CN113061566A - Cell large-scale culture method and microcarrier used by same - Google Patents
Cell large-scale culture method and microcarrier used by same Download PDFInfo
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- 238000012136 culture method Methods 0.000 title claims description 17
- 239000000017 hydrogel Substances 0.000 claims abstract description 37
- 239000000499 gel Substances 0.000 claims abstract description 27
- 238000000034 method Methods 0.000 claims abstract description 26
- 239000002738 chelating agent Substances 0.000 claims abstract description 17
- BHPQYMZQTOCNFJ-UHFFFAOYSA-N Calcium cation Chemical compound [Ca+2] BHPQYMZQTOCNFJ-UHFFFAOYSA-N 0.000 claims abstract description 16
- 229910001424 calcium ion Inorganic materials 0.000 claims abstract description 16
- 239000011159 matrix material Substances 0.000 claims abstract description 16
- 229920001661 Chitosan Polymers 0.000 claims abstract description 15
- 229920001436 collagen Polymers 0.000 claims abstract description 12
- 238000001879 gelation Methods 0.000 claims abstract description 12
- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical group CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 claims abstract description 11
- 238000006243 chemical reaction Methods 0.000 claims abstract description 11
- 239000000661 sodium alginate Substances 0.000 claims abstract description 11
- 235000010413 sodium alginate Nutrition 0.000 claims abstract description 11
- 229940005550 sodium alginate Drugs 0.000 claims abstract description 11
- 108010035532 Collagen Proteins 0.000 claims abstract description 9
- 102000008186 Collagen Human genes 0.000 claims abstract description 9
- SQDAZGGFXASXDW-UHFFFAOYSA-N 5-bromo-2-(trifluoromethoxy)pyridine Chemical compound FC(F)(F)OC1=CC=C(Br)C=N1 SQDAZGGFXASXDW-UHFFFAOYSA-N 0.000 claims abstract description 6
- 229920001287 Chondroitin sulfate Polymers 0.000 claims abstract description 6
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- 108010067306 Fibronectins Proteins 0.000 claims abstract description 6
- 239000000648 calcium alginate Substances 0.000 claims abstract description 6
- 235000010410 calcium alginate Nutrition 0.000 claims abstract description 6
- 229960002681 calcium alginate Drugs 0.000 claims abstract description 6
- OKHHGHGGPDJQHR-YMOPUZKJSA-L calcium;(2s,3s,4s,5s,6r)-6-[(2r,3s,4r,5s,6r)-2-carboxy-6-[(2r,3s,4r,5s,6r)-2-carboxylato-4,5,6-trihydroxyoxan-3-yl]oxy-4,5-dihydroxyoxan-3-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylate Chemical compound [Ca+2].O[C@@H]1[C@H](O)[C@H](O)O[C@@H](C([O-])=O)[C@H]1O[C@H]1[C@@H](O)[C@@H](O)[C@H](O[C@H]2[C@H]([C@@H](O)[C@H](O)[C@H](O2)C([O-])=O)O)[C@H](C(O)=O)O1 OKHHGHGGPDJQHR-YMOPUZKJSA-L 0.000 claims abstract description 6
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- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 claims description 10
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- 108010037362 Extracellular Matrix Proteins Proteins 0.000 claims description 10
- FKNQFGJONOIPTF-UHFFFAOYSA-N Sodium cation Chemical compound [Na+] FKNQFGJONOIPTF-UHFFFAOYSA-N 0.000 claims description 10
- 210000002744 extracellular matrix Anatomy 0.000 claims description 10
- 238000002156 mixing Methods 0.000 claims description 9
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- 238000002360 preparation method Methods 0.000 claims description 7
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 claims description 6
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 claims description 6
- 238000012258 culturing Methods 0.000 claims description 6
- 239000004017 serum-free culture medium Substances 0.000 claims description 6
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 claims description 5
- 229910000019 calcium carbonate Inorganic materials 0.000 claims description 5
- 210000004748 cultured cell Anatomy 0.000 claims description 5
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- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 4
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- 230000001419 dependent effect Effects 0.000 claims description 4
- 230000005686 electrostatic field Effects 0.000 claims description 4
- 239000010452 phosphate Substances 0.000 claims description 4
- 229910052708 sodium Inorganic materials 0.000 claims description 4
- 239000001569 carbon dioxide Substances 0.000 claims description 3
- 229910002092 carbon dioxide Inorganic materials 0.000 claims description 3
- 238000005119 centrifugation Methods 0.000 claims description 3
- 230000035755 proliferation Effects 0.000 claims description 3
- 229910000030 sodium bicarbonate Inorganic materials 0.000 claims description 3
- 235000017557 sodium bicarbonate Nutrition 0.000 claims description 3
- 239000001509 sodium citrate Substances 0.000 claims description 3
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 claims description 3
- BVKZGUZCCUSVTD-UHFFFAOYSA-M Bicarbonate Chemical compound OC([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-M 0.000 claims description 2
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 claims description 2
- KRKNYBCHXYNGOX-UHFFFAOYSA-K Citrate Chemical compound [O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-K 0.000 claims description 2
- 230000002378 acidificating effect Effects 0.000 claims description 2
- 238000003501 co-culture Methods 0.000 claims description 2
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- 238000003306 harvesting Methods 0.000 claims description 2
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- 239000002609 medium Substances 0.000 claims description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 2
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 claims 1
- 239000011575 calcium Substances 0.000 claims 1
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- 239000007921 spray Substances 0.000 claims 1
- 239000000463 material Substances 0.000 abstract description 8
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- FHVDTGUDJYJELY-UHFFFAOYSA-N 6-{[2-carboxy-4,5-dihydroxy-6-(phosphanyloxy)oxan-3-yl]oxy}-4,5-dihydroxy-3-phosphanyloxane-2-carboxylic acid Chemical compound O1C(C(O)=O)C(P)C(O)C(O)C1OC1C(C(O)=O)OC(OP)C(O)C1O FHVDTGUDJYJELY-UHFFFAOYSA-N 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
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- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 229940072056 alginate Drugs 0.000 description 1
- 235000010443 alginic acid Nutrition 0.000 description 1
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/0068—General culture methods using substrates
- C12N5/0075—General culture methods using substrates using microcarriers
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2500/00—Specific components of cell culture medium
- C12N2500/05—Inorganic components
- C12N2500/10—Metals; Metal chelators
- C12N2500/12—Light metals, i.e. alkali, alkaline earth, Be, Al, Mg
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- C12N2531/00—Microcarriers
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2533/00—Supports or coatings for cell culture, characterised by material
- C12N2533/70—Polysaccharides
- C12N2533/72—Chitin, chitosan
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Abstract
The application discloses a microcarrier, which comprises gel molecules and an outer matrix molecule, wherein the gel molecules and the outer matrix molecule are mixed and then induced to carry out a gelation reaction to generate a hydrogel microcarrier; wherein the gel molecule is sodium alginate or calcium alginate, and the outer matrix molecule is one or more of collagen, matrigel protein, chondroitin sulfate, fibronectin and chitosan. The main component of the microcarrier in the application contains calcium alginate composite. The material has the greatest advantage that the material is easy to expand and deform in the presence of the calcium ion chelating agent, so that the contact area between the material and cells is reduced, the cells can fall off under slight physical shearing force, and the separation of the cells and microcarriers is realized. The whole process can realize cell separation without the participation of pancreatin.
Description
Technical Field
The application relates to the technical field of cell culture, in particular to a cell large-scale culture method and a used microcarrier.
Background
With the development of stem cell technology, cell therapy offers the possibility of radical treatment of many major diseases such as tumors, liver and kidney organ failure, neuro-endocrine system diseases such as parkinson, diabetes, and the like. However, the cell product used for clinical implantation needs to avoid the introduction of various proteins and enzymes in the preparation process, which brings difficulties to the large-scale cell culture work. Especially aiming at the large-scale culture of the anchorage dependent cells, the pancreatin digestion is needed to collect the cultured cells, the introduction of the pancreatin increases the subsequent process flow of cell product purification, and increases the production cost of the product.
Disclosure of Invention
The purpose of the application is to overcome the defects of the prior art and provide a cell large-scale culture method without trypsinization and a microcarrier for realizing the culture method.
In order to achieve the technical purpose, the technical scheme adopted by the application is as follows:
in a first aspect, a microcarrier comprises a gel molecule and an extracellular matrix molecule, wherein the gel molecule and the extracellular matrix molecule are mixed and then induce a gelation reaction to produce a hydrogel microcarrier; wherein the gel molecule is sodium alginate or calcium alginate, and the outer matrix molecule is one or more of collagen, matrigel protein, chondroitin sulfate, fibronectin and chitosan.
The dry powder of the hydrogel microcarrier can swell and deform when soaked in a solution containing a sodium ion and calcium ion chelating agent.
Specifically, the calcium ion chelating agent is any one of phosphate, carbonate, bicarbonate, citrate or EDTA.
In a second aspect, there is provided a method for preparing a microcarrier, which comprises the steps of:
respectively preparing a gel molecule solution and an outer matrix molecule solution;
mixing calcium carbonate powder with the gel molecular solution, and then mixing with the outer matrix molecular solution to form a premixed solution;
promoting the pre-mixed liquid to carry out gelation reaction by a fluid granulation technology to generate a hydrogel microcarrier;
and drying and sterilizing the hydrogel microcarrier to obtain dry powder of the microcarrier.
Preferably, the concentration of the gel molecule solution is 10-50g/L, and the concentration of the outer matrix molecule solution is 1-50 g/L.
More preferably, the concentration of the gel molecule solution in the premix is 10g/L or more.
Specifically, the fluid granulation technology is any one of a high-voltage electrostatic field technology, a gas orifice spraying technology, a micro-fluidic technology or an emulsification technology.
Further, after the hydrogel microcarrier is generated, adding 1-10g/L of chitosan solution, adding the outer matrix molecule solution, and performing polyelectrolyte complexation reaction to generate the hydrogel microcarrier with a shell-core structure. The volume ratio of the hydrogel microcarrier to the chitosan solution is 1: 2-10; the duration time of the polyelectrolyte complexation reaction is 2-60 minutes.
Further, after the hydrogel microcarrier is formed, the hydrogel microcarrier is placed in an acidic environment to produce carbon dioxide, resulting in a hydrogel microcarrier having a porous structure.
In a third aspect, a method for large-scale cell culture comprises the following steps:
preparing microcarrier and seed cell separately;
co-culturing the microcarrier and the seed cells by using a serum-free culture medium which does not contain a sodium ion and calcium ion chelating agent, so that the seed cells are adhered to the surface of the microcarrier for proliferation;
replacing serum-free medium containing sodium ion and calcium ion chelating agent when harvesting cells;
after the microcarrier is expanded and deformed, collecting cells in a physical mode;
wherein the microcarrier is the microcarrier or the microcarrier is the microcarrier obtained by the preparation method.
Preferably, the microcarrier and seed cell co-culture process needs periodic shaking to promote the adhesion of the seed cell to the microcarrier surface.
Further preferably, the culture process after replacement of the serum-free medium containing the sodium ion and calcium ion chelating agent requires periodic shaking to promote uniform suspension of the microcarriers to which the cells are adhered in the medium.
Specifically, the serum-free culture medium containing the sodium ion and calcium ion chelating agent comprises 100mmol/L sodium bicarbonate and 55mmol/L sodium citrate.
Optionally, the physically collecting cells comprises at least one of:
promoting the cells to be separated from the surface of the microcarrier by shaking or stirring;
separating the microcarriers and cells by a screen;
cells were enriched by centrifugation.
Preferably, the stirring speed is less than or equal to 100 r/min.
Alternatively, the seed cells are anchorage-dependent cultured cells.
Compared with the prior art, the method has the following advantages:
1. the main component of the microcarrier in the application contains calcium alginate composite. The material has the greatest advantage that in the presence of a calcium ion chelating agent (such as phosphate radical, carbonate radical, bicarbonate radical, citrate radical, EDTA and the like), the material is easy to expand and deform, so that the contact area between the material and cells is reduced, the cells can fall off under slight physical shearing force, and the separation of the cells and microcarriers is realized. The whole process can realize cell separation without the participation of pancreatin.
2. The microcarrier material is simultaneously compounded with a plurality of extracellular matrix materials to provide adhesion sites for cells, so that the cells can be adhered and proliferated on the surface of the microcarrier.
Drawings
FIG. 1 is a flow chart of a method of preparing a microcarrier according to the present application.
FIG. 2 is a flow chart of the cell mass culture method of the present application.
Detailed Description
The present application is described in further detail below with reference to the attached drawings and the detailed description.
The microcarriers of the present application consist essentially of gel molecules and extracellular matrix molecules. The gel molecules mainly refer to components that are cross-linked and polymerized to form a gel-like substance through a gelation reaction, and in this embodiment, alginate is preferably used, and specifically, sodium alginate and calcium alginate may be used. The extracellular matrix molecules mainly refer to components which can simulate the extracellular matrix, such as collagen, matrigel, chondroitin sulfate, fibronectin and chitosan, and can be single components or composite components according to the types of cultured cells; the presence of the extracellular matrix molecules allows cells to readily adhere to the surface of the microcarriers for proliferation. The microcarrier is easy to expand and deform in the presence of the calcium ion chelating agent, the contact area between the surface of the microcarrier subjected to expansion and deformation and cells is reduced, and the cells are easy to fall off from the surface of the microcarrier.
The preparation method of the microcarrier of the application is as follows:
respectively preparing a gel molecule solution and an outer matrix molecule solution;
mixing calcium carbonate powder with the gel molecular solution, and then mixing with the outer matrix molecular solution to form a premixed solution;
promoting the pre-mixed liquid to carry out gelation reaction by a fluid granulation technology to generate a hydrogel microcarrier;
and drying and sterilizing the hydrogel microcarrier to obtain dry powder of the microcarrier.
Further, the concentration of the gel molecule solution and the outer matrix solution affects the size of the hydrogel microcarrier, and a person skilled in the art can determine a specific value according to the experimental result, but preferably, the concentration of the gel molecule solution in the premix is kept above 10g/L to ensure that the gelation reaction is smoothly performed. The fluid granulation technology can be selected from high-voltage electrostatic field technology, gas orifice spraying technology, micro-fluidic technology or emulsification technology, and the emulsification technology can be further subdivided into emulsification-external gelation method, emulsification-internal gelation method and film emulsification method.
The preparation of microcarriers and the use of microcarriers in cell-scale cultivation methods according to the present application are further described below by way of specific examples.
Example one
Referring to fig. 1, the microcarrier is prepared as follows:
A. solution preparation: sodium alginate solution with concentration of 10-50 g/L; chitosan solution with the concentration of 1-10 g/L;
B. preparing a collagen solution, a chitosan solution, a matrigel solution, a chondroitin sulfate solution or a fibronectin solution with the concentration of 2-50 g/L;
C. mixing the sodium alginate solution prepared in the step A with calcium carbonate powder to prepare uniform suspension, and uniformly mixing the suspension with one or more of the collagen solution, the chitosan solution, the matrigel solution, the chondroitin sulfate solution or the fibronectin solution prepared in the step B, wherein the concentration of the sodium alginate in the mixed solution is kept above 10 g/L;
D. c, forming uniform liquid drops by the mixed liquid prepared in the step C through a fluid granulation technology, and carrying out a gelation reaction by calcium ion crosslinking and temperature change to obtain a spherical hydrogel microcarrier;
E. the spherical hydrogel microcarrier obtained in the step D can also be used as a core to further react with the chitosan solution prepared in the step A with the concentration of 1-10g/L, the volume ratio of the carrier to the chitosan solution is 1:2-1:10, the microcarrier is in a uniform suspension state in the chitosan solution, polyelectrolyte complex reaction is carried out for 2-60 minutes, namely a hydrogel film is formed on the surface of the core of the spherical hydrogel microcarrier, and the spherical hydrogel carrier with a core-shell structure is obtained after the physiological saline solution is cleaned;
F. and D, implanting the spherical hydrogel microcarrier prepared in the step D or the step E into an acid environment, and fully reacting with calcium carbonate powder in the carrier to generate carbon dioxide to generate the hydrogel microcarrier with a pore structure, wherein the pore structure of the hydrogel microcarrier can increase the surface area of the microcarrier, is favorable for increasing the cell adhesion area, and is also favorable for improving the material exchange efficiency in the hydrogel microcarrier.
G. And F, drying the spherical hydrogel carrier obtained in the step F in vacuum to obtain a dried microcarrier, and sterilizing the microcarrier by using high-pressure steam to obtain microcarrier dry powder for later use.
Example two
【1】 Preparing a sodium alginate solution with the concentration of 30 g/L;
【2】 Preparing a collagen solution with the concentration of 5g/L, and adjusting the concentration to be neutral by using NaOH;
【3】 Preparing a gel bath solution, wherein the gel bath contains calcium chloride (the concentration is 20 g/L);
【4】 Mixing the sodium alginate solution prepared in the step (1) with the collagen solution prepared in the step (2) in a volume ratio of 2: 1;
【5】 Forming stable jet flow by the mixed solution of the sodium alginate and the collagen prepared in the step (4) under a high-voltage electrostatic field, spraying the stable jet flow into the gel bath prepared in the step (3), and carrying out gelation reaction for 30 minutes at 37 ℃ in the gel bath to obtain the sodium alginate-collagen blended hydrogel microspheres;
【6】 And (5) sterilizing the sodium alginate-collagen blended hydrogel microspheres prepared in the step (5) under high pressure, and drying the sodium alginate-collagen blended hydrogel microspheres into microcarrier dry powder by a fluidized bed dryer for later use.
EXAMPLE III
A. The microcarrier obtained in the first embodiment or the second embodiment is cultured together with the cells to be cultured, and the cells to be cultured are uniformly adhered to the surface of the microcarrier by periodic shaking.
B. Preparing a serum-free medium containing specific ions: the serum-free medium contains sodium ions and one or more anions selected from phosphate radical, carbonate radical, bicarbonate radical, citrate radical and EDTA.
C. And D, culturing the microcarrier loaded with the cells obtained in the step A in a bioreactor, replacing the culture solution with the serum-free culture medium containing the sodium ion and calcium ion chelating agent prepared in the step B after the cells are expanded to a satisfactory degree, and periodically shaking after the culture solution is completely replaced to enable the microcarrier to be uniformly suspended in the solution prepared in the step B.
D. The microcarrier is expanded and deformed, cells are shed from the surface of the carrier into the solution under slight shearing force, the cells are physically separated through a screen with the aperture of 100 microns, the microcarrier is intercepted, and the cells in the supernatant are collected by centrifugation.
The process of the cell scale culture method is shown in FIG. 2, and the culture method is suitable for all anchorage-dependent cultured cells.
Example four
Taking the liver cell line HepG2 as an example, the cell scale culture method is used for culturing. The process is as follows:
the liver cell line HepG2 was inoculated onto microcarriers and cultured using a bioreactor.
Preparing a serum-free medium containing specific ions: contains 100mmol/L sodium bicarbonate and 55mmol/L sodium citrate.
Culturing liver cells on the microcarrier for 7 days, collecting the microcarrier, cleaning with normal saline, transferring to the serum-free culture medium containing specific ions prepared in the previous step, culturing in a bioreactor for 10 hours, stirring at the speed of 50 r/min, and collecting the cells after the microcarrier expands and the cells automatically fall off.
And (3) intercepting the expanded microcarrier by using a screen with the particle size of 100 microns in the mixture in the bioreactor, and centrifuging and collecting the cell suspension to obtain a cell product.
In summary, the cell mass culture method and the microcarrier used in the method are that cells are cultured on the surface of a microcarrier prepared from an extracellular matrix component such as sodium alginate and chitosan, the microcarrier and the cells are separated after the cells are cultured in a reactor for a certain time, specifically, a serum-free culture medium containing a sodium ion chelating agent and a calcium ion chelating agent is added into the microcarrier to expand the microcarrier, the adhesion degree of the cells and the surface of the microcarrier is reduced, the cells are separated, the expanded microcarrier is filtered and trapped by a screen, and the filtrate containing the cells is centrifuged to collect the cells.
The above embodiments are only preferred embodiments of the present application, but not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present application should be construed as equivalents and are included in the scope of the present application.
Claims (17)
1. A microcarrier comprising a gel molecule and an extracellular matrix molecule, wherein the gel molecule and the extracellular matrix molecule are mixed and then induced to undergo a gelation reaction to produce a hydrogel microcarrier; wherein the gel molecule is sodium alginate or calcium alginate, and the outer matrix molecule is one or more of collagen, matrigel protein, chondroitin sulfate, fibronectin and chitosan.
2. The microcarrier of claim 1, wherein the hydrogel microcarrier swells and deforms when soaked in a solution comprising a chelating agent for sodium and calcium ions.
3. The microcarrier of claim 2, wherein the calcium chelating agent is any one of phosphate, carbonate, bicarbonate, citrate, or EDTA.
4. A method for producing a microcarrier according to any one of claims 1 to 3, comprising the steps of:
respectively preparing a gel molecule solution and an outer matrix molecule solution;
mixing calcium carbonate powder with the gel molecular solution, and then mixing with the outer matrix molecular solution to form a premixed solution;
promoting the pre-mixed liquid to carry out gelation reaction by a fluid granulation technology to generate a hydrogel microcarrier;
and drying and sterilizing the hydrogel microcarrier to obtain dry powder of the microcarrier.
5. The method according to claim 4, wherein the concentration of the gel molecule solution is 10 to 50g/L, and the concentration of the outer matrix molecule solution is 1 to 50 g/L.
6. The method according to claim 4, wherein the concentration of the gel molecule solution in the premix is 10g/L or more.
7. The method of claim 4, wherein the fluid granulation technique is any one of high voltage electrostatic field technique, gas orifice spray technique, micro-fluidic technique, or emulsification technique.
8. The preparation method according to claim 4, wherein the hydrogel microcarrier is prepared by adding 1-10g/L of chitosan solution to perform polyelectrolyte complexation reaction to obtain the hydrogel microcarrier with a core-shell structure.
9. The method of claim 8, wherein the volume ratio of hydrogel microcarrier to chitosan solution is 1: 2-10; the duration time of the polyelectrolyte complexation reaction is 2-60 minutes.
10. The method of claim 4 or 8, wherein after the hydrogel microcarrier is formed, the hydrogel microcarrier is placed in an acidic environment to produce carbon dioxide, thereby forming a hydrogel microcarrier having a porous structure.
11. A cell scale culture method is characterized by comprising the following steps:
preparing microcarrier and seed cell separately;
co-culturing the microcarrier and the seed cells by using a serum-free culture medium which does not contain a sodium ion and calcium ion chelating agent, so that the seed cells are adhered to the surface of the microcarrier for proliferation;
replacing serum-free medium containing sodium ion and calcium ion chelating agent when harvesting cells;
after the microcarrier is expanded and deformed, collecting cells in a physical mode;
wherein the microcarrier is the microcarrier according to any one of claims 1 to 3, or the microcarrier is the microcarrier obtained by the preparation method according to any one of claims 4 to 10.
12. The culture method according to claim 11, wherein the co-culture of the microcarrier and the seed cell requires periodic shaking to promote the adhesion of the seed cell to the microcarrier surface.
13. The culture method according to claim 11, wherein the culture process after replacing the serum-free medium containing the sodium ion and calcium ion chelating agent requires periodic shaking to promote uniform suspension of the microcarriers to which the cells are adhered in the medium.
14. The culture method according to claim 11, wherein the serum-free medium containing the sodium ion-and calcium ion-chelating agent comprises 100mmol/L sodium bicarbonate and 55mmol/L sodium citrate.
15. The culture method of claim 11, wherein physically collecting the cells comprises at least one of:
promoting the cells to be separated from the surface of the microcarrier by shaking or stirring;
separating the microcarriers and cells by a screen;
cells were enriched by centrifugation.
16. The culture method according to claim 15, wherein the stirring speed is 100r/min or less.
17. The culture method of claim 11, wherein the seed cells are anchorage-dependent cultured cells.
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