invention further comprises: porous culture The porous culture is placed under the viscous hydrogel and embedded in the micropatterned template.
- the viscous hydrogel is formed by crosslinking by at least one selected from the group consisting of natural biomaterials and synthetic biomaterials.
- the natural biomaterial is at least one selected from the group consisting of gelatin, gelatin derivatives, alginate, alginate derivatives, agar, matrigel, collagen, proteoglycan , glycoproteins, hyaluronic acid, laminin and fibronectin.
- the synthetic biomaterial is at least one selected from the group consisting of: polypropylene, polystyrene, polyacrylamide, polylactic acid, polyhydroxy acid, polylactic acid alkyd copolymer , polydimethylsiloxane, polyacid liver, polyacrylate, polyamide, polyamino acid, polyacetal, polycyanoacrylate, polyurethane, polypyrrole, polyester, polymethacrylate , polyethylene, polycarbonate and polyethylene oxide.
- the micropatterned template is formed from polymethyl methacrylate.
- the viscous hydrogel is formed of gelatin-methyl methacrylate and a photoinitiator, wherein the photoinitiator is 2-hydroxy-4-(2-hydroxyethoxy) ) -2-methylpropiophenone.
- the gelatin-methyl methacrylate is a polymer of gelatin and methyl methacrylate in a ratio of lg of the gelatin to 1 ml of the methyl methacrylate.
- the viscous hydrogel is prepared by the following steps: dissolving the gelatin-methyl methacrylate in a DMEM culture solution to obtain a gelatin-methyl methacrylate solution, wherein The gelatin-methyl methacrylate solution has a gelatin-methyl methacrylate content of 5 g of the gelatin-methyl methacrylate per 100 ml of the gelatin-methyl methacrylate solution;
- the photoinitiator is dissolved in anhydrous ethanol to obtain a photoinitiator solution, wherein the photoinitiator solution is contained in an amount of 10 g of the photoinitiator per 100 ml of the photoinitiator solution.
- the viscous hydrogel contains at least one selected from the group consisting of an extracellular matrix component, a growth factor, and a chemical signal molecule.
- the extracellular matrix is applied to the upper surface of the viscous hydrogel.
- the extracellular matrix component is at least one selected from the group consisting of: Protoplast, gelatin, matrigel, proteoglycan, glycoprotein, hyaluronic acid, laminin or fibronectin.
- the viscous hydrogel is embedded with growth factors and chemical signal molecules.
- the viscous hydrogel has a thickness of 1-100 ⁇ m.
- the present invention also provides the use of the above-described cell colony in vitro culture apparatus for regulating stem cell self-renewal.
- the present invention also provides the use of the above-described cell colony in vitro culture apparatus for regulating the differentiation of stem cells.
- stem cells include pluripotent stem cells and other types of stem cells.
- a cell colony in vitro culture apparatus provided by the present invention comprises a micropatterned template having a plurality of micropattern cavities and a viscous hydrogel under the micropatterned template; the viscous hydrogel The glue is integrally bonded to the micropatterned template, the viscous hydrogel forming a bottom of the micropattern cavity; the shape and volume of each of the micropattern cavities of the micropatterned template is determined
- the physical space cell growth density, culture time and culture mode of cell colony growth determine the growth mode of cells (single or multi-layer).
- the cell colony in vitro culture apparatus may further comprise a hard substrate under the viscous hydrogel, or a porous layer located under the viscous hydrogel and embedded in the micropatterned template. to cultivate.
- the viscous hydrogel is made of a natural biomaterial and/or a synthetic biomaterial that can be crosslinked to form a hydrogel
- the natural biomaterial being selected from at least one of the following materials Species: gelatin, gelatin derivatives, alginate, alginate derivatives, agar, matrigel, collagen, proteoglycans, glycoproteins, hyaluronic acid, laminin and fibronectin; At least one of the following materials: polypropylene, polystyrene, polyacrylamide, polylactic acid, polyhydroxy acid, polylactic acid copolymer, polydimethylsiloxane, polyacid liver, polyacrylate , polyamides, polyamino acids, polyacetals, polycyanoacrylates, polyurethanes, polypyrroles, polyesters, polymethacrylates, polyethylenes, polycarbonates and polyethylene oxides.
- the material used as the micropatterned template is specifically polymethyl methacrylate.
- the material for the viscous hydrogel specifically gelatin-methyl methacrylate and photoinitiator Irgacure 2959 (12959, 2-hydroxy-4-(2-hydroxyethoxy) -2-methylpropiophenone) composition.
- the gelatin-methyl methacrylate is a polymer of gelatin and methyl methacrylate in a ratio of lg of the gelatin to 1 ml of the methyl methacrylate.
- the gelatin-methyl methacrylate can be specifically prepared as follows: lg gelatin powder is added to 10 mL of DPBS solution, heated in a water bath at 50 ° C, and uniformly stirred until completely melted, to 0.5 mL.
- the viscous hydrogel may be specifically prepared according to the method comprising the following steps: 1) dissolving the gelatin-methyl methacrylate in a DMEM culture solution to obtain gelatin-methyl methacrylate. a solution, wherein the gelatin-methyl methacrylate solution has a gelatin-methyl methacrylate content of 5 g of the gelatin-methyl methacrylate per 100 ml of the gelatin-methyl methacrylate solution; 2) dissolving the photoinitiator 2-hydroxy-4-(2-hydroxyethoxy)-2-methylpropiophenone in absolute ethanol to obtain a photoinitiator solution, as described in the photoinitiator solution
- the content of 2-hydroxy-4-(2-hydroxyethoxy)-2-methylpropiophenone is 10 g of the 2-hydroxy-4-(2-hydroxyethoxy group) per 100 ml of the photoinitiator solution. -2-methylpropiophenone; 3) The photoinitiator solution is mixed with the gelatin-methyl
- the DMEM broth is commercially available, such as the product of Invitrogen under the product number 11965092.
- an extracellular matrix component in preparing the viscous hydrogel, may also be coated on the upper surface of the viscous hydrogel as needed.
- the extracellular matrix component comprises collagen, gelatin, matrigel, proteoglycan, glycoprotein, hyaluronic acid, laminin, fibronectin.
- growth factors and chemical signals can also be incorporated into the viscous hydrogel.
- the viscous hydrogel has a thickness of usually about 1 to 100 ⁇ m.
- the cell colony in vitro culture apparatus may be constructed as follows: the micropatterned template device is placed in the porous culture, and a viscous hydrogel-based solution containing a crosslinking agent is described The bottom surface of the micropatterned template is uniformly injected into the porous culture, and the micropatterned template and the porous culture surface are connected into a whole by the crosslinking action of the crosslinking agent and the adhesion force of the viscous hydrogel.
- a cell colony in vitro culture device is formed which is a micropatterned template-substrate hydrogel layer-culture surface from top to bottom.
- homogenous growth of cell colonies can be achieved by using the cell colony in vitro culture apparatus provided by the present invention.
- the pluripotent stem cells are human embryonic stem cells; the directed differentiation into human embryonic stem cells differentiates into hepatocyte-like cells.
- the present invention also provides a method of regulating stem cell self-renewal.
- the method comprises: culturing the stem cells using a cell colony in vitro culture device as described above.
- the present invention also provides a method of regulating the directed differentiation of stem cells.
- the method comprises: culturing the stem cells using a cell colony in vitro culture device as described above.
- stem cells include pluripotent stem cells and other types of stem cells.
- the present invention also provides a method of forming a cell monolayer micropattern.
- the method comprises: culturing the cell using the cell colony in vitro culture device of the present invention as described above to form a monolayer cell micropattern, wherein the cell is at least one selected from the group consisting of a stem cell and an adult cell. kind.
- the invention also provides a method of forming a multi-layered microclusters of cells.
- the method comprises: culturing the cell using the cell colony in vitro culture apparatus of the present invention as described above to form a multi-layer cell microcluster, wherein the cell is at least selected from the group consisting of a stem cell and an adult cell.
- the method comprises: culturing the cell using the cell colony in vitro culture apparatus of the present invention as described above to form a multi-layer cell microcluster, wherein the cell is at least selected from the group consisting of a stem cell and an adult cell.
- the cell colony in vitro culture device provided by the invention integrates the micropatterned template and the viscous hydrogel to realize the shape and size of the cell colony, the single or multi-layer growth mode of the cell, the mechanical strength and chemical adhesion of the adherent substrate.
- the regulation of various factors such as components, growth factors and chemical signals, co-cultured cells, etc., and the means of regulation of each factor are independent of each other and do not interfere with each other.
- the cell colony in vitro culture device provided by the invention can realize the in-situ progress of cell proliferation culture and induced differentiation culture, thereby greatly enlarging the operation and reducing the treatment and damage of the cells.
- the preparation process of the cell colony in vitro culture device provided by the invention is convenient to use with the commercial culture, and the original operation steps and details of the cell culture need not be changed, and the aseptic environment can be maintained.
- the cell colony in vitro culture device provided by the invention fully utilizes the micro-patterning technology, and can conveniently construct a plurality of cell co-culture environments, and provides a convenient and practical means for high-throughput and high-content model construction and screening research. .
- FIG. 1 is a schematic diagram of a micro-patterned template design according to an embodiment of the present invention, wherein four circles of different diameters in the figure represent a diameter of 300 ⁇ , 500 ⁇ , ⁇ or sequentially according to a diameter from small to large.
- 2 is a schematic structural view of a cell colony in vitro culture apparatus according to an embodiment of the present invention, wherein 2 is a micropatterned template, 3 is a base hydrogel layer, 4 is a cell colony, and 6 is a micropattern cavity; 3 is a schematic structural view of a cell colony in vitro culture apparatus according to another embodiment of the present invention, wherein 2 is a micropatterned template, 3 is a base hydrogel layer, 4 is a cell colony, 5 is a hard substrate, and 6 is a hard substrate.
- FIG. 4 is a schematic view showing the structure of a cell colony in vitro culture apparatus according to still another embodiment of the present invention, wherein 1 is culture, 2 is a micropatterned template, 3 is a base hydrogel layer, and 4 is a cell colony. 6 is a micro-patterned cavity;
- FIG. 5 is a schematic structural view of a cell colony in vitro culture apparatus according to still another embodiment of the present invention, wherein 1 is a culture, 2 is a micro-patterned template, and 3 is a base hydrogel layer, 4
- For cell colonies 5 is a hard substrate, 6 is a micro-patterned cavity;
- Figure 6 is a diagram showing the growth of monolayer cell micropatterns in a cell colony in vitro culture apparatus according to an embodiment of the present invention, wherein (A) is a micropatterned hydrogel substrate after disassembling the upper micropattern template, (B) For the formation of a homogeneous monolayer cell micropattern for murine fibroblasts (NIH/3T3), (C) for single-layer cell micropattern live/dead fluorescent staining, showing good cell activity (green fluorescence indication) Living cells, red fluorescence indicates dead cells);
- Figure 7 is a diagram showing the formation of NIH/3T3 homogeneous multi-layer cell microclusters in a cell colony in vitro culture apparatus according to an embodiment of the present invention, wherein (A) is a multilayer microscopic cluster observed under a light microscope. (B) A slice diagram showing the growth of multi-layer cell microclusters by three-dimensional reconstruction of laser confocal microscopy, (C) Observing the growth of multi-layer cell microclusters by three-dimensional reconstruction of laser confocal microscopy Figure
- Figure 8 is a diagram showing the formation of hESC multi-layer cell microclusters in a cell colony in vitro culture apparatus according to an embodiment of the present invention, wherein (A) is a macroscopic observation of hESC multi-layer cell microclusters by alkaline phosphatase staining, The pluripotent cells were purple-red by alkaline phosphatase-positive staining, (B) microscopic observation of hESC multi-layer microclusters, the micro-pattern diameter was 800 micrometers, (C) microscopic observation of hESC multilayer microclusters In the case of alkaline phosphatase staining, a purple-red positive stain showed pluripotent hESC.
- A is a macroscopic observation of hESC multi-layer cell microclusters by alkaline phosphatase staining, The pluripotent cells were purple-red by alkaline phosphatase-positive staining
- B microscopic observation of hESC
- the invention provides a cell colony in vitro culture device.
- the cell colony in vitro culture apparatus of the present invention comprises: a micropatterned template and a viscous hydrogel.
- the micropatterned template has a micropatterned cavity defining a cell colony growth space, and a viscous hydrogel is formed on a lower surface of the micropatterned template.
- the cell colony can be uniformly grown by using the cell colony in vitro culture apparatus of the present invention.
- the cell colony in vitro culture apparatus of the present invention utilizes a micropatterned template and a viscous hydrogel to achieve the shape and size of the cell colony, the mechanical strength of the adherent substrate, and The chemical components, growth factors and chemical signals, co-cultured cells and other factors are regulated, and the control means for each factor are independent of each other and do not interfere with each other; the cell proliferation culture and induced differentiation culture can be realized in situ, so that the operation Larger tube size, at the same time, can reduce the treatment and damage of cells; preparation process cartridge, easy to use with commercial culture, without changing the original operation steps and details of cell culture and maintaining a good aseptic environment Make full use of micro-patterning technology, and be able to construct a variety of cell co-culture environments at the same time, providing convenient and practical means for high-throughput and high-content model construction
- the cell colony in vitro culture apparatus may comprise: a micropatterned template 2 and a viscous hydrogel 3.
- the micropatterned template 2 may have a micro-patterned cavity 6 which may be formed on a lower surface of the micro-patterned template 2, wherein the micro-patterned cavity 6 may define Cell colony 4 growth space.
- the cell colony in vitro culture apparatus of the present invention may further comprise: a hard substrate 5 which may be located on a lower surface of the viscous hydrogel 3.
- the material of the hard substrate 5 is not particularly limited, and it may be made of a material such as glass or plastic.
- the cell colony in vitro culture apparatus of the present invention may further comprise: a porous culture 1 which may be located under the viscous hydrogel 3 and embedded in the template 2 .
- the viscous hydrogel 3 may be formed by crosslinking by at least one selected from the group consisting of natural biomaterials and synthetic biomaterials.
- the above natural biomaterial may be at least one selected from the group consisting of gelatin, gelatin derivatives, alginate, alginate derivatives, agar, matrigel, collagen, proteoglycan , glycoproteins, hyaluronic acid, laminin and fibronectin.
- the synthetic biomaterial may be at least one selected from the group consisting of polypropylene, polystyrene, polyacrylamide, polylactic acid, polyhydroxy acid, and polylactic acid copolymer.
- polydimethylsiloxane polyacid liver, polyacrylate, polyamide, polyamino acid, polyacetal, polycyanoacrylate, polyurethane, polypyrrole, polyester, polymethacrylate , polyethylene, polycarbonate and polyethylene oxide.
- the micropatterned template 2 may be formed of polymethyl methacrylate.
- the viscous hydrogel 3 can be formed from gelatin-methyl methacrylate and a photoinitiator, wherein the photoinitiator can be 2-hydroxy-4-(2-hydroxyethoxy)-2- Methyl phenylacetone.
- the gelatin-methyl methacrylate may be a polymer of gelatin and methyl methacrylate in a ratio of lg gelatin to 1 ml of methyl methacrylate.
- the viscous hydrogel 3 can be prepared by the following steps:
- the gelatin-methyl methacrylate is dissolved in a DMEM culture solution to obtain a gelatin-methyl methacrylate solution, wherein the gelatin-methyl methacrylate solution has a gelatin-methyl methacrylate content of 5 g of gelatin-methyl methacrylate per 100 ml of gelatin-methyl methacrylate solution;
- the photoinitiator is dissolved in absolute ethanol to obtain a photoinitiator solution, wherein the photoinitiator solution has a photoinitiator content of 10 g of photoinitiator per 100 ml of the photoinitiator solution;
- the photoinitiator solution and the gelatin-methyl methacrylate solution are mixed at a volume ratio of 1:20; finally, gelatin-methyl methacrylate is crosslinked by ultraviolet rays to obtain a viscous 7j gel 3 .
- the viscous hydrogel 3 may contain at least one selected from the group consisting of an extracellular matrix component, a growth factor, and a chemical signal molecule.
- the extracellular matrix may be applied to the upper surface of the viscous hydrogel 3.
- the extracellular matrix may be at least one selected from the group consisting of collagen, gelatin, matrigel, proteoglycan, glycoprotein, hyaluronic acid, laminin or fibronectin.
- growth factors and chemical signal molecules can be incorporated into the viscous hydrogel 3.
- the viscous hydrogel 3 may have a thickness of 1-100 ⁇ m.
- the viscous hydrogel 3 can serve as a growth substrate for supporting cultured cells (e.g., pluripotent stem cells) or feeder cells (e.g., mouse embryonic fibroblasts).
- cultured cells e.g., pluripotent stem cells
- feeder cells e.g., mouse embryonic fibroblasts
- the present invention also provides a cell colony in vitro culture apparatus. It should be noted that the features and effects described below for the cell colony in vitro culture apparatus are of course applicable to the above-described cell colony in vitro culture apparatus.
- the cell colony in vitro culture apparatus comprises a micropatterned template 2 having a plurality of micropattern cavities 6 and a viscous hydrogel 3 located under the micropatterned template 2;
- the hydrogel 3 is crosslinked with the micropatterned template 2 as a whole; the shape and volume of each micropatterned cavity 6 of the micropatterned template 2 determines the physical space in which the cell colony 4 grows.
- the cell colony in vitro culture apparatus of the present invention may further comprise a hard substrate 5 located under the viscous hydrogel 3 or under the viscous hydrogel 3 and embedded in the micropatterned template 2, as needed. Porous culture 1.
- the upper micro-patterning template 2 can be selectively disassembled or fixedly used to adapt Period of cell monolayer growth, multi-layer growth, cell co-culture or pluripotent stem cell differentiation.
- the micropatterned template 2 can be obtained by micro-patterning methods well known in the art, such as laser cutting and engraving, micro-patterning mold forming, etc., combined with computer-aided design and manufacturing techniques, can adjust cell colonies. 4 growth space and micro-patterned template 2 overall shape and size.
- the shape and size of various micro-pattern cavities 6 can be conveniently implemented by computer-aided design and manufacturing techniques, such as circular, square, rectangular, diamond, trapezoidal, and various types.
- the size is l ( ⁇ m ⁇ 2cm, which determines the physical space for cell colony 4 growth; the overall size and shape of the micropatterned template 2 is usually determined by the size of the culture 1 used, and the shape is mostly regular. Round, diameter equal to or slightly smaller than the diameter of the culture 1, can be easily prepared by computer-aided design and manufacturing technology micro-patterned template 2 of the required size and shape; ⁇ graphical template 2 thickness to save material for easy use Suitable, generally ranging from 10 (m to 2 cm).
- the viscous hydrogel 3 is a growth substrate that supports cultured cells (e.g., totipotent stem cells) or feeder cells (e.g., mouse embryonic fibroblasts).
- the viscous hydrogel 3 is made of a natural biomaterial and/or a synthetic biomaterial that can be crosslinked to form a hydrogel, wherein natural biomaterials such as gelatin and its derivatives, alginate and Its derivatives, agar, matrigel, collagen, proteoglycan, glycoprotein, hyaluronic acid, laminin, fibronectin, etc., synthetic biomaterials including polypropylene, polystyrene, polyacrylamide, polylactic acid, polyhydroxyl Acid, polylactic acid alkyd copolymer, polydimethylsiloxane, polyacid liver, polyacrylate, polyamide, polyamino acid, polyacetal, polycyanoacrylate, polyurethane, polypyrrole, Polyester, polymethacryl
- the material forming the substrate may be a mixture of one or more of the above materials.
- the type and amount of the crosslinking agent depend on the type and amount of the substrate biomaterial, and it is premised on sufficient crosslinking.
- the material used as the micro-patterning template 2 may specifically be polymethyl methacrylate; used as a material of the viscous hydrogel 3, specifically by gelatin-methyl methacrylate And photoinitiator Irgacure 2959 (12959, 2-hydroxy-4-(2-hydroxyethoxy)-2-methylpropiophenone); the gelatin-methyl methacrylate is gelatin and methyl methacrylate A polymer having a ratio of lg of the gelatin to 1 ml of the methyl methacrylate was used.
- the gelatin-methyl methacrylate can be specifically prepared as follows: lg gelatin powder is added to 10 mL of DPBS solution, heated in a water bath at 50 ° C, and uniformly stirred until completely melted, to 0.5 mL / Min slowly added lmL methyl methacrylate, reacted for 2h, then removed, the solution was cooled to room temperature, diluted with 40mL DPBS solution, dialyzed in deionized water at 60 °C for 1 week with a molecular weight cutoff of 8000-12000, centrifuged The supernatant was freeze-dried for 1 week to form a white fluffy foamy solid, which was stored at -80 °C.
- the viscous hydrogel 3 can be prepared in a manner comprising the following steps:
- gelatin-methyl methacrylate solution Dissolving the gelatin-methyl methacrylate in DMEM culture solution to obtain gelatin-methyl methacrylate solution
- the gelatin-methyl methacrylate solution in the gelatin-methyl methacrylate solution is contained in an amount of 5 g of the gelatin-methyl methacrylate per 100 ml of the gelatin-methyl methacrylate solution;
- the DMEM broth is commercially available, such as the product of Invitrogen under the product number 11965092.
- an extracellular matrix component may be coated on the upper surface of the viscous hydrogel 3 as needed; the extracellular matrix component includes collagen, gelatin, matrigel, proteoglycan, glycoprotein, Hyaluronic acid, laminin or fibronectin; growth factors and chemical signals can also be chimeric in the viscous hydrogel 3.
- the amount of the viscous hydrogel 3 material is determined according to the use area of the culture 1, and the conditions of the culture 1 or the hard substrate 5 are uniformly covered, generally ranging from 1 ⁇ l to 10 ⁇ 1. .
- the method for constructing the cell colony in vitro culture apparatus of the present invention includes, but is not limited to, the following four types:
- the device After designing and preparing the micro-patterned template 2 requiring size and shape, the device is placed in the porous culture 1, and the substrate biomaterial is uniformly injected into the culture 1 along the bottom surface of the template to be cross-linked, and the cross-linking effect and viscosity of the cross-linking agent are utilized.
- the viscous action of the hydrogel 3 connects the micropatterned template 2 and the surface of the porous culture 1 to form a whole. After the crosslinking is sufficient, the excess crosslinking agent and bubbles are washed away, and used immediately or at a low temperature for use.
- the sheet-like hard substrate 5 and the micro-patterned template 2 are vertically aligned or adhered from the bottom, and then the base biomaterial is uniformly injected and cross-linked along the gap therebetween to form a hard base-water from bottom to top.
- the three-layer monolithic structure of the gel-template, the three-layer monolithic structure is embedded in the culture 1, and the residual cross-linking agent and the bubbles are thoroughly washed away and used immediately or at a low temperature. Save for use.
- the cell colony in vitro culture device can achieve homogenous growth of cell colonies, and can be used for regulating self-renewal and directed differentiation of pluripotent stem cells and other types of stem cells.
- This mainly includes regulation of physical factors such as colony shape, size, and mechanical strength of the substrate, and chemical elements such as substrate components, growth factors, and chemical signals, and biological elements such as co-cultured cells.
- the method for realizing the physical factors such as the shape, size, and mechanical strength of the substrate is as follows:
- the template material is excised to form a micro-pattern cavity 6, which constitutes a physical space for the growth of the pluripotent stem cell colony 4, and thus the colony
- the shape and size are determined by the characteristic shape and size of the micropatterned template cavity 6.
- the shape and size of various micro-pattern cavities 6 can be conveniently realized by computer-aided design and manufacturing techniques, and different colony shapes, sizes, and distributions under the same culture conditions can be realized by cutting cavities 6 of different physical properties on one template. The comparison is important for high-throughput research and screening.
- the shape of the template cavity 6 described above is such as a circle, a square, a rectangle, a diamond, a trapezoid, and various irregular shapes, and the size is l ( ⁇ m ⁇ 2cm).
- the mechanical strength of the substrate is mainly determined by the type of the material of the base hydrogel 3 (the material of the viscous hydrogel 3), the amount thereof, and the degree of crosslinking thereof.
- the material of the base hydrogel 3 The greater the amount and the higher the degree of crosslinking, the greater the mechanical strength.
- the mechanical strength of the substrate can be intervened by a process such as artificial regulation or spontaneous regulation during use of the cell colony in vitro culture device, and artificially controlling the mechanical strength of the substrate mainly through the substrate hydrogel material.
- One or more cross-linking implementations such as gelatin-methyl methacrylate as the base hydrogel material, can be physically crosslinked by ultraviolet light, blue light, etc., and chemical cross-linking methods such as genipin and glutaraldehyde. Conveniently achieving secondary or multiple cross-linking in time and in situ, the result is generally an increase in the mechanical strength of the substrate hydrogel material; spontaneous regulation of the mechanical strength of the substrate hydrogel material is primarily through cell culture during cell culture - Material interactions and material degradation are achieved, the result of which is generally a reduction in the mechanical strength of the substrate hydrogel material.
- the combination of spontaneous regulation and artificial regulation can achieve the mechanical strength of the substrate hydrogel material in situ, at any time, multiple times to adjust and control, to meet the requirements of pluripotent stem cell self-renewal and directed differentiation, such as
- pluripotent stem cell self-renewal and directed differentiation such as
- the lower basal mechanical strength is conducive to the self-renewal and pluripotency maintenance of stem cells.
- the pluripotent stem cells proliferate to form a homogenous set, they can be induced to differentiate, such as to hepatocyte-like cells, cardiomyocytes, Pancreatic cells are differentiated in the same direction, and the higher mechanical strength of the substrate at this time favors the directed differentiation of stem cells.
- self-renewal and directed differentiation induction culture of pluripotent stem cell colonies can be achieved in situ by the present cell colony in vitro culture device and its regulation method.
- the amount of the base hydrogel material to be used depends on the area of use of the culture 1, and is based on the condition of uniformly covering the surface of the culture substrate 1 or the hard substrate 5, generally ranging from ⁇ l Oml.
- the type and amount of the cross-linking agent depend on the type and amount of the base biomaterial, and it is premised on sufficient cross-linking.
- the specific method of the present invention for regulating chemical components such as substrate components, growth factors, chemical signal molecules, and the like is as follows: due to different biological and physical and chemical properties of the upper micropatterned template 2 and the lower viscous hydrogel 3, With different vertical heights and subsequent operations such as blowing and suction, the surface of the base hydrogel material is generally a direct attachment surface for the growth of pluripotent stem cells or feeder cells, which can be achieved by coating various biomaterials on the surface of the substrate hydrogel.
- Modification and regulation such as coating collagen, gelatin, matrigel, proteoglycan, glycoprotein, hyaluronic acid, laminin, fibronectin and other extracellular matrix components to improve cell adhesion and better maintain Self-renewal of stem cells, or promotion of directional differentiation of pluripotent stem cells.
- the coating material herein is not limited to the above kinds of materials, and the specific kind and amount to be used depend on the kind of cultured cells and the purpose of cultivation.
- the present invention can conveniently realize the compounding and sustained release of various growth factors and chemical signal molecules in a cell colony culture apparatus.
- growth factors and chemical signal molecules can be chimeric in the base hydrogel layer 3 by factor immobilization, thereby realizing bionic, efficient, and controllable sustained release of various factors, and improving factors.
- the effect efficiency and time of action provide a convenient and practical model for in vitro simulation of in vivo development and have important economic benefits.
- the growth factor and the chemical signal molecule may also participate in the cell culture process in the form of soluble molecules, and the sequential and controllable factor addition may be conveniently performed to meet the requirements of the program-induced differentiation culture. .
- the selection of growth factors and chemical signal molecules herein depends on the type of cultured cells and the purpose of the culture.
- the present invention can be applied to various uses such as proliferation culture and differentiation culture of pluripotent stem cells, and a convenient cell co-culture route is an important guarantee for achieving the above functions.
- the specific methods of the present invention for regulating biological elements such as co-cultured cells include, but are not limited to, the following two categories:
- the feeder layer cells may be selectively grown in the cell colony in vitro culture device to be attached to the surface hydrogel surface of the micropattern cavity 6 or the micropattern template 2, Then, the pluripotent stem cells are planted on the cell colony in vitro culture device to form a physiological environment co-cultured with the feeder cells, which is beneficial to the self-renewal and pluripotency maintenance of the pluripotent stem cells.
- the selection and planting density of the feeder cells herein are well known in the art, and the selection depends on the type of pluripotent stem cells.
- the stem cell differentiation differentiation induction culture can be performed in situ, and the helper cell co-culture can be selectively performed at this time, and the co-culture method includes retaining the micro-pattern Templates and disassembly of micro-graphic templates.
- the helper cells can be directly implanted in the cell colony in vitro culture device while retaining the template, and the helper cells are preferentially attached to the non-cavity surface of the micropatterned template 2 to form helper cells.
- helper cells implanted at this time are preferentially attached to the blank surface of the basal hydrogel layer 3 which is not occupied by the pluripotent stem cell colony 4, forming a contact co-culture environment of the helper cells and the pluripotent stem cell colony.
- the selection of the above two methods may be based on factors such as the type of the substrate hydrogel material, the direction of differentiation of the totipotent stem cells, and the type of the helper cells.
- the type of helper cells and planting density are well known in the art, and are mainly determined by factors such as the number of pluripotent stem cell colonies, cell density, and stem cell differentiation direction.
- the present invention also provides the use of the above-described cell colony in vitro culture apparatus for regulating stem cell self-renewal.
- the present invention also provides the use of the above-described cell colony in vitro culture apparatus for regulating the differentiation of stem cells.
- the present invention also provides a method of regulating stem cell self-renewal.
- the method may comprise: culturing the stem cells using the cell colony in vitro culture device as described above.
- the present invention also provides a method of regulating the directed differentiation of pluripotent stem cells and other types of stem cells.
- the method may comprise: culturing the stem cells using the cell colony in vitro culture device as described above.
- stem cells are not particularly limited and may include pluripotent stem cells and other types of stem cells.
- the present invention also provides a method of forming a cell monolayer micropattern.
- the method comprises: culturing the cell using the cell colony in vitro culture device of the present invention as described above to form a monolayer cell micropattern, wherein the cell is at least one selected from the group consisting of a stem cell and an adult cell. kind.
- the invention also provides a method of forming a multi-layered microclusters of cells.
- the method comprises: culturing the cell using the cell colony in vitro culture apparatus of the present invention as described above to form a multi-layer cell microcluster, wherein the cell is at least selected from the group consisting of a stem cell and an adult cell.
- the method comprises: culturing the cell using the cell colony in vitro culture apparatus of the present invention as described above to form a multi-layer cell microcluster, wherein the cell is at least selected from the group consisting of a stem cell and an adult cell.
- the cell colony in vitro culture device of the present invention is particularly suitable for the cultivation of pluripotent stem cells, and can also be used for the cultivation of various other cells.
- the invention is illustrated by the following specific examples, which are to be construed as merely limiting the scope of the invention.
- the experimental methods used in the following examples are all conventional methods unless otherwise specified.
- the materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
- Example 1 Production of a cell colony in vitro culture device
- a cell colony in vitro culture apparatus was prepared as follows:
- the upper micropatterned template 2 was prepared by laser cutting.
- the specific operation is as follows: Using a Rayjet laser engraving machine, a polymethyl methacrylate plate (Zumbo Technology) having a thickness of 0.5 cm is cut to form an upper micropatterned template 2 .
- the micro-patterned template is designed by software AutoCAD: The template is circular, the diameter is 22.1mm, and the center is evenly distributed in the square. 8 ⁇ 8 cavities with a diameter of 300 ⁇ , 500 ⁇ , ⁇ or 1500 ⁇ are used as micro Graphical cells (such as pluripotent stem cells) culture space, the distance between the centers of two adjacent cavities is 1500 ⁇ .
- the main processing parameters of the laser engraving machine are: cutting energy 100%, cutting times 2, cutting line speed 10%.
- the processed polymethyl methacrylate template was rinsed twice with deionized water, ultrasonically cleaned for 30 min, and then heat treated to improve the flatness: a flat and clean heating plate was set at a temperature of 90 ° C and heat treated under pressure. Polymethyl methacrylate template for 45 min.
- the heat-treated polymethyl methacrylate template was rinsed with sterile three-steamed water for 30 minutes, wiped clean with a sterile cleaning paper, and then UV-sterilized for 45 minutes on both sides to obtain a micro-patterned template 2 . spare.
- the photoinitiator (12959, 2-hydroxy-4-(2-hydroxyethoxy)-2-methylpropiophenone, 106797-53-9, Yingli, China) was formulated to a concentration of 0.1 g with absolute ethanol. /ml photoinitiator solution, 0.2 ⁇ filter membrane was sterilized, dispensed in 1ml/tube and stored at 4 °C in the dark.
- the gelatin-methyl methacrylate was prepared as follows: lg gelatin (G6144, Sigma, USA) The powder was added to 10 mL of DPBS solution, heated in a water bath at 50 ° C, and evenly stirred until completely melted. Slowly add 1 mL of A at 0.5 mL/min. Methyl acrylate (276685, Sigma, USA), after 2h reaction, remove the solution, cool the solution to room temperature, dilute with 40mL DPBS solution, use dialysis bag with molecular weight cutoff of 8000-12000 in deionized water 60
- gelatin-methyl methacrylate prepared above was dissolved in DMEM medium (1 1965092, Invitrogen, USA), and dissolved at 60 ° C for 3 hours to form a gelatin-methyl methacrylate solution at a concentration of 0.05 g/ml. And then use
- the 0.2 ⁇ filter was filtered and dispensed into 1 ml/tube for use.
- the prepared photoinitiator solution having a concentration of 0.1 g/ml and a gelatin-methyl methacrylate solution having a concentration of 0.05 g/ml are mixed at 1:20, and stored in the dark, and then correlated. The operation is carried out in the dark.
- the micropatterned template 2 prepared above was embedded in the culture hole of 12-well plate culture 1 (Corning) in a ratio of 1:1, and the micro-graphic template was placed under the dark conditions.
- 2 bottom A 70 ⁇ l sterile gelatin-methyl methacrylate/photoinitiator solution was slowly injected, at which point a uniform layer of substrate biomaterial was formed between the bottom surface of the template and the culture surface.
- Example 2 Formation of multi-layer microclusters using the cell-raising device of Example 1 (taking NIH/3T3 cells as an example)
- NIH/3T3 cells were used as an example, and the multi-layer microclusters were formed by the cell colony in vitro culture apparatus prepared in Example 1 according to the following procedure:
- the MH/3T3 cells routinely cultured in a 75 cm 2 flask were digested with 0.25% trypsin and collected by centrifugation, and then planted in the culture apparatus prepared in Example 1 at a density of 4*10 6 /mL, and 1 mL of cells per well. After the suspension, after 24 hours, the upper film of the micro-pattern template was peeled off with tweezers, and the results are shown in Fig. 6. As can be seen from Figure 6, a micropatterned homogeneous cell cluster having approximately three layers of cell layers was formed. Hoechst nuclear staining showed homogeneity of microclusters and multi-layer cell distribution.
- Example 3 Culture of human embryonic stem cells using the cell colony in vitro culture apparatus of Example 1.
- MEF medium DMEM (11965092, Invitrogen) 225ml per 250ml MEF medium, fetal bovine serum (10099141, Invitrogen) 25ml, glutamate dipeptide (Glutamax I) (35050061, Invitrogen) 2.5ml, MEM non-essential amino acids (MEM NEAA ) (11 140050, Invitrogen) 2.5 ml, penicillin/streptomycin (Pen/strep) (100X (15140122, Invitrogen) 2.5 ml.
- Human embryonic stem cell culture medium knock out DMEM (10829018, Invitrogen) per 250ml culture medium
- Knockout serum replacer ( 10828028, Invitrogen ) 50ml, Glutamax I ( 35050061 , Invitrogen ) 2.5ml, MEM Non-essential amino acids ( MEM NEAA ) ( 11140050 , Invitrogen ) 2.5ml , Penicillin / Streptomyces Pen/strep (15140122, Invitrogen) 2.5ml, human basic fibroblast growth factor (hbFGF) (25 g/ml) (233-FB-025, R&D) 0.08ml.
- hbFGF human basic fibroblast growth factor
- mice were treated with pregnant horse serum gonadotropin (HOR-272, Shanghai Gaochuang) to make estrus and superovulation.
- HOR-272 pregnant horse serum gonadotropin
- mice Male and female mice were caged overnight at a ratio of 2:1.
- mice from 12.5-14.5 days of gestation were taken out as materials for further experiments described below.
- Pregnancy mice with the step of cervical dislocation (4) disinfected in a beaker containing 75% alcohol, dissected on a clean bench, first cut open the abdominal skin, expose the uterus, and then change the other
- a surgical instrument is used to lift the proximal cervix with an ophthalmology, separate the uterine mesentery, cut the uterine horn, remove the uterus, and place it in a culture containing PBS.
- mouse embryonic fibroblasts (MEF) were prepared.
- the inactivated MEF (hereinafter referred to as In-MEF) was uniformly planted in a cell colony in vitro culture apparatus constructed in Example 1 at a cell density of 2.5 ⁇ 10 4 /cm 2 , such as a cell colony stored at a low temperature aseptic storage.
- In-MEF The inactivated MEF
- the PBS was washed twice and then incubated at 37 ° C for 10 min before use.
- the cell colony in vitro culture device is planted with In-MEF, it is placed After standing in the incubator for 24 hours, the upper layer of the micro-pattern template was removed with tweezers, and the lml/well MEF medium was added to the cultured well wall twice to remove the cells on the surface of the template, leaving a micro-patterned cavity.
- In-MEF cells 1.5 ml/well of MEF culture solution was added along the wall of the culture well, and In-MEF cell culture was carried out in an incubator, and the results are shown in Fig. 7. As can be seen from Fig. 7, a single layer of MEF cell micropatterns was formed, and live/dead fluorescent staining showed good cell activity.
- Human embryonic stem cells H9 (Wicell) with a confluency of about 60-70% in a conventional culture in a six-well plate culture are uniformly passaged to a cell colony in vitro culture apparatus, and human embryonic stem cells H9 in a culture well of a six-well plate are placed.
- the cells were transferred to a culture well containing a 12-well plate of an In-MEF cell colony in vitro culture apparatus, that is, human embryonic stem cells H9 were planted at a ratio of about 3:1.
- human embryonic stem cell H9 was placed in a 5% carbon dioxide, 37 ° C incubator for 10 min, and the supernatant was slightly aspirated to remove the human embryonic stem cells H9 on the template surface, leaving the person in the micropatterned cavity.
- 1.5 ml/well human embryonic stem cell H9 medium was added along the cultured well wall, and MEF and human embryonic stem cell H9 were co-cultured in a 5% carbon dioxide, 37 ° C incubator, and changed daily, observed, and alkaline.
- the biological characteristics of human embryonic stem cell H9 were identified by phosphatase activity assay, specific surface antigen expression assay (such as SSEA-3, SSEA-4, T A-1-60), nuclear transcription factor assay (such as OCT4).
- the above-mentioned MEF cell culture and passage, conventional culture, passage and detection methods of human embryonic stem cells ⁇ 9 are well known in the art, and are preferably carried out using the current classical operation specifications.
- step 1 after co-cultivation for about 3 to 4 days, cultured human embryonic stem cells ⁇ 9 can be observed, and 8x8 homogenous human embryonic stem cells having a diameter of 300 ⁇ m, 500 ⁇ m, ⁇ or 1500 ⁇ are formed in the micropatterned cavity. ⁇ 9 colonies (identified by the biological characteristics). Then, using the obtained human embryonic stem cell ⁇ 9, the in situ human embryonic stem cell ⁇ 9 induced differentiation culture was carried out in the cell colony in vitro culture apparatus prepared in Example 1, and the cells were continuously differentiated while proliferating to form a multi-layer cell microclusters. For example, hepatic-like cells are induced to differentiate, and the specific steps are as follows:
- Programmed induced differentiation is guided by in vivo liver development and is divided into defined endoderm induction and hepatic progenitor cells. Stages of induction and induction of hepatocyte maturation.
- the induction medium at each stage was prepared based on the component culture solution.
- the composition of the component culture solution was: 50% IMDM medium (Iscove's modified Dulbecco's medium) and 50% F12 NUT-MIX, 7 g/ml insulin, 15 g/ml. Transferrin, 45 ( ⁇ mol/L thioglycerol and 5 mg/ml bovine serum albumin.
- the induction start time is recorded as the first day, and the differentiation method is: Days 1 ⁇ 2, with the addition of 10 ng/mL Stem cells were cultured with activin and 12 ng/ml human fibroblast growth factor 2 (FGF2), and changed daily. On days 3 to 5, 100 ng/mL activin, 20 ng/mL FGF2, 10 ng was added.
- Days 1 ⁇ 2 with the addition of 10 ng/mL Stem cells were cultured with activin and 12 ng/ml human fibroblast growth factor 2 (FGF2), and changed daily. On days 3 to 5, 100 ng/mL activin, 20 ng/mL FGF2, 10 ng was added.
- FGF2 human fibroblast growth factor 2
- LY294003 PI3K inhibitor component culture medium induced human embryonic stem cell H9 to differentiate into endoderm cells, changing daily; 6 ⁇ 8 days, adding 50 ng/
- the component culture medium of mL human fibroblast growth factor 10 (FGF10) induces the directed differentiation of the anterior definitive endoderm, and the fluid changes every day. On the 9th to 10th day, 50 ng/mL FGF10, 10-7 mol/L is added.
- Xanthate and ⁇ /L SB431542 TGF-Smads signaling pathway inhibitor
- the component culture medium induces directional differentiation to hepatic progenitor cells, and daily changes; on the 11th to 20th day, 30 ng/mL human fibroblast growth factor 4 (FGF4), 50 ng/mL hepatocyte growth factor (HGF) and 50 ng/mL epithelial growth factor (EGF) component culture medium induced differentiation into hepatocyte-like cells, changing every 2 to 3 days.
- FGF4 human fibroblast growth factor 4
- HGF hepatocyte growth factor
- EGF epithelial growth factor
- the cells differentiated by the above-described induced human embryonic stem cell H9 were able to express the liver-specific gene AFP and secrete albumin, thereby determining that human embryonic stem cell H9 was differentiated into hepatocyte-like cells.
- the human embryonic stem cell H9 cultured in the above step 1) was observed to form 8 ⁇ 8 homogenous human embryonic stem cells ⁇ 9 episodes of ⁇ in the micropatterned cavity, and induced by in situ co-culture in a cell colony in vitro culture apparatus. Differentiated culture, as described herein, is induced by hepatocyte-like cell co-culture.
- the human embryonic stem cell ⁇ 9 obtained in the above step 1) is subjected to in situ co-culture to induce differentiation culture in the cell colony in vitro culture apparatus prepared in Example 1, and the specific steps are as follows:
- Human Hepatic Stellate Cells (cat #5300, Sciencell) were cultured in a 37 ° C, 5% C0 2 saturated humidity incubator under the conditions of adding 10% FBS and 200 U/mL cyan DMEM medium of mycin.
- the upper micropatterned template was removed with tweezers, and cultured twice with human embryonic stem cell H9 whole culture solution, at which time the substrate hydrogel layer and micropatterned human embryonic stem cells were left in the culture. H9 colony.
- Hepatic stellate cells were resuspended in human embryonic stem cell H9 whole broth, planted in culture at a cell density of 2.5 ⁇ 10 4 /cm 2 , and changed after one day. At this time, stellate cells were preferentially attached to the stellate cells.
- the basal hydrogel layer forms a two-cell co-culture environment around human embryonic stem cell H9 colonies.
- induced embryonic stem cells were used to induce differentiation of human embryonic stem cells H9 into hepatocyte-like cells.
- the composition of the induced culture medium was: 50% IMDM medium and 50% F12 NUT-MIX, 7 g/ml insulin, 15 g was added.
- /ml transferrin, 45 ( ⁇ mol/L thioglycerol, 5 mg/ml bovine serum albumin, 50 ng/mL FGF4, 50 ng/mL HGF and 50 ng/mL EGF, change every 2 to 3 days.
- the cell colony in vitro culture apparatus was placed in a 37 ° C, 5% C0 2 saturated humidity incubator, and 1.5 ml per well was added.
- Example 4 Formation of hESC multi-layer cell microclusters using the cell colony in vitro culture apparatus of Example 1.
- hESC was cultured as described in Example 3.
- the human embryonic stem cell H9 (Wicell) with a confluency of about 60-70% in a conventional culture in a six-well plate culture is collected by conventional digestion, shearing, and centrifugation, and further digested into a single cell by an enzyme such as Accutase (sigma).
- the cells were seeded at a density of 2*10 6 /ml, and li ⁇ g/ml ROCK blocker Y27632 was added to the culture medium to increase the survival rate of hESC single cells.
- li ⁇ g/ml ROCK blocker Y27632 was added to the culture medium to increase the survival rate of hESC single cells.
- a homogeneous multi-layered hESC microclusters of high pluripotency are formed, and the results are shown in FIG.
- alkaline phosphatase positive staining showed that the cells were pluripotent.
- the cell colony in vitro culture device of the invention has a controllable colony shape and size, physical and chemical properties adjustable medium bottom, and can effectively realize a plurality of cultures such as a single layer cell colony, a multi-layer cell cluster, and a cell co-culture. In a way, and can be effectively used to regulate self-renewal and directed differentiation of pluripotent stem cells and other types of stem cells.
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US14/363,507 US20140295553A1 (en) | 2011-12-07 | 2012-12-07 | In-vitro cell colony culture device and use thereof |
EP12855702.2A EP2796541A1 (en) | 2011-12-07 | 2012-12-07 | In-vitro cell colony cultivation device and use thereof |
JP2014545085A JP2015501640A (ja) | 2011-12-07 | 2012-12-07 | 細胞コロニーの体外培養装置及びその使用 |
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WO2019078278A1 (ja) * | 2017-10-18 | 2019-04-25 | 国立大学法人京都大学 | 心筋細胞に分化させるための多能性幹細胞の製造方法 |
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WO2020203769A1 (ja) * | 2019-03-29 | 2020-10-08 | 積水化学工業株式会社 | 細胞培養用足場材料、細胞培養用容器、細胞培養用繊維及び細胞の培養方法 |
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IN2014KN01409A (zh) | 2015-10-23 |
CN103146572B (zh) | 2016-01-06 |
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CN103146572A (zh) | 2013-06-12 |
US20140295553A1 (en) | 2014-10-02 |
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