CN118459836A - Preparation method of gelatin microcarrier with streamline shape - Google Patents
Preparation method of gelatin microcarrier with streamline shape Download PDFInfo
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- CN118459836A CN118459836A CN202310073456.XA CN202310073456A CN118459836A CN 118459836 A CN118459836 A CN 118459836A CN 202310073456 A CN202310073456 A CN 202310073456A CN 118459836 A CN118459836 A CN 118459836A
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- 108010010803 Gelatin Proteins 0.000 title claims abstract description 161
- 239000008273 gelatin Substances 0.000 title claims abstract description 161
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- 235000011852 gelatine desserts Nutrition 0.000 title claims abstract description 161
- 238000002360 preparation method Methods 0.000 title claims abstract description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 62
- 238000001914 filtration Methods 0.000 claims abstract description 57
- 239000000243 solution Substances 0.000 claims abstract description 56
- 238000003756 stirring Methods 0.000 claims abstract description 56
- 238000006243 chemical reaction Methods 0.000 claims abstract description 54
- 239000004005 microsphere Substances 0.000 claims abstract description 41
- 238000004132 cross linking Methods 0.000 claims abstract description 20
- 239000011148 porous material Substances 0.000 claims abstract description 20
- 239000003995 emulsifying agent Substances 0.000 claims abstract description 19
- 239000007864 aqueous solution Substances 0.000 claims abstract description 16
- 238000001816 cooling Methods 0.000 claims abstract description 9
- 239000003960 organic solvent Substances 0.000 claims abstract description 6
- 239000003431 cross linking reagent Substances 0.000 claims abstract description 5
- 238000004108 freeze drying Methods 0.000 claims abstract description 4
- 238000005406 washing Methods 0.000 claims abstract description 4
- 230000001954 sterilising effect Effects 0.000 claims abstract description 3
- 238000004140 cleaning Methods 0.000 claims description 24
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- 238000000034 method Methods 0.000 claims description 11
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- 239000004215 Carbon black (E152) Substances 0.000 claims description 4
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- 229930195733 hydrocarbon Natural products 0.000 claims description 4
- 150000002430 hydrocarbons Chemical class 0.000 claims description 4
- FPQQSJJWHUJYPU-UHFFFAOYSA-N 3-(dimethylamino)propyliminomethylidene-ethylazanium;chloride Chemical compound Cl.CCN=C=NCCCN(C)C FPQQSJJWHUJYPU-UHFFFAOYSA-N 0.000 claims description 2
- XDTMQSROBMDMFD-UHFFFAOYSA-N Cyclohexane Chemical compound C1CCCCC1 XDTMQSROBMDMFD-UHFFFAOYSA-N 0.000 claims description 2
- AZKVWQKMDGGDSV-BCMRRPTOSA-N Genipin Chemical compound COC(=O)C1=CO[C@@H](O)[C@@H]2C(CO)=CC[C@H]12 AZKVWQKMDGGDSV-BCMRRPTOSA-N 0.000 claims description 2
- KWYHDKDOAIKMQN-UHFFFAOYSA-N N,N,N',N'-tetramethylethylenediamine Chemical compound CN(C)CCN(C)C KWYHDKDOAIKMQN-UHFFFAOYSA-N 0.000 claims description 2
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 claims description 2
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 claims description 2
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- WSFSSNUMVMOOMR-NJFSPNSNSA-N methanone Chemical compound O=[14CH2] WSFSSNUMVMOOMR-NJFSPNSNSA-N 0.000 claims description 2
- 239000002480 mineral oil Substances 0.000 claims description 2
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- 239000001788 mono and diglycerides of fatty acids Substances 0.000 claims description 2
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- 229920000136 polysorbate Polymers 0.000 claims description 2
- 239000010453 quartz Substances 0.000 claims description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N silicon dioxide Inorganic materials O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 2
- 235000015112 vegetable and seed oil Nutrition 0.000 claims description 2
- 239000008158 vegetable oil Substances 0.000 claims description 2
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- 238000007710 freezing Methods 0.000 abstract description 6
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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
- C12N5/0068—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
- C08J3/00—Processes of treating or compounding macromolecular substances
- C08J3/02—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques
- C08J3/03—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques in aqueous media
- C08J3/07—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques in aqueous media from polymer solutions
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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
- C08J3/00—Processes of treating or compounding macromolecular substances
- C08J3/24—Crosslinking, e.g. vulcanising, of macromolecules
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- 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
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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
- C08J2389/00—Characterised by the use of proteins; Derivatives thereof
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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
- C12N2531/00—Microcarriers
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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
- C12N2533/00—Supports or coatings for cell culture, characterised by material
- C12N2533/50—Proteins
- C12N2533/54—Collagen; Gelatin
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- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Medicinal Chemistry (AREA)
- Polymers & Plastics (AREA)
- Biomedical Technology (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Microbiology (AREA)
- Materials Engineering (AREA)
- Cell Biology (AREA)
- Dispersion Chemistry (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention relates to a preparation method of a gelatin microcarrier with a streamline shape, which comprises the following steps: adding gelatin aqueous solution into oil phase containing emulsifying agent, stirring and emulsifying, cooling and filtering to obtain gelatin microsphere, washing with organic solvent, dispersing gelatin microsphere with water, adding cross-linking agent for reaction, cooling and freezing the reaction solution rapidly, preserving heat and cross-linking for 12-48 h, thawing to obtain gelatin microcarrier solution, filtering and washing, re-dispersing into water, freeze-drying and sterilizing. The invention takes the gelatin microsphere as the raw material, forms ice crystals by controlling the heat conduction direction and the speed to produce the gelatin microcarrier with streamline shape, has the advantages of adjustable grain diameter and proper pore size, and the streamline shape can help to reduce the flow resistance, thereby being suitable for large-scale cell culture application.
Description
Technical Field
The invention relates to the field of gelatin microcarriers, in particular to a preparation method of a gelatin microcarrier with a streamline shape.
Background
Cell mass culture is important in biotechnology and has been widely used for the production of various vaccines, enzymes, hormones, antibodies, interferons, nucleic acids, and other products. Successful development and use of microcarriers has enabled large-scale high-yield culture of anchorage-dependent cells. In microcarrier culture, cells are grown as a monolayer on the surface of the pellet or as multiple layers in the pores of a macroporous structure. Microcarriers are typically suspended in the medium by gentle agitation. By using microcarriers for cell culture in suspension culture and fluid bed systems, the cell yield per milliliter of culture medium can be over one hundred million, and the space utilization is very high. Therefore, microcarrier culture is currently recognized as the most promising technology for large-scale culture of animal cells, and has been widely used for culturing various types of cells, such as mesenchymal stem cells and Vero cells.
Traditional cellular microcarriers are polystyrene microcarriers and cross-linked dextran microcarriers. The compatibility of the polystyrene microsphere and the cell is common, so that the adhesion of the cell on the surface of the polystyrene microcarrier is slower; and the polystyrene microspheres can be uniformly suspended in the culture medium at a high rotating speed, and the collision among the microspheres is easy to damage cells due to the high shearing force. The cross-linked dextran carrier has certain toxicity to cells, and for adherent cells, the growth speed is still not fast, and the cross-linked dextran carrier can not be completely hydrolyzed, so that the harvesting difficulty of cultured cells is high.
Gelatin is protein obtained by partial hydrolysis and denaturation of collagen, has a chemical structure similar to that of collagen, is favorable for cell proliferation and adhesion, has good biocompatibility, and the degradation product is nontoxic and is an ideal biological tissue engineering material. The preparation of the cell microcarrier by using the gelatin is quite suitable, compared with the traditional microcarrier, the gelatin-based microcarrier has better biocompatibility, can be completely subjected to enzymolysis, is convenient for cell harvest, and has higher yield and expandability. In order to reduce the stirring speed of the culture medium and reduce the cell damage caused by microsphere collision, the microcarrier with the streamline shape of water drops has more advantages. Thus, the invention develops a preparation method of a gelatin microcarrier with a streamline shape.
Disclosure of Invention
The invention aims to provide a preparation method of a porous gelatin microcarrier, which is used for solving the problem of a cell microcarrier in the prior art.
The invention provides a preparation method of a porous gelatin microcarrier, which comprises the following steps:
(1) Preparing a gelatin aqueous solution with the concentration of 1-20%, heating the aqueous solution to 45-65 ℃ in the preparation process, and continuously stirring;
(2) Preparing a mixed solution of an oil phase and an emulsifier: mixing the emulsifier with the oil phase according to the concentration of 0.1-5%, and heating to 45-65 ℃ in a stirring state;
(3) Slowly adding the gelatin aqueous solution prepared in the step (1) into the mixed solution of the oil phase and the emulsifier prepared in the step (2), continuously stirring in the adding process, maintaining the temperature of the system at 45-65 ℃, and continuously stirring for 5-60 min after the adding is completed to obtain W/O type emulsion, wherein the mass ratio of the gelatin aqueous solution to the mixed solution of the oil phase and the emulsifier is 1:2-1:10;
(4) Cooling the W/O emulsion obtained in the step (3) to 1-15 ℃ and continuously stirring to solidify gelatin gel to obtain gelatin emulsion;
(5) Directly filtering the gelatin emulsion obtained in the step (4) or adding 20-200% of organic solution to demulsifie and then filtering, washing the filtered gelatin microspheres for 2-6 times by using the organic solvent, and finally filtering and collecting the gelatin microspheres;
(6) Slowly dispersing the gelatin microspheres obtained in the step (5) into deionized water under the stirring state, filtering and cleaning for 2-6 times, and finally filtering and collecting;
The gelatin microspheres collected by filtration are directly dispersed into deionized water without drying, so that the dispersion rate of the gelatin microspheres in water can be greatly accelerated, and the condition that the microspheres are easy to adhere after being dried is avoided;
The gelatin microspheres are directly dispersed into deionized water for filtration without crosslinking after being washed by an organic solvent, and because the gelatin microspheres are very fragile when being dispersed into water without crosslinking, the adhesion is extremely easy to occur in the general filtration process; the non-crosslinked direct filtration avoids the adverse effect of crosslinking the microspheres before filtration on the pore formation of the final gelatin microcarrier;
(7) Dispersing the gelatin microspheres obtained in the step (6) into deionized water, adding a cross-linking agent, reacting for 0-24 h at the temperature of 1-15 ℃, and then pouring the reaction solution into a flat container or a cylindrical container;
(8) Immersing the container filled with the reaction solution in the step (7) in a cooling liquid for 2-600 min, wherein the temperature of the cooling liquid is-80 to-5 ℃, taking out the gelatin solution after the gelatin solution is completely frozen, and placing the gelatin solution in an environment of-25 to-5 ℃ for continuous crosslinking for 12-48 h;
Immersing a high-heat-conductivity flat container or a cylindrical container in a low-temperature cooling liquid to quickly establish a cooling gradient so as to freeze gelatin solution in the container, wherein the pore size of the surface of the prepared gelatin porous microcarrier is 10-40 mu m, the pore size is proper, the pore size distribution is uniform, and the microcarrier has a streamline shape and is very suitable for cell suspension culture;
(9) After the crosslinking in the step (8) is completed, placing the container in normal temperature air or immersing the container in an aqueous solution at 10-40 ℃, carrying out deicing, filtering and recovering the microcarrier, and cleaning the microcarrier for 2-6 times by using deionized water;
(10) Redispersing the gelatin microcarrier obtained in the step (9) in water, freeze-drying and sterilizing to obtain the streamline porous gelatin microcarrier.
Further, the gelatin of the gelatin aqueous solution in the step (1) is a type A and/or a type B gelatin.
Further, the oil phase in the step (2) is one or a combination of more of vegetable oil, mineral oil, petroleum ether and hydrocarbon, and the hydrocarbon is hexane, heptane, cyclohexane, toluene or xylene.
Further, the emulsifier in the step (2) is one or a combination of more of tween, glyceryl monostearate and span.
Further, the organic solvent in the step (5) is one or a combination of more of ethanol, isopropanol and acetone.
Further, the temperature of deionized water in the step (6) to the step (7) is 1-15 ℃.
Further, the cross-linking agent in the step (7) is one or more of formaldehyde, glutaraldehyde, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide, tetramethyl ethylenediamine and genipin.
Further, the container in the step (7) is made of one or a combination of several of glass, quartz, metal and alloy, the container is made of high-heat-conductivity material, so that the problem that the hole forming effect is poor or the hole cannot be formed due to the low-heat-conductivity material such as plastic, rubber and the like is avoided, the cross section of the flat container is flat, particularly rectangular, diamond or oval, the long side of the inner cavity of the cross section is L, the short side is D, the length of the short side is less than or equal to 20mm, the cross section of the cylindrical container is circular, the diameter of the inner cavity circle is less than or equal to 20mm, and the problem that the hole structure is poor or the hole cannot be formed due to the fact that the length of the short side and the diameter of the circle are larger than 20mm is avoided.
Further, the pore diameter of the filter screen is 20-300 mu m.
The technical scheme of the invention has the beneficial effects that:
The invention takes gelatin as raw material, adopts emulsion method to prepare spheres, and prepares porous gelatin microcarrier by controlling heat conduction direction and rate to form ice crystals;
The prepared gelatin microcarrier is in a streamline porous structure, accords with hydrodynamic characteristics, has the advantages of proper pore size and high porosity, is easier to stably suspend in a culture medium in a stirring state, and is more beneficial to cell wall-attached growth;
The porous gelatin microcarrier is of a non-rigid structure, the microcarrier does not damage cells in the stirring process, the natural polypeptide structure is completely nontoxic to the cells and has good compatibility with the cells, so that the attachment rate of the cells on the surface of the gelatin microcarrier is high, and the cell suspension culture experiment proves that the microcarrier greatly improves the expansion speed of the cells;
gelatin microcarriers are completely degradable with specific enzymes (e.g. collagenase) to allow easier harvesting of adherent cells.
Drawings
FIGS. 1 to 3 are scanning electron micrographs of gelatin microcarriers obtained in example 1 of the present invention;
FIG. 4 is a scanning electron microscope image of a gelatin microcarrier obtained in example 2 of the present invention;
FIG. 5 is a scanning electron microscope image of a gelatin microcarrier obtained in comparative example 1 of the present invention;
FIG. 6 is a scanning electron microscope image of a gelatin microcarrier obtained in comparative example 2 of the present invention;
FIG. 7 is a schematic cross-sectional view of three flat containers of the present invention;
Description of the embodiments
For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments of the present invention.
Examples
(1) 200G of gelatin and 1.8L of deionized water are weighed and added into a 5L reaction kettle together, the reaction kettle is heated to 45 ℃ and the stirring speed is set to 60rpm, and stirring is continued until the gelatin is completely dissolved, and the temperature is kept for later use;
(2) Weighing 12L of liquid paraffin and 40g of emulsifier span 80, adding the liquid paraffin and the emulsifier span 80 into a jacketed reaction kettle, heating the jacketed reaction kettle to 45 ℃, setting the stirring speed to 300rpm, maintaining stirring for 30min, and preserving heat for later use;
(3) Restarting the jacketed reaction kettle, setting the stirring speed to be 200rpm, setting the temperature to be 45 ℃, simultaneously pumping the gelatin solution in the 5L reaction kettle in the step (1) into the jacketed reaction kettle at the speed of 200ml/min through a peristaltic pump, closing a feed inlet of the jacketed reaction kettle after the gelatin solution is added, and maintaining stirring for 50min to prepare W/O gelatin emulsion;
(4) The 45 ℃ hot oil circulating pump of the jacket reaction kettle is turned off, hot oil in the jacket is removed, then the 5 ℃ low-temperature circulating pump is turned on to enable the jacket reaction kettle to be rapidly cooled, and after cooling is completed, stirring is continuously maintained for 50min to enable gelatin gel to be solidified, so that gelatin emulsion is obtained;
(5) The rotating speed of a jacket reaction kettle is increased to 400rpm, 14L of absolute ethyl alcohol at 5 ℃ is added into the jacket reaction kettle at the speed of 10L/min through a flow pump, stirring is maintained for 5min after the adding is finished to demulsify gelatin emulsion, the demulsifying solution is pumped into a negative pressure filtering tank through the flow pump, the bottom of the filtering tank is a filtering screen with the aperture of 100 mu m, the negative pressure is set to be 0.03MPa, a valve at the bottom of the filtering tank is closed after the filtering is finished, 25L of absolute ethyl alcohol at 5 ℃ is added again for cleaning and filtering, and gelatin microsphere powder is obtained at the bottom of the filtering tank after repeated cleaning and filtering for 5 times;
(6) Turning off the negative pressure in a negative pressure filter tank, re-adding 50L of water at 5 ℃ into the filter tank, starting stirring to disperse gelatin microspheres into the water at a speed of 100rpm, turning off stirring after the dispersion is finished, starting a valve at the bottom of the filter tank to filter gelatin microsphere solution by means of gravity, repeating the water cleaning operation for 3 times after the filtration is finished, and obtaining gelatin microspheres at the bottom of the filter tank;
(7) Re-dispersing gelatin microspheres by adding 10L of water at 5 ℃ into a filter tank, adding 100ml of glutaraldehyde solution (25% aqueous solution), and then pumping the solution into a 10L flat stainless steel container, wherein the length of the short side of the inner cavity of the cross section of the flat stainless steel container is D=40 mm;
(8) Immersing the container in cooling liquid at-20deg.C for 1 hr, completely freezing gelatin dispersion liquid in the container, taking out the flat container from the cooling liquid, and standing at-20deg.C for continuous crosslinking for 48 hr;
(9) Immersing the flat container in warm water at 37 ℃ for 15min after the crosslinking is finished to completely ice the internal ice cubes to obtain a gelatin microcarrier solution, then opening a container cover to pour the internal gelatin microcarrier solution into a 20L filter tank for negative pressure suction filtration, wherein the pore diameter of the filter screen of the filter tank is 50 mu M, the negative pressure is 0.2MPa, adding 20L of water for repeated cleaning for 2 times after the crosslinking is finished, then adding 0.1M/L glycine solution into the gelatin microcarrier collected by the filter screen for soaking for 2h, and cleaning for 3 times by using water after the filter screen is filtered to obtain the gelatin microcarrier;
(10) And re-dispersing the gelatin microcarrier into water, and then freeze-drying to obtain the streamline porous gelatin microcarrier, wherein the gelatin microcarrier is in a streamline structure as shown in a figure 1, the surface pore structure of the microcarrier is uniform, and the pore size is distributed at 10-40 mu m as shown in a figure 2-3.
Example 2
(1) Weighing 500g of gelatin and 1.5L of deionized water, adding the gelatin and the 1.5L of deionized water into a 5L reaction kettle, heating the reaction kettle to 45 ℃, setting the stirring speed to 60rpm, continuously stirring until the gelatin is completely dissolved, and preserving heat for later use;
(2) Weighing 12L of liquid paraffin and 40g of emulsifier span 80, adding the liquid paraffin and the emulsifier span 80 into a jacketed reaction kettle, heating the jacketed reaction kettle to 45 ℃, setting the stirring speed to 300rpm, maintaining stirring for 30min, and preserving heat for later use;
(3) Restarting the jacketed reaction kettle, setting the stirring speed to be 200rpm, setting the temperature to be 45 ℃, simultaneously pumping the gelatin solution in the 5L reaction kettle in the step (1) into the jacketed reaction kettle at the speed of 200ml/min through a peristaltic pump, closing a feed inlet of the jacketed reaction kettle after the gelatin solution is added, and maintaining stirring for 50min to prepare W/O gelatin emulsion;
(4) The 45 ℃ hot oil circulating pump of the jacket reaction kettle is turned off, hot oil in the jacket is removed, then the 5 ℃ low-temperature circulating pump is turned on to enable the jacket reaction kettle to be rapidly cooled, and after cooling is completed, stirring is continuously maintained for 50min to enable gelatin gel to be solidified, so that gelatin emulsion is obtained;
(5) The rotating speed of a jacket reaction kettle is increased to 400rpm, 14L of absolute ethyl alcohol at 5 ℃ is added into the jacket reaction kettle at the speed of 10L/min through a flow pump, stirring is maintained for 5min after the adding is finished to demulsify gelatin emulsion, the demulsifying solution is pumped into a negative pressure filtering tank through the flow pump, the bottom of the filtering tank is a filtering screen with the aperture of 100 mu m, the negative pressure is set to be 0.03MPa, a valve at the bottom of the filtering tank is closed after the filtering is finished, 25L of absolute ethyl alcohol at 5 ℃ is added again for cleaning and filtering, and gelatin microspheres are obtained at the bottom of the filtering tank after repeated cleaning and filtering are carried out for 5 times;
(6) Turning off the negative pressure in a negative pressure filter tank, re-adding 50L of water at 5 ℃ into the filter tank, starting stirring to disperse gelatin microspheres into the water at a speed of 100rpm, turning off stirring after the dispersion is finished, starting a valve at the bottom of the filter tank to filter gelatin microsphere solution by means of gravity, repeating the water cleaning operation for 3 times after the filtration is finished, and obtaining gelatin microspheres at the bottom of the filter tank;
(7) Re-adding 10L of water at 5 ℃ into a filtering tank to re-disperse gelatin microspheres, adding 100ml of glutaraldehyde solution (25% aqueous solution), and then pumping the solution into a 10L flat stainless steel container, wherein the thickness of the inner cavity of the stainless steel container is 40mm;
(8) Immersing the container in-20deg.C cooling liquid for 1 hr after covering, completely freezing gelatin dispersion liquid in the container, taking out the flat container from the cooling liquid, and placing at-20deg.C for continuous crosslinking for 48 hr;
(9) Immersing the flat container in warm water at 37 ℃ for 15min after the crosslinking is finished to completely ice the internal ice cubes to obtain a gelatin microcarrier solution, then opening a container cover to pour the internal gelatin microcarrier solution into a 20L filter tank for negative pressure suction filtration, wherein the pore diameter of the filter screen of the filter tank is 50 mu M, the negative pressure is 0.2MPa, adding 20L of water for repeated cleaning for 2 times after the crosslinking is finished, then adding 0.1M/L glycine solution into the gelatin microcarrier collected by the filter screen for soaking for 2h, and cleaning for 3 times by using water after the filter screen is filtered to obtain the gelatin microcarrier;
(10) The gelatin microcarrier is redispersed in water and then freeze-dried to obtain the gelatin microcarrier, as shown in figure 4, with irregular pore size and shape.
Comparative example 1
(1) 200G of gelatin and 1.8L of deionized water are weighed and added into a 5L reaction kettle together, the reaction kettle is heated to 45 ℃ and the stirring speed is set to 60rpm, and stirring is continued until the gelatin is completely dissolved, and the temperature is kept for later use;
(2) Weighing 12L of liquid paraffin and 40g of emulsifier span 80, adding the liquid paraffin and the emulsifier span 80 into a jacketed reaction kettle, heating the jacketed reaction kettle to 45 ℃, setting the stirring speed to 300rpm, maintaining stirring for 30min, and preserving heat for later use;
(3) Restarting the jacketed reaction kettle, setting the stirring speed to be 200rpm, setting the temperature to be 45 ℃, simultaneously pumping the gelatin solution in the 5L reaction kettle in the step (1) into the jacketed reaction kettle at the speed of 200ml/min through a peristaltic pump, closing a feed inlet of the jacketed reaction kettle after the gelatin solution is added, and maintaining stirring for 50min to prepare W/O gelatin emulsion;
(4) The 45 ℃ hot oil circulating pump of the jacket reaction kettle is turned off, hot oil in the jacket is removed, then the 5 ℃ low-temperature circulating pump is turned on to enable the jacket reaction kettle to be rapidly cooled, and after cooling is completed, stirring is continuously maintained for 50min to enable gelatin gel to be solidified, so that gelatin emulsion is obtained;
(5) The rotating speed of a jacket reaction kettle is increased to 400rpm, 14L of absolute ethyl alcohol at 5 ℃ is added into the jacket reaction kettle at the speed of 10L/min through a flow pump, stirring is maintained for 5min after the adding is finished to demulsify gelatin emulsion, the demulsifying solution is pumped into a negative pressure filtering tank through the flow pump, the bottom of the filtering tank is a filtering screen with the aperture of 100 mu m, the negative pressure is set to be 0.03MPa, a valve at the bottom of the filtering tank is closed after the filtering is finished, 25L of absolute ethyl alcohol at 5 ℃ is added again for cleaning and filtering, and gelatin microspheres are obtained at the bottom of the filtering tank after repeated cleaning and filtering are carried out for 5 times;
(6) Turning off the negative pressure in a negative pressure filter tank, re-adding 50L of water at 5 ℃ into the filter tank, starting stirring to disperse gelatin microspheres into the water at a speed of 100rpm, turning off stirring after the dispersion is finished, starting a valve at the bottom of the filter tank to filter gelatin microsphere solution by means of gravity, repeating the water cleaning operation for 3 times after the filtration is finished, and obtaining gelatin microspheres at the bottom of the filter tank;
(7) Re-dispersing gelatin microspheres by adding 10L of 5 ℃ water to a filter tank, adding 100ml of glutaraldehyde solution (25% aqueous solution), and then pouring the solution into 5 2L stainless steel flat trays with the height of the solution in the tray being about 50mm;
(8) Placing the flat chassis in a refrigerator with low temperature of-80 ℃ for 6-10h, completely freezing gelatin dispersion liquid in the container, taking out the flat chassis from the refrigerator, and placing the flat chassis in the refrigerator with low temperature of-20 ℃ for continuous crosslinking for 48h;
(9) Placing the flat chassis at normal temperature after the crosslinking is finished, applying flowing air to completely ice the ice cubes in the flat chassis to obtain a gelatin microcarrier solution, pouring the gelatin microcarrier solution into a 20L filter tank for negative pressure suction filtration, wherein the pore diameter of the filter screen of the filter tank is 50 mu M, the negative pressure is 0.2MPa, adding 20L of water for repeated cleaning for 2 times after the crosslinking is finished, then adding 0.1M/L glycine solution into the gelatin microcarrier collected by the filter screen for soaking for 2 hours, filtering by the filter screen, and cleaning for 3 times by using water to obtain the gelatin microcarrier;
(10) The gelatin microcarrier was redispersed in water and then freeze-dried to obtain a gelatin microcarrier, as shown in figure 5, with a partially non-uniform size and a non-uniform pore size and shape distribution.
Comparative example 2
(1) 200G of gelatin and 1.8L of deionized water are weighed and added into a 5L reaction kettle together, the reaction kettle is heated to 45 ℃ and the stirring speed is set to 60rpm, and stirring is continued until the gelatin is completely dissolved, and the temperature is kept for later use;
(2) Weighing 12L of liquid paraffin and 40g of emulsifier span 80, adding the liquid paraffin and the emulsifier span 80 into a jacketed reaction kettle, heating the jacketed reaction kettle to 45 ℃, setting the stirring speed to 300rpm, maintaining stirring for 30min, and preserving heat for later use;
(3) Restarting the jacketed reaction kettle, setting the stirring speed to be 200rpm, setting the temperature to be 45 ℃, simultaneously pumping the gelatin solution in the 5L reaction kettle in the step (1) into the jacketed reaction kettle at the speed of 200ml/min through a peristaltic pump, closing a feed inlet of the jacketed reaction kettle after the gelatin solution is added, and maintaining stirring for 50min to prepare W/O gelatin emulsion;
(4) The 45 ℃ hot oil circulating pump of the jacket reaction kettle is turned off, hot oil in the jacket is removed, then the 5 ℃ low-temperature circulating pump is turned on to enable the jacket reaction kettle to be rapidly cooled, and after cooling is completed, stirring is continuously maintained for 50min to enable gelatin gel to be solidified, so that gelatin emulsion is obtained;
(5) The rotating speed of a jacket reaction kettle is increased to 400rpm, 14L of absolute ethyl alcohol at 5 ℃ is added into the jacket reaction kettle at the speed of 10L/min through a flow pump, stirring is maintained for 5min after the adding is finished to demulsify gelatin emulsion, the demulsifying solution is pumped into a negative pressure filtering tank through the flow pump, the bottom of the filtering tank is a filtering screen with the aperture of 100 mu m, the negative pressure is set to be 0.03MPa, a valve at the bottom of the filtering tank is closed after the filtering is finished, 25L of absolute ethyl alcohol at 5 ℃ is added again for cleaning and filtering, and gelatin microspheres are obtained at the bottom of the filtering tank after repeated cleaning and filtering are carried out for 5 times;
(6) Turning off the negative pressure in a negative pressure filter tank, re-adding 50L of water at 5 ℃ into the filter tank, starting stirring to disperse gelatin microspheres into the water at a speed of 100rpm, turning off stirring after the dispersion is finished, starting a valve at the bottom of the filter tank to filter gelatin microsphere solution by means of gravity, repeating the water cleaning operation for 3 times after the filtration is finished, and obtaining gelatin microspheres at the bottom of the filter tank;
(7) Re-dispersing gelatin microspheres by adding 10L of water at 5 ℃ into a filter tank, adding 100ml of glutaraldehyde solution (25% aqueous solution), and then pumping the solution into 10 1L cylindrical plastic (polypropylene) containers, wherein the diameter of the inner cavity of each plastic container is 40mm;
(8) Immersing the container in-20deg.C cooling liquid for 6 hr after covering, completely freezing gelatin dispersion liquid in the container, taking out the flat container from the cooling liquid, and placing at-20deg.C for continuous crosslinking for 48 hr;
(9) Immersing the flat container in warm water at 37 ℃ for 15min after the crosslinking is finished to completely ice the internal ice cubes to obtain a gelatin microcarrier solution, then opening a container cover to pour the internal gelatin microcarrier solution into a 20L filter tank for negative pressure suction filtration, wherein the pore diameter of the filter screen of the filter tank is 50 mu M, the negative pressure is 0.2MPa, adding 20L of water for repeated cleaning for 2 times after the crosslinking is finished, then adding 0.1M/L glycine solution into the gelatin microcarrier collected by the filter screen for soaking for 2h, and cleaning for 3 times by using water after the filter screen is filtered to obtain the gelatin microcarrier;
(10) The gelatin microcarriers were redispersed in water and then freeze-dried to obtain irregular gelatin microcarriers as shown in figure 6.
In a preferred embodiment of the present invention, the flat container in the step (7) is rectangular, oval or diamond, the long side of the inner cavity of the cross section is L, the short side is D, and the length of the short side is less than or equal to 20mm, as shown in FIG. 7.
In summary, the gelatin microcarrier prepared in example 1 of the present invention has a streamline structure, the microcarrier has a uniform size distribution, the pore size and shape are uniformly distributed, the gelatin microcarrier prepared in example 2 has no streamline structure, the pore size and size distribution are not uniform, the pore-forming effect of the preparation method of comparative examples 1-2 using stainless steel flat plates and plastic containers is poor, and the microcarrier surface pore size is large due to air freezing, and a large number of lamellar structures appear.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.
Claims (9)
1. The preparation method of the gelatin microcarrier with the streamline shape is characterized by comprising the following steps:
(1) Preparing a gelatin aqueous solution with the concentration of 1-20%, heating the aqueous solution to 45-65 ℃ in the preparation process, and continuously stirring until gelatin is completely dissolved;
(2) Preparing a mixed solution of an oil phase and an emulsifier: mixing the emulsifier with the oil phase according to the concentration of 0.1-5%, and heating to 45-65 ℃ in a stirring state;
(3) Slowly adding the gelatin aqueous solution prepared in the step (1) into the mixed solution of the oil phase and the emulsifier prepared in the step (2), continuously stirring in the adding process, maintaining the temperature of the system at 45-65 ℃, and continuously stirring for 5-60 min after the adding is completed to obtain W/O type emulsion, wherein the mass ratio of the gelatin aqueous solution to the mixed solution of the oil phase and the emulsifier is 1:2-1:10;
(4) Cooling the W/O emulsion obtained in the step (3) to 1-15 ℃ and continuously stirring to solidify gelatin gel to obtain gelatin emulsion;
(5) Directly filtering the gelatin emulsion obtained in the step (4) or adding 20-200% of organic solution to demulsifie and then filtering, washing the filtered gelatin microspheres for 2-6 times by using the organic solvent, and finally filtering and collecting the gelatin microspheres;
(6) Slowly dispersing the gelatin microspheres obtained in the step (5) into deionized water under the stirring state, filtering and cleaning for 2-6 times, and finally filtering and collecting;
(7) Dispersing the gelatin microspheres obtained in the step (6) into deionized water, adding a cross-linking agent, reacting for 0-24 h at the temperature of 1-15 ℃, and then pouring the reaction solution into a flat container or a cylindrical container;
(8) Immersing the container filled with the reaction solution in the step (7) in a cooling liquid for 2-600 min, wherein the temperature of the cooling liquid is-80 to-5 ℃, taking out the gelatin solution after the gelatin solution is completely frozen, and placing the gelatin solution in an environment of-25 to-5 ℃ for continuous crosslinking for 12-48 h;
(9) After the crosslinking in the step (8) is completed, placing the container in normal temperature air or immersing the container in an aqueous solution at 10-40 ℃, carrying out deicing, filtering and recovering the microcarrier, and cleaning the microcarrier for 2-6 times by using deionized water;
(10) Redispersing the gelatin microcarrier obtained in the step (9) in water, freeze-drying and sterilizing to obtain the streamline porous gelatin microcarrier.
2. The method for preparing a porous gelatin microcarrier according to claim 1, wherein the gelatin of the aqueous gelatin solution in step (1) is form a and/or form B.
3. The method of claim 1, wherein the oil phase in step (2) is one or more of vegetable oil, mineral oil, petroleum ether, and hydrocarbon, and the hydrocarbon is hexane, heptane, cyclohexane, toluene, or xylene.
4. The method for preparing porous gelatin microcarrier according to claim 1, wherein the emulsifier in step (2) is one or a combination of several of tween, glyceryl monostearate and span.
5. The method of preparing a porous gelatin microcarrier according to claim 1, wherein the organic solvent in step (5) is one or a combination of several of ethanol, isopropanol and acetone.
6. The method of claim 1, wherein the deionized water in steps (6) to (7) is at a temperature of 1-15 ℃.
7. The method for preparing a porous gelatin microcarrier according to claim 1, wherein the cross-linking agent in step (7) is one or more of formaldehyde, glutaraldehyde, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide, tetramethyl ethylenediamine and genipin.
8. The method for preparing the porous gelatin microcarrier according to claim 1, wherein the container material in the step (7) is one or a combination of a plurality of glass, quartz, metal and alloy, the cross section of the flat container is flat, particularly rectangular, diamond-shaped or elliptic, the long side of the inner cavity of the cross section is L, the short side is D, the length of the short side is less than or equal to 20mm, the inner cavity of the cross section of the cylindrical container is circular, and the diameter of the circle is less than or equal to 20mm.
9. The method for preparing a porous gelatin microcarrier according to claim 1, wherein the pore size of the filter screen is 20-300 μm.
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