CN116102770B - Cell culture bracket, preparation method thereof and cell culture method - Google Patents
Cell culture bracket, preparation method thereof and cell culture method Download PDFInfo
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- CN116102770B CN116102770B CN202310059955.3A CN202310059955A CN116102770B CN 116102770 B CN116102770 B CN 116102770B CN 202310059955 A CN202310059955 A CN 202310059955A CN 116102770 B CN116102770 B CN 116102770B
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- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 1
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- 210000002901 mesenchymal stem cell Anatomy 0.000 description 1
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- -1 natural silk) Chemical compound 0.000 description 1
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/0062—General methods for three-dimensional culture
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L13/00—Meat products; Meat meal; Preparation or treatment thereof
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J9/00—Working-up of macromolecular substances to porous or cellular articles or materials; After-treatment thereof
- C08J9/26—Working-up of macromolecular substances to porous or cellular articles or materials; After-treatment thereof by elimination of a solid phase from a macromolecular composition or article, e.g. leaching out
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J2305/00—Characterised by the use of polysaccharides or of their derivatives not provided for in groups C08J2301/00 or C08J2303/00
- C08J2305/08—Chitin; Chondroitin sulfate; Hyaluronic acid; Derivatives thereof
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J2389/00—Characterised by the use of proteins; Derivatives thereof
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J2405/00—Characterised by the use of polysaccharides or of their derivatives not provided for in groups C08J2401/00 or C08J2403/00
- C08J2405/08—Chitin; Chondroitin sulfate; Hyaluronic acid; Derivatives thereof
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J2489/00—Characterised by the use of proteins; Derivatives thereof
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2513/00—3D culture
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2533/00—Supports or coatings for cell culture, characterised by material
- C12N2533/50—Proteins
- C12N2533/54—Collagen; Gelatin
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2533/00—Supports or coatings for cell culture, characterised by material
- C12N2533/70—Polysaccharides
- C12N2533/80—Hyaluronan
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- Polymers & Plastics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Materials Engineering (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Medicinal Chemistry (AREA)
- Cell Biology (AREA)
- Nutrition Science (AREA)
- Food Science & Technology (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention provides a cell culture bracket, a preparation method thereof and a cell culture method, and relates to the technical field of cell culture. The cell culture scaffold is prepared through the steps of raw material modification, structure repair, performance repair and freeze drying. The cell culture bracket disclosed by the invention breaks through the technical problems of inedibility, high cost, low density and the like of the cell bracket in the market, has a beautifying effect after being eaten, and breaks through the key neck clamping problem of the traditional bracket. The cell culture bracket can be used for culturing various different types of cells, has extremely high culture efficiency, breaks through the limitation of cell culture conditions, and can realize the culture of cells in any form in any container in a conventional cell culture environment.
Description
Technical Field
The invention belongs to the technical field of cell culture, and particularly relates to a cell culture bracket, a preparation method thereof and a cell culture method.
Background
The biological cultivated meat is the closest to traditional meat in all meat analogue species, has all physicochemical properties of real meat, which makes it of high commercial value. Although the method is well seen by the market, the traditional cell culture scheme cannot meet the requirement of the cultured meat on the cell number due to the need of large-scale cell culture in the manufacturing process, and the cell culture is needed to be carried out through a microcarrier or a bracket to provide a large amount of cells, but the current microcarrier or bracket has poor batch effect of cell attachment efficiency on one hand and unstable cell proliferation efficiency, and on the other hand, the bracket is complex in material, expensive and not edible, so that the method forms a global neck problem in the aspects of manufacturing cost, biosafety and eating for the manufacturing of the biological cultured meat, and is a key technical problem for limiting the mass production of the cultured meat.
Disclosure of Invention
The invention aims to provide a cell culture bracket, a preparation method thereof and a cell culture method, wherein the cell culture bracket is edible, and has high culture efficiency and low cost.
The invention provides a preparation method of a cell culture bracket, which comprises the following steps: hydrolyzing natural polymer fiber protein to obtain dispersion;
denaturing the dispersion liquid by using alkali solution to obtain a denatured material;
Neutralizing the modified material by using an acid solution, and standing for 24 hours to obtain a structural repair material;
Mixing the structural repair material with a biocompatible material for incubation to obtain a performance repair material; the biocompatible material comprises collagen and hyaluronic acid;
and filling the performance repairing material into a mould, and freeze-drying to obtain the cell culture scaffold.
Preferably, the hydrolysis comprises ultrasonic hydrolysis at 80 ℃.
Preferably, the alkali solution comprises aqueous sodium hydroxide solution with the concentration of 0.5-5 mol/L.
Preferably, the denaturation includes a reaction at 0 to 80 ℃ for 5min to 2h using the alkali solution.
Preferably, the incubation includes a first incubation for 24 hours after mixing the structural repair material with the aqueous collagen solution, and a second incubation for 24 hours after taking out the first incubation material and mixing with the aqueous hyaluronic acid solution.
Preferably, the temperature of the first incubation and the second incubation are both 37 ℃;
The concentration of the collagen in the collagen aqueous solution is 2-5 mug/L;
the concentration of the hyaluronic acid in the hyaluronic acid aqueous solution is 2-12 g/L.
The invention also provides a cell culture scaffold prepared by the preparation method.
The invention also provides a cell culture method based on the cell culture bracket, which comprises the following steps: placing the cell suspension in the cell culture bracket, standing for 2 hours at 37 ℃, and continuing to stand for 2 hours at 37 ℃ after blowing.
Preferably, during the cell proliferation phase, the new medium is replaced every 24 hours;
when the large-scale cell expansion is to be continuously carried out, a new bracket is required to be added for cell expansion;
When the cell differentiation treatment is performed, the cell differentiation medium is replaced for culture.
The invention also provides application of the cell culture scaffold in cultivating meat products and performing tissue engineering research.
The beneficial effects are that: the invention provides a cell culture bracket and a preparation method thereof, and the cell culture bracket is prepared by the steps of raw material modification, structure repair, performance repair and freeze drying. The cell culture bracket contains full-valence amino acid, has the effects of maintaining beauty and keeping young, can be eaten, breaks through the key problem that the current microcarrier and bracket are not edible, and lays a material foundation for the commercial development of cell agriculture and tissue engineering. The cell scaffold breaks through the limit of cost, and the cost of the scaffold required by high-density cell culture of a 100mL culture system is less than 1 yuan.
The cell culture bracket can be used for culturing various different types of cells, for example, on the premise of single-layer bracket cell culture, the culture efficiency is more than 26 times of that of a traditional equal-area culture dish; the culture efficiency of the equal space is 260 times to 1000 times or even higher than that of the traditional culture dish by utilizing multi-layer and three-dimensional culture; the invention breaks through the upper limit of the cell culture density of the current carrier or bracket, and can be used for growing or proliferating 450 cells within the area of 0.1mm 2, in addition, the cell culture bracket developed by the invention breaks through the limitation of cell culture conditions, and can realize the culture of cells in any container and any form in the conventional cell culture environment.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions of the prior art, the drawings that are needed in the embodiments will be briefly described below, it being obvious that the drawings in the following description are only some embodiments of the present invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a schematic diagram of the overall development of the cell culture scaffold of the present invention;
FIG. 2 is a stent treatment diagram of the present invention;
FIG. 3 is a diagram of cell proliferation culture;
Fig. 4 is a graph of stent efficiency validation.
Detailed Description
The invention provides a preparation method of a cell culture bracket, which comprises the following steps: hydrolyzing natural polymer fiber protein to obtain dispersion;
denaturing the dispersion liquid by using alkali solution to obtain a denatured material;
Neutralizing the modified material by using an acid solution, and standing for 24 hours to obtain a structural repair material;
incubating the structural repair material with a biocompatible material to obtain a performance repair material; the biocompatible material comprises collagen and hyaluronic acid;
and filling the performance repairing material into a mould, and freeze-drying to obtain the cell culture scaffold.
The invention hydrolyzes natural high molecular fibrin, preferably comprising natural silk or biological modified silk (the biological modified silk is silk formed by modifying silkworm feeding ingredients). The hydrolysis of the invention preferably comprises ultrasonic hydrolysis, and specifically comprises ultrasonic hydrolysis for 0.5-3 hours at 80 ℃ and 50-60 kHz. The invention hydrolyzes the material by an ultrasonic instrument to hydrolyze the solid material into a vaporific dispersion state.
After the dispersion liquid is obtained, the invention uses alkali solution to denature the dispersion liquid to obtain denatured materials. The alkali solution of the present invention is preferably a strong alkali solution, more preferably comprises an aqueous sodium hydroxide solution, and the concentration is preferably 0.5 to 5mol/L, more preferably 1 to 5mol/L, and most preferably 2mol/L. The dispersion liquid is denatured by the strong alkali solution, wherein the temperature of the denaturation is preferably 0-80 ℃, more preferably 20-80 ℃ and most preferably 20 ℃; the reaction time is preferably 5min to 2h, more preferably 30min. In the denaturation treatment process, the higher the alkali concentration is, the higher the material treatment efficiency is, but the material damage degree is also increased, and the longer the treatment time is, the more complete the material treatment is; in addition, the temperature has a catalytic effect on the material denaturation treatment, the higher the temperature is, the more complete the material treatment is, but the more severe the structural damage is, and the treatment under the low-temperature condition can maintain the basic structure of the material. In the embodiment of the invention, the optimal denaturation conditions, namely 2mol/L, 30min and 20 ℃ are selected in consideration of factors such as cell binding sites, shearing force, protein activity and biocompatibility, the strong alkali concentration is relatively low at the moment, but covalent bond sites which are favorable for cell attachment in a bracket can be fully exposed, the denaturation damage degree of materials is small, the subsequent recovery is facilitated, meanwhile, the mechanical strength and shearing force of material treatment are similar to those of fresh meat, and physical indexes and chewing sense of real meat are endowed to the cultivated meat in the manufacturing process of the later-stage cultivated meat.
After the modified material is obtained, the structural repair material is obtained by neutralizing the modified material with an acid solution and then standing for 24 hours. The denatured material is preferably acid-base neutralized by weak acid, more preferably titrated neutralized by weak acid, and then allowed to stand for 24 hours. The weak acid of the present invention preferably comprises glacial acetic acid.
After the structural repair material is obtained, the structural repair material is incubated with the biocompatible material to obtain the performance repair material; the biocompatible material includes collagen and hyaluronic acid. The incubation of the invention is preferably carried out in stages under aseptic conditions, firstly the structural repair material is mixed with the collagen aqueous solution, then the structural repair material is incubated for 24 hours for the first time, and the first incubation material is taken out and mixed with the hyaluronic acid aqueous solution for the second incubation for 24 hours. The structure repairing material is physically modified by the biocompatible material, and hyaluronic acid is used as a main material for performance repairing. The first incubation is preferably carried out in an aqueous solution of collagen, the temperature of the first incubation is preferably 37 ℃, the type of collagen is preferably type I collagen, and the concentration is 2-5 mug/L. According to the invention, after the material subjected to the first incubation is taken out, the material is placed in a hyaluronic acid aqueous solution for the second incubation, the temperature of the second incubation is preferably 37 ℃, the concentration of hyaluronic acid in the hyaluronic acid aqueous solution is preferably 2-12 g/L, and through a material-hyaluronic acid coverage and hyaluronic acid water absorption-cell swelling screening experiment, the best density of 8g/L hyaluronic acid covering material is verified, and meanwhile, the influence on the swelling of cells is minimum, so that the method is an optimal scheme.
After the performance repairing material is obtained, the cell culture bracket is obtained by filling the performance repairing material into a mold and freeze-drying the material. According to the invention, different standard molds can be arranged for brackets with different sizes and models, for example, a cell culture plate is used as the mold, and when the cell culture plate is filled, the bracket and the hyaluronic acid solution with the concentration of 8g/L are filled into the mold, so that the bracket is in a full and uniform dispersion state in the solution, the mold is transferred to a-20 ℃ for freezing treatment for 24 hours, and then the freezing mold is transferred to a freeze dryer for freeze drying. The procedure for setting up the freeze-dryer in the examples of the present invention is shown in table 1:
table 1 freeze dryer program settings
After the freeze drying is finished, the invention preferably further comprises the steps of packaging the stent by a packaging film (a sterilized plastic film), and then carrying out vacuum packaging treatment by a vacuum device to obtain the stent.
The invention also provides a cell culture scaffold prepared by the preparation method.
The cell culture scaffold of the present invention may be used to culture a variety of cells, such as mesenchymal stem cells, myoblasts, adipose precursor cells, fibroblasts, etc., and the examples are described by taking fibroblasts as an example, but it is not intended to be construed as merely limiting the scope of the present invention. The cell culture bracket can be used for culturing a plurality of different forms, such as monolayer bracket cell culture, multilayer and three-dimensional culture and the like.
The invention also provides a cell culture method based on the cell culture bracket, as shown in figure 1, comprising the following steps: placing the cell suspension in the cell culture bracket, standing for 2 hours at 37 ℃, and continuing to stand for 2 hours at 37 ℃ after blowing.
The cell type to be cultured is not particularly limited, but the cultured cells (fibroblasts) are exemplified in the examples, but they are not limited to the full scope of the present invention. Specifically, before cells are inoculated, the cultured cells are required to be passaged, the basal medium in the culture plate is firstly sucked and removed, PBS containing double antibody (a green streptomycin mixture) preheated at 37 ℃ is used for cleaning, the blowing force in the cleaning process can be properly increased so as to wash away dead cells, 0.25% of trypsin is added into the culture plate, the culture plate is preferably covered at the bottom of the culture plate, after digestion for 2min at 37.0 ℃, the culture plate is placed under a microscope for observation, after the interstitial tissue is retracted, the cells are rounded and the cell gap is increased, the digestion is stopped by adding 3 times of basal medium (DMEM medium) or 1 time of fetal bovine serum, and the bottom wall of the culture plate is repeatedly blown by a 10mL liquid-transfering gun so as to separate the cells from the culture plate, and after the cells are separated from the bottle wall, cell suspension is formed. Transferring the cell suspension into a centrifuge tube, centrifuging at 1000rpm for 5min, removing supernatant, adding the culture medium again to remove trypsin, adding the culture medium again to resuspend the cells, placing the bracket into an equal volume sterile container (the invention uses a sterile plastic bottle as an example), adding the cell suspension into the bracket, adsorbing the cells onto the surface of the bracket under the water absorption effect of hyaluronic acid, standing at 37 ℃ for 2h, blowing the cell suspension by a pipette, standing at 37 ℃ for 2h, and repeating the above operation for 2 times.
After the inoculation, a cell proliferation stage using the cell culture scaffold is carried out, preferably, a new culture medium is replaced every 24 hours, efficient proliferation of cells is maintained until the cells can form a structure with a membrane in the scaffold, if the large-scale expansion of the cells is required to be continuously carried out, the new scaffold can be added for cell expansion, if the cell differentiation treatment is required to be carried out, the cell differentiation culture medium is replaced for culture, and flexible application of the scaffold can be realized according to different research or industrial requirements.
The invention also provides application of the cell culture scaffold in cultivating meat products and performing tissue engineering research.
For further explanation of the present invention, a cell culture scaffold, a method for preparing the same and a method for culturing cells according to the present invention will be described in detail with reference to the accompanying drawings and examples, which should not be construed as limiting the scope of the present invention.
Example 1
1. Stent development
(1) Material structure modification treatment
The method comprises the steps of firstly carrying out hydrolysis treatment on natural high molecular fibrin (such as natural silk), carrying out hydrolysis on the material at 80 ℃ by an ultrasonic instrument to hydrolyze solid materials into a vaporous dispersion state, and carrying out 125 different denaturation treatments on the solid materials by sodium hydroxide: the treatment concentration comprises 0.5mol/L, 1mol/L, 2mol/L, 4mol/L and 5mol/L, the treatment time comprises 5min, 15min, 30min, 1h and 2h, the treatment temperature comprises 0 ℃, 20 ℃, 40 ℃, 60 ℃ and 80 ℃, and in the denaturation treatment process, the higher the strong alkali concentration is, the higher the material treatment efficiency is, but the material damage degree is increased, the longer the treatment time is, and the material treatment is more complete; in addition, the temperature has a catalytic effect on the material denaturation treatment, the higher the temperature is, the more complete the material treatment is, but the more severe the structural damage is, and the treatment under the low-temperature condition can maintain the basic structure of the material.
Performance testing was performed after the denaturation treatment, including: cell binding sites, shear forces, biocompatibility, and the like. The cell binding site is detected by the cell attachment position in the cell culture process, and the shearing force is detected by a shearing force measuring instrument in the subsequent efficiency detection method, wherein the shearing force ranges from 3.25kg to 4.25kg, and is similar to meat in view of the application of the method in meat cultivation. The biocompatibility test was also performed by detecting the cell attachment and proliferation capacity by cell culture, as in the subsequent culture efficiency verification section.
The results indicated that the treatment combinations (1 mol/L, 80 ℃,2 h), (2 mol/L, 20 ℃, 30 min), (5 mol/L, 0 ℃,5 min) showed good cell binding sites, shear force, biocompatibility, etc., and the treatment combinations of 2mol/L, 20 ℃, 30min were optimal, and the materials of the treatment combinations were subjected to the subsequent tests.
(2) Material structure repair
The denatured material was acid-base neutralized with glacial acetic acid (1 mol/L), the pH value of the material in solution was first checked, then titrated with weak acid for neutralization, and then allowed to stand for 24h.
(3) Performance restoration
Taking out the neutralized material, and carrying out physical modification on the scaffold by using collagen, hyaluronic acid and other materials with high biocompatibility: the scaffold is placed in type I collagen (2-5 mug/ml) for incubation for 24 hours (sterile environment at 37 ℃), and then the scaffold is taken out and sequentially transferred into 8g/L hyaluronic acid solution for co-incubation for 24 hours (sterile environment at 37 ℃).
(4) Freeze drying
The scaffolds and 8g/L hyaluronic acid solution were filled into 24 well plates (1 cm 3 per well) of a mold to ensure that the scaffolds were in a filled and uniformly dispersed state in the solution, the mold was transferred into-20 ℃ for freezing treatment for 24 hours, and then the frozen mold was transferred into a freeze dryer for freeze drying (table 1).
After freeze-drying is completed, the stent is packaged by a packaging film (sterilized plastic film), and then vacuum packaging treatment is performed by vacuum equipment, so that the stent is obtained.
(5) The edible property verification of the material is carried out by grinding raw materials, feeding the mice in the form of food additives for 15 days, and detecting weight and analyzing blood (whole blood) components of the mice after feeding, namely, the material has no unexpected toxicological effect on the feeding process of the mice, so that the edible property of the material is proved.
2. Bracket incubation and cell inoculation
The cultured fibroblast cells are passaged, the basic culture medium in the culture plate is firstly sucked and removed, PBS which is preheated at 37 ℃ and contains double antibody (green streptomycin mixture) is used for cleaning, the blowing force in the cleaning process can be properly increased so as to wash away dead cells, 0.25% of trypsin is added into the culture plate, the culture plate is preferably covered at 37.0 ℃ for digestion for 2min, the culture plate is placed under a microscope for observation, after the interstitial tissue of the cells is retracted, the cells are rounded and the cell gap is increased, 3 times of basic culture medium (DMEM culture medium) or 1 time of fetal bovine serum is added for stopping digestion, and a 10mL liquid-transferring gun is used for repeatedly blowing the bottom wall of the culture plate so as to separate the cells from the culture plate, and after the cells are separated from the bottle wall, cell suspension is formed. Transferring the cell suspension into a centrifuge tube, centrifuging at 1000rpm for 5min, removing supernatant, adding the culture medium again to remove trypsin, adding the culture medium again to resuspend the cells, placing the bracket into an equal volume sterile container (taking a sterile plastic bottle as an example in the invention), adding the cell suspension into the bracket, adsorbing the cells onto the surface of the bracket under the water absorption effect of hyaluronic acid, standing at 37 ℃ for 2h, blowing the cell suspension by a pipetting gun, standing at 37 ℃ for 2h, and repeating the above operation for 2 times.
3. Cell culture
In the cell proliferation stage, a new culture medium is replaced every 24 hours, the efficient proliferation of cells is maintained until the cells can form a structure with a membrane in the scaffold, if the large-scale expansion of the cells is required to be continuously carried out, the new scaffold can be added for cell expansion, if the cell differentiation treatment is required to be carried out, the cell differentiation culture medium can be replaced for culture, and the flexible application of the scaffold can be realized according to different research or industrial requirements.
4. Efficiency verification
Immunofluorescence labeling is performed on the cells which proliferate in an adherent manner, and the proliferation/differentiation efficiency of the cells is verified. PBST: PBS solution containing 0.5% Triton X-100 (vol%). Sealing liquid: PBST solution containing 10% goat serum or horse serum. Secondary antibody diluent: 1g of Bovine Serum Albumin (BSA) was weighed and dissolved in 100ml of PBS, i.e., 1% BSA in PBS.
The specific operation is as follows: 1) The cells were removed, the medium was discarded, and washed 2 times with PBS.
2) 4% Paraformaldehyde was added and the mixture was fixed at room temperature for 15 minutes. Or adding absolute methanol for fixing for 5-10 minutes at room temperature or fixing for 2 minutes at-20 ℃.
3) The PBS was washed 3 times for 5 minutes.
4) Permeabilization was performed with PBST solution for 10 minutes at room temperature.
5) The PBS was washed 1 time, 5 minutes.
6) PBST containing 10% goat serum or horse serum is added, and the mixture is blocked for 30 to 60 minutes at room temperature.
7) The blocking solution was discarded and primary antibody (1: 50-1: 200 Diluted in blocking solution, incubated at 4℃overnight or 37℃for 2 hours.
8) The PBS with 1% BSA was washed 3 times for 5 minutes.
9) Secondary antibodies (1: 50-1: 200 Diluted in secondary antibody diluent, and placed in a dark place at 37 ℃ for 1 hour.
103 Times with PBS for 5 minutes.
11 DAPI/Hoechst was added at the final concentration and left at room temperature for 5 minutes.
12 3 Times with PBS for 5 minutes.
13 500 Μl PBS or PBS glycerol (1: 1) Photographing under a fluorescence microscope or storing in a dark place.
14 Observing the growth and proliferation forms of cells on the scaffold by a fluorescence microscope, and verifying the scaffold cell culture efficiency.
The verification case results show that: the area of 0.1mm 2 can be used for carrying out attachment proliferation on 450 cells (figures 2-4), which is 25 times of the number of cells cultured in a traditional culture dish with the same area. In the three-dimensional culture process, the number of cells which can be attached and proliferated by the equal volume of the bracket is increased by 200-1000 times compared with that of a traditional culture dish (the thickness of a single-layer bracket is 30 mu m, the three-dimensional culture height of the cells is 30 mu m-50 mu m, at least 150 layers of brackets can be accommodated in the 1cm height in the multi-layer three-dimensional culture, the ultimate increase of the cell culture efficiency is 3750 times, and the cell three-dimensional culture efficiency is 200-1000 times of that of the traditional culture dish when the three-dimensional culture dish is used in different cases).
Although the foregoing embodiments have been described in some, but not all, embodiments of the invention, it should be understood that other embodiments may be devised in accordance with the present embodiments without departing from the spirit and scope of the invention.
Claims (4)
1. A method for preparing a cell culture scaffold, comprising the steps of: hydrolyzing natural polymer fiber protein to obtain dispersion; the hydrolysis comprises ultrasonic hydrolysis at 80 ℃; the natural high molecular fibrin comprises natural silk or biological modified silk;
Denaturing the dispersion liquid by using alkali solution to obtain a denatured material; the alkali solution comprises sodium hydroxide aqueous solution with the concentration of 0.5-5 mol/L; the denaturation comprises the steps of reacting for 5 minutes to 2 hours at the temperature of 0 ℃ to 80 ℃ by utilizing the alkali solution;
Neutralizing the modified material by using an acid solution, and standing for 24 hours to obtain a structural repair material;
Mixing the structural repair material with a biocompatible material for incubation to obtain a performance repair material; the biocompatible material comprises collagen and hyaluronic acid; the incubation comprises the steps of mixing the structural repair material with a collagen aqueous solution, then carrying out first incubation for 24 hours, taking out the first incubation material, mixing the first incubation material with a hyaluronic acid aqueous solution, and carrying out second incubation for 24 hours; the temperature of the first incubation and the second incubation is 37 ℃; the mass concentration of the collagen in the collagen aqueous solution is 2-5 mug/L; the concentration of hyaluronic acid in the hyaluronic acid aqueous solution is 2-12 g/L;
and filling the performance repairing material into a mould, and freeze-drying to obtain the cell culture scaffold.
2. A cell culture scaffold prepared by the method of claim 1.
3. A cell culture method based on the cell culture scaffold of claim 2, comprising the steps of: placing the cell suspension in the cell culture bracket, standing for 2 hours at 37 ℃, and continuing to stand for 2 hours at 37 ℃ after blowing;
During the cell proliferation phase, changing the new culture medium every 24 hours;
when the large-scale cell expansion is to be continuously carried out, a new bracket is required to be added for cell expansion;
When the cell differentiation treatment is performed, the cell differentiation medium is replaced for culture.
4. Use of the cell culture scaffold of claim 2 for cultivating meat products and/or for tissue engineering studies.
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| CN112940304A (en) * | 2021-03-16 | 2021-06-11 | 杭州基智生物科技有限公司 | Three-dimensional cell culture scaffold, fibroblast gel and preparation method thereof |
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| CN112940304A (en) * | 2021-03-16 | 2021-06-11 | 杭州基智生物科技有限公司 | Three-dimensional cell culture scaffold, fibroblast gel and preparation method thereof |
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