CN114516974A - Preparation method and application of porous gelatin microcarrier - Google Patents

Preparation method and application of porous gelatin microcarrier Download PDF

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CN114516974A
CN114516974A CN202210239078.3A CN202210239078A CN114516974A CN 114516974 A CN114516974 A CN 114516974A CN 202210239078 A CN202210239078 A CN 202210239078A CN 114516974 A CN114516974 A CN 114516974A
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gelatin
solution
microcarrier
porous
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CN114516974B (en
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王向东
靳荣华
赵娜
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Shaanxi Future Meat Meal Health Technology Co ltd
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    • 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
    • C08J9/28—Working-up of macromolecular substances to porous or cellular articles or materials; After-treatment thereof by elimination of a liquid phase from a macromolecular composition or article, e.g. drying of coagulum
    • 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
    • C08J9/0014—Use of organic additives
    • C08J9/0023—Use of organic additives containing oxygen
    • 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
    • C08J9/0066—Use of inorganic compounding ingredients
    • 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
    • C08J9/0095—Mixtures of at least two compounding ingredients belonging to different one-dot groups
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0068—General culture methods using substrates
    • C12N5/0075—General culture methods using substrates using microcarriers
    • 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
    • C08J2201/00—Foams characterised by the foaming process
    • C08J2201/04—Foams characterised by the foaming process characterised by the elimination of a liquid or solid component, e.g. precipitation, leaching out, evaporation
    • C08J2201/048—Elimination of a frozen liquid phase
    • 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
    • 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

Abstract

The invention discloses a preparation method and application of a porous gelatin microcarrier, which comprises the step of adding an alkaline solution into a gelatin solution to prepare the porous gelatin microcarrier, wherein the alkaline solution can ionize OH－The ratio of the alkaline solution to the gelatin solution is 1: 10-40 by volume, and the mass percentage concentration of the gelatin solution is 5-20%. The invention only adopts pure gelatin, and the alkaline solution is added firstly, then the cross-linking agent is added at room temperature and stirredAnd then crosslinking and curing, and emulsifying with the oil phase solution to form the porous gelatin microcarrier. The obtained pore diameter is uniform, and the porosity and the pore diameter can be regulated and controlled according to the addition amount of the alkaline solution and the like. The obtained porous gelatin microcarrier is especially suitable for cell culture.

Description

Preparation method and application of porous gelatin microcarrier
Technical Field
The invention belongs to the technical field of biological materials, and particularly relates to a preparation method and application of a porous gelatin microcarrier.
Background
The microcarrier is microbeads with the diameter of 60-250 mu m and can be suitable for adherent cell growth, and the microcarrier mainly comprises natural glucan or various synthetic polymers. Since the first microcarrier developed by Van Wezel with DEAE-sephadex a 50, there are over a dozen types of microcarrier commercial products available on the international market, including liquid microcarriers, macroporous gelatin microcarriers, polystyrene microcarriers, PHEMA microcarriers, chitin microcarriers, polyurethane foam microcarriers, alginate gel microcarriers, and magnetic microcarriers. Among the numerous microcarriers, gelatin microcarriers are of interest because of their good cell adhesion properties.
Gelatin is a polypeptide mixture prepared by degrading collagen molecules, and comprises eighteen different amino acids which form a gelatin peptide chain, wherein the 3 'end of the amino group at the 5' end of carboxyl. The gelatin is formed by condensing alpha-amino acid, the main chain unit of the gelatin is CH-CO-NH-, and the polar groups in the side chain can be divided into acidic groups, basic groups and neutral groups.
There are methods for preparing porous microcarriers from gelatin. CN113750076A discloses a chitin-gelatin-based composite porous microsphere as a drug carrier, a preparation method and an application thereof, wherein the preparation method comprises the following steps: synthesizing gelatin microspheres; preparing a chitin solution and adjusting the pH value to be neutral; synthesizing chitin-gelatin composite porous microspheres (mixing a chitin solution and gelatin microspheres in a mass ratio of 1: 10-5: 10, dropwise adding the mixture into an oil phase, and carrying out water bath reaction); cooling to 0 +/-2 ℃, and adding glutaraldehyde for crosslinking reaction; standing and removing supernatant; adding petroleum ether to wash away redundant liquid paraffin, standing, and removing the upper white emulsion; adding deionized water, washing until the upper layer has no oil drop, and lyophilizing. CN107073165B provides a preparation method capable of obtaining a porous microcarrier having specific properties and particle size distribution, and provides a gelatin porous microcarrier having excellent cell proliferation characteristics. According to the present invention, there are provided a method for preparing a porous microcarrier of recombinant gelatin comprising an emulsification step using a content of an emulsifier having a specific HLB value, and a porous microcarrier of gelatin having a specific pore.
The patent expands the preparation method of the porous gelatin microcarrier, but has the technical problems of complex preparation process, low method controllability, high cost caused by the need of specially-made emulsifying agent or modified gelatin and the like. Therefore, the method for preparing the porous gelatin microcarrier is developed, has simple and feasible preparation process, controllable method, low cost and high biological safety, is easy for industrial production and has extremely high value.
Disclosure of Invention
The invention aims to provide a preparation method of a porous gelatin microcarrier, which has the advantages of simple and feasible preparation process, controllable method, low cost, high biological safety and easy industrial production.
In view of the above objects, the present invention meets this need in the art by providing a method for preparing a porous gelatin microcarrier.
In one aspect, the present invention relates to a method for preparing a porous gelatin microcarrier, comprising adding an alkaline solution to a gelatin solution to prepare a porous gelatin microcarrier; the alkaline solution can ionize to obtain OH－The solution of (1); the ratio of the alkaline solution to the gelatin solution is 1: 10-40 by volume.
Further, in the method for preparing a porous gelatin microcarrier provided by the present application, the alkaline solution comprises a NaOH solution.
Further, in the preparation method of the porous gelatin microcarrier provided by the application, OH in the alkaline solution－The concentration was 0.25 mol/L.
Further, in the method for preparing a porous gelatin microcarrier provided by the present application, the gelatin comprises type a gelatin or type B gelatin; the mass percentage concentration of the gelatin solution is 5-20%.
Further, in the preparation method of the porous gelatin microcarrier, after the alkaline solution is added into the gelatin solution, the cross-linking agent with the mass percentage concentration of 2.5-25% is added for cross-linking and fixing for 1-24 hours; the cross-linking agent is saturated straight-chain aliphatic dialdehyde; the ratio of the cross-linking agent to the gelatin solution is 1:10 by volume.
Further, in the preparation method of the porous gelatin microcarrier provided by the application, glutaraldehyde with the mass percentage concentration of 2.5% is selected as the cross-linking agent; the cross-linking fixing time is 1-12 h.
Further, in the method for preparing a porous gelatin microcarrier provided by the present application, the method further comprises: and (3) obtaining an aqueous phase solution after the crosslinking and fixing are finished, mixing the aqueous phase solution with the organic phase solution, stirring and emulsifying to generate W/O type droplets, reacting at room temperature for 24 hours, collecting emulsion, cleaning, freeze drying and sterilizing.
Further, in the preparation method of the porous gelatin microcarrier provided by the application, the organic phase solution is edible oil, and the edible oil comprises sunflower seed oil.
Specifically, the preparation method of the porous gelatin microcarrier provided by the application comprises the following steps:
heating in water bath at 60-80 deg.c, adding gelatin to swell, and dissolving in deionized water to final concentration of 5-20%. Adding OH when the temperature is reduced to 37 DEG C－Adding an alkaline solution with the concentration of 0.25mol/L into the premixed solution, stirring, and then adding a cross-linking agent for cross-linking and fixing to obtain an aqueous phase solution;
the ratio of the alkaline solution to the gelatin solution is 1: 10-40 by volume; the gelatin is selected from type A gelatin or type B gelatin; the mass percentage concentration of the gelatin solution is 5-20%; the alkaline solution is NaOH solution; the cross-linking agent is selected from saturated straight-chain aliphatic dialdehyde with the mass percentage concentration of 2.5-25%, the ratio of the cross-linking agent to the gelatin solution is 1:10 in terms of volume ratio, and the cross-linking time is 1-24 h; preferably, the cross-linking agent is glutaraldehyde with the mass percentage concentration of 2.5%, the ratio of the glutaraldehyde to the gelatin solution is 1:10 in terms of volume ratio, and the cross-linking time is 1-12 h.
Mechanically stirring the organic phase solution at the rotating speed of 700rpm for 1h, and removing bubbles generated in the solution; the organic phase solution is edible oil; preferably, the organic phase solution is sunflower seed oil. Mixing the water phase solution and the organic phase solution, stirring at the speed of 1000rpm, and fully stirring for 30min to generate W/O type liquid drops; the ratio of the aqueous phase solution to the organic phase solution is 1:100 by volume. Reacting at room temperature for 24h, collecting emulsion, filtering the upper organic phase, and collecting microcarrier. And cleaning the collected microcarrier with a cleaning agent to remove the surface organic phase of the microcarrier material. Freeze drying and sterilizing to obtain the porous gelatin microcarrier.
The invention adopts alkaline solution to prepare the porous gelatin microcarrier, and achieves the aim of controlling the aperture and the porosity. The stem cell micro-tissue obtained by the obtained porous gelatin micro-carrier is more compact and closer to the animal tissue character through a stem cell micro-tissue culture test. The invention thus further claims a microcarrier prepared by the above method and the use of the microcarrier in cell culture.
Compared with the prior art, the invention has the following beneficial effects or advantages:
the invention provides a preparation method of a porous gelatin microcarrier. According to the method, the alkaline solution is added, the reaction rate of the gelatin solution for forming the gel is controlled, the crosslinking degree and the crosslinking time of the gel are further controlled, and the uniform regulation of the pore diameter and the porosity of the porous gelatin microcarrier is realized. The method has the advantages of no need of modifying gelatin or using special reagents, simple and easy preparation method, controllable method, low cost, high biological safety, and easy industrial production.
Drawings
FIG. 1 is a TEM image of a porous gelatin microcarrier prepared in example 1.
FIG. 2 is a TEM image of the porous gelatin microcarrier obtained in example 2.
FIG. 3 is a TEM image of the porous gelatin microcarrier prepared in example 3.
FIG. 4 is a TEM image of the porous gelatin microcarrier obtained in example 4.
FIG. 5 is an image of HE sections of stem cell micro-tissues derived from umbilical cord of 5-day cattle cultured. FIG. 5a is a commercial image of HE slices from umbilical cord derived stem cells of 5-day cattle cultured on microcarriers; FIG. 5b is the image of the HE section of the stem cell micro-tissue from the umbilical cord of 5-day cattle cultured by the porous gelatin microcarrier prepared in example 4.
Detailed Description
The following examples are given to illustrate the technical aspects of the present invention, but the present invention is not limited to the following examples.
Example 1
This example provides a test for the preparation of porous gelatin microcarriers starting from type A gelatin.
In the test, the gelatin solution is prepared by heating in water bath at 60-80 ℃, adding type A gelatin for swelling, and fully dissolving with deionized water to obtain a final concentration of 5%; the alkaline solution is 0.25M NaOH solution; the cross-linking agent is glutaraldehyde with the mass percentage concentration of 2.5%; the organic phase is sunflower seed oil.
Heating in water bath at 60-80 deg.c, adding type A gelatin to swell, and dissolving in deionized water to reach final concentration of 5%. When the temperature dropped to 37 ℃, 1/40, a 0.25M NaOH solution in volume of gelatin solution was added and stirred. 1/10, 2.5% glutaraldehyde by volume of the gelatin solution, was then added for crosslinking fixation for 1h to give an aqueous solution.
Mechanically stirring sunflower seed oil at 700rpm for 1 hr to remove bubbles generated in the solution to obtain organic phase solution.
The ratio of the aqueous phase solution to the organic phase solution is 1:100 by volume, the aqueous phase solution and the organic phase solution are mixed, the stirring speed is 1000rpm, and the W/O type liquid drops are generated after fully stirring for 30 min. Reacting at room temperature for 24h, collecting emulsion, filtering the upper organic phase, and collecting microcarrier.
And washing the collected microcarriers. Firstly, petroleum ether is used for ultrasonic cleaning, acetone which is fully precooled is used for ultrasonic cleaning for the second time, precipitated microcarrier particles are collected, drying is carried out at 37 ℃, deionized water is added again for ultrasonic cleaning, and ultrasonic cleaning is carried out for 5 times. Wetting the washed microcarrier in a small amount of water, transferring the microcarrier into a freezing device at the temperature of minus 20 ℃ for pre-freezing, and transferring the microcarrier into a freeze dryer for freeze drying for 24 hours at the temperature of minus 40 ℃ after freezing. And (5) irradiating and sterilizing by Co 60.
The aperture of the micropores on the obtained porous gelatin microcarrier is 1-20 microns, the porosity reaches 75-85%, and the image is shown in figure 1.
Example 2
This example provides a test of preparing a porous gelatin microcarrier with a ratio of 1:20 alkaline solution to gelatin solution.
In the test, the gelatin solution is prepared by heating in water bath at 60-80 ℃, adding type A gelatin for swelling, and fully dissolving with deionized water to obtain a final concentration of 20%; the alkaline solution is 0.25M NaOH solution; the cross-linking agent is glutaraldehyde with the mass percentage concentration of 25%; the organic phase is sunflower seed oil.
Heating in water bath at 60-80 deg.c, adding type A gelatin to swell, and dissolving in deionized water to obtain final concentration of 20%. When the temperature dropped to 37 ℃, 1/20, a 0.25M NaOH solution in volume of gelatin solution was added and stirred. 1/10, 25% glutaraldehyde by volume of the gelatin solution was then added for cross-linking fixation for 24h to give an aqueous solution.
Mechanically stirring sunflower seed oil at 700rpm for 1h to remove bubbles generated in the solution to obtain organic phase solution.
The ratio of the aqueous phase solution to the organic phase solution is 1:100 by volume, the aqueous phase solution and the organic phase solution are mixed, the stirring speed is 1000rpm, and the W/O type liquid drops are generated after fully stirring for 30 min. Reacting at room temperature for 24h, collecting emulsion, filtering the upper organic phase, and collecting microcarrier.
And washing the collected microcarrier. Firstly, petroleum ether is used for ultrasonic cleaning, acetone which is fully precooled is used for ultrasonic cleaning for the second time, precipitated microcarrier particles are collected, drying is carried out at 37 ℃, deionized water is added again for ultrasonic cleaning, and ultrasonic cleaning is carried out for 5 times. Wetting the washed microcarrier in a small amount of water, transferring the microcarrier into a freezing device at the temperature of minus 20 ℃ for pre-freezing, and transferring the microcarrier into a freeze dryer for freeze drying for 24 hours at the temperature of minus 40 ℃ after freezing. And (5) irradiating and sterilizing by using Co 60.
The aperture of the micropores on the obtained porous gelatin microcarrier is 20-60 microns, the porosity reaches 85-95%, and the image is shown in figure 2.
Example 3
This example provides a test for the preparation of a porous gelatin microcarrier with a gelatin solution concentration of 10% by mass.
In the test, the gelatin solution is prepared by heating in water bath at 60-80 ℃, adding type A gelatin for swelling, and fully dissolving with deionized water to obtain a final concentration of 10%; the alkaline solution is 0.25M NaOH solution; the cross-linking agent is glutaraldehyde with the mass percentage concentration of 2.5%; the organic phase is sunflower seed oil.
Heating in water bath at 60-80 deg.c, adding type-A gelatin to swell, and dissolving in deionized water to obtain final concentration of 10%. When the temperature dropped to 37 ℃, 1/10, a 0.25M NaOH solution in volume of gelatin solution was added and stirred. 1/10, 2.5% glutaraldehyde by volume of the gelatin solution was then added for crosslinking fixation for 12h to give an aqueous solution.
Mechanically stirring sunflower seed oil at 700rpm for 1h to remove bubbles generated in the solution to obtain organic phase solution.
The ratio of the aqueous phase solution to the organic phase solution is 1:100 by volume, the aqueous phase solution and the organic phase solution are mixed, the stirring speed is 1000rpm, and the W/O type liquid drops are generated after fully stirring for 30 min. Reacting at room temperature for 24h, collecting emulsion, filtering the upper organic phase, and collecting microcarrier.
And washing the collected microcarriers. Firstly, petroleum ether is used for ultrasonic cleaning, acetone which is fully precooled is used for ultrasonic cleaning for the second time, precipitated microcarrier particles are collected, drying is carried out at 37 ℃, deionized water is added again for ultrasonic cleaning, and ultrasonic cleaning is carried out for 5 times. Wetting the washed microcarrier in a small amount of water, transferring the microcarrier into a freezing device at the temperature of minus 20 ℃ for pre-freezing, and transferring the microcarrier into a freeze dryer for freeze drying for 24 hours at the temperature of minus 40 ℃ after freezing. And (5) irradiating and sterilizing by Co 60.
The aperture of the micropores on the obtained porous gelatin microcarrier is 50-100 microns, the porosity reaches 85-95%, and the image is shown in figure 3.
Example 4
This example provides a test for the preparation of porous gelatin microcarriers starting from type B gelatin.
In the test, the gelatin solution is prepared by heating in water bath at 60-80 ℃, adding B-type gelatin for swelling, and fully dissolving with deionized water to obtain a final concentration of 10%; the alkaline solution is 0.25M NaOH solution; the cross-linking agent is glutaraldehyde with the mass percentage concentration of 25%; the organic phase is sunflower seed oil.
Heating in water bath at 60-80 deg.c, adding type B gelatin to swell, and dissolving in deionized water to obtain final concentration of 10%. When the temperature dropped to 37 ℃, 1/10, a 0.25M NaOH solution in volume of gelatin solution was added and stirred. 1/10, 2.5% glutaraldehyde by volume of the gelatin solution was then added for crosslinking fixation for 12h to give an aqueous solution.
Mechanically stirring sunflower seed oil at 700rpm for 1 hr to remove bubbles generated in the solution to obtain organic phase solution.
The ratio of the aqueous phase solution to the organic phase solution is 1:100 by volume, the aqueous phase solution and the organic phase solution are mixed, the stirring speed is 1000rpm, and the mixture is fully stirred for 30min to generate W/O type liquid drops. Reacting at room temperature for 24h, collecting emulsion, filtering the upper organic phase, and collecting the microcarrier.
And washing the collected microcarrier. Firstly, petroleum ether is used for ultrasonic cleaning, acetone which is fully precooled is used for ultrasonic cleaning for the second time, precipitated microcarrier particles are collected, drying is carried out at 37 ℃, deionized water is added again for ultrasonic cleaning, and ultrasonic cleaning is carried out for 5 times. Wetting the washed microcarrier in a small amount of water, transferring the microcarrier into a freezing device at the temperature of minus 20 ℃ for pre-freezing, and transferring the microcarrier into a freeze dryer for freeze drying for 24 hours at the temperature of minus 40 ℃ after freezing. And (5) irradiating and sterilizing by using Co 60.
The aperture of the micropores on the obtained porous gelatin microcarrier is 50-100 microns, the porosity reaches 85-95%, and the image is shown in figure 4.
Example 5
This example provides a comparative experiment of cell micro-tissue culture of the porous gelatin microcarriers obtained in example 4 and commercially available microcarriers.
The model of the commercial microcarrier is F01-100/3D Table
Figure BDA0003543565350000101
Micro slide curve
Figure BDA0003543565350000102
After the microcarriers prepared in example 4 were sterilized by Co 60 irradiation, 125mL cell reactors were selected and the experiments were divided into two groups:
experimental group 1: 100mg of commercial microcarriers + 2.5X 104cells/mL bovine umbilical cordMesenchymal stem cells +50mL of alpha-MEM culture medium containing 5% bovine platelet lysate; experimental group 2: 100mg of the microcarrier prepared in example 4 + 2.5X 104cells/mL bovine umbilical cord mesenchymal stem cells +50mL of alpha-MEM culture medium containing 5% bovine platelet lysate were subjected to cell culture. After 120h of culture, the cells form a compact cluster structure and the number of the cells is large. After further culturing for 5 days, HE sections of the obtained stem cell microtissue were stained, and HE staining results are shown in fig. 5.
The HE section of the microtissue shown in FIG. 5 shows that the stem cell microtissue obtained by culturing the microcarrier provided by the invention (FIG. 5b) is denser and more similar to the animal tissue characteristics compared with the commercial microcarrier (FIG. 5 a).
As described above, the present invention can be preferably implemented, and the above-mentioned embodiments only describe the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various changes and modifications of the technical solution of the present invention made by those skilled in the art without departing from the design spirit of the present invention shall fall within the protection scope defined by the present invention.

Claims (10)

1. A preparation method of a porous gelatin microcarrier is characterized by comprising the steps of adding an alkaline solution into a gelatin solution to prepare the porous gelatin microcarrier;
the alkaline solution can ionize to obtain OH－The solution of (1); the ratio of the alkaline solution to the gelatin solution is 1: 10-40 by volume.
2. The method for preparing a porous gelatin microcarrier according to claim 1, wherein the alkaline solution comprises a NaOH solution.
3. The method for preparing a porous gelatin microcarrier according to claim 1, wherein the OH in the alkaline solution－The concentration of (2) is 0.25 mol/L.
4. The method for preparing a porous gelatin microcarrier according to claim 1, wherein the gelatin comprises type a gelatin or type B gelatin; the mass percentage concentration of the gelatin solution is 5-20%.
5. The preparation method of the porous gelatin microcarrier according to claim 1, wherein after the alkaline solution is added into the gelatin solution, a cross-linking agent with the mass percentage concentration of 2.5-25% is added for cross-linking and fixing for 1-24 h; the cross-linking agent is saturated straight-chain aliphatic dialdehyde; the ratio of the cross-linking agent to the gelatin solution is 1:10 by volume.
6. The method for preparing the porous gelatin microcarrier according to claim 5, wherein glutaraldehyde with a mass percentage concentration of 2.5% is used as the cross-linking agent; the cross-linking fixing time is 1-12 h.
7. The method for preparing a porous gelatin microcarrier of claim 5, further comprising: and (3) obtaining an aqueous phase solution after the crosslinking and fixing are finished, mixing the aqueous phase solution with the organic phase solution, stirring and emulsifying to generate W/O type droplets, reacting at room temperature for 24 hours, collecting emulsion, cleaning, freeze drying and sterilizing.
8. The method for preparing a porous gelatin microcarrier according to claim 7, wherein the organic phase solution is edible oil, and the edible oil comprises sunflower seed oil.
9. A microcarrier prepared by the method of any one of claims 1-8.
10. Use of a microcarrier according to claim 9 in cell culture.
CN202210239078.3A 2022-03-11 2022-03-11 Preparation method and application of porous gelatin microcarrier Active CN114516974B (en)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060222680A1 (en) * 2005-03-31 2006-10-05 Chunlin Yang Method of preparing crosslinked collagen microspheres
CN102597230A (en) * 2009-11-13 2012-07-18 株式会社日立高新技术 Particle-containing cell aggregate
CN102604128A (en) * 2012-04-18 2012-07-25 吉林大学 Preparation method for gelatin nanosphere
CN103816573A (en) * 2014-03-01 2014-05-28 湖南科技大学 Preparation method of porous gelatin/hyaluronic acid composite microspheres

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060222680A1 (en) * 2005-03-31 2006-10-05 Chunlin Yang Method of preparing crosslinked collagen microspheres
CN102597230A (en) * 2009-11-13 2012-07-18 株式会社日立高新技术 Particle-containing cell aggregate
CN102604128A (en) * 2012-04-18 2012-07-25 吉林大学 Preparation method for gelatin nanosphere
CN103816573A (en) * 2014-03-01 2014-05-28 湖南科技大学 Preparation method of porous gelatin/hyaluronic acid composite microspheres

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