CN114516974B - Preparation method and application of porous gelatin microcarrier - Google Patents

Preparation method and application of porous gelatin microcarrier Download PDF

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CN114516974B
CN114516974B CN202210239078.3A CN202210239078A CN114516974B CN 114516974 B CN114516974 B CN 114516974B CN 202210239078 A CN202210239078 A CN 202210239078A CN 114516974 B CN114516974 B CN 114516974B
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CN114516974A (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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Abstract

The invention discloses a preparation method and application of a porous gelatin microcarrier, which comprises the steps 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 adopts only pure gelatin, and forms the porous gelatin microcarrier by adding alkaline solution, adding cross-linking agent, cross-linking and solidifying at room temperature under stirring, and emulsifying with oil phase solution. The pore size obtained by the method is uniform, and the porosity and the pore size can be regulated and controlled according to the addition amount of the alkaline solution and the like. The 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
Microcarriers are microbeads with a diameter of 60-250 μm, which are suitable for adherent cell growth, and which mainly consist of natural dextran or various synthetic polymers. Since the advent of the first microcarrier developed by Van Wezel using DEAE-Sephadex A50, the types of microcarrier commercial products sold in the International market have reached more than ten, including liquid microcarriers, macroporous gelatin microcarriers, polystyrene microcarriers, PHEMA microcarriers, chitin microcarriers, polyurethane foam microcarriers, alginate gel microcarriers, and magnetic microcarriers. Among the many microcarriers, gelatin microcarriers have been of interest due to their good cell adhesion properties.
Gelatin is a polypeptide mixture prepared by degrading collagen molecules, and eighteen different amino acids are shared to form a gelatin peptide chain, and the amino group at the 5 '-end of the carboxyl at the 3' -end. Gelatin is formed by condensing alpha-amino acid, wherein the main chain unit of the gelatin is CH-CO-NH-, and the polar groups in the side chains can be divided into acidic groups, alkaline groups and neutral groups.
Methods for preparing porous microcarriers by gelatin are currently available. CN113750076a discloses a chitin-gelatin based composite porous microsphere as a drug carrier and a preparation method and application thereof, wherein the preparation method comprises the following steps: synthesizing gelatin microspheres; preparing chitin solution and regulating pH value to neutrality; synthesizing chitin-gelatin composite porous microspheres (mixing chitin solution and gelatin microspheres in a mass ratio of 1:10-5:10, dripping 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 out excessive liquid paraffin, standing, and removing upper white emulsion; and adding deionized water for washing until the upper layer is free of oil drops, and freeze-drying. CN107073165B provides a preparation method capable of obtaining a porous microcarrier with unique properties and particle size distribution, and provides a gelatin porous microcarrier with excellent cell proliferation properties. According to the present invention, there are provided a method for preparing a porous microcarrier of gelatin comprising a process of emulsification using the content of an emulsifier having a specific HLB value, and a porous microcarrier of gelatin having a specific void.
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 research and development of the method for preparing the porous gelatin microcarrier has the advantages of simple and easy preparation process, controllable method, low cost, high biological safety and high value, and is easy for industrial production.
Disclosure of Invention
The invention aims to provide a preparation method of a porous gelatin microcarrier, which has the advantages of simple and easy preparation process, controllable method, low cost, high biological safety and easy industrial production.
Based on the above objects, the present invention meets this need in the art by providing a method for preparing a porous gelatin microcarrier.
On the one handThe invention relates to a preparation method of a porous gelatin microcarrier, which comprises the steps of adding an alkaline solution into a gelatin solution to prepare the porous gelatin microcarrier; the alkaline solution can ionize OH － Is a solution of (a); the proportion of the alkaline solution to the gelatin solution is 1:10-40 by volume.
Further, in the preparation method of the porous gelatin microcarrier provided by the 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 0.25mol/L.
Further, in the preparation method of the porous gelatin microcarrier provided by the 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 provided by the application, 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 fixation 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 crosslinking fixation time is 1-12 h.
Further, in the preparation method of the porous gelatin microcarrier provided by the application, the method further comprises the following steps: and (3) obtaining an aqueous phase solution after the crosslinking and fixing are completed, mixing the aqueous phase solution with an organic phase solution, stirring and emulsifying to generate W/O type liquid drops, reacting for 24 hours at room temperature, 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 60deg.CAdding gelatin for swelling at 80 ℃ and fully dissolving by using deionized water, wherein the final concentration is 5% -20%. Cooling to 37deg.C, adding OH － Adding an alkaline solution with the concentration of 0.25mol/L into the premix solution, stirring, and then adding a cross-linking agent for cross-linking and fixing to obtain a water phase solution;
the proportion 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 saturated straight-chain aliphatic dialdehyde, the mass percentage concentration is 2.5-25%, the ratio of the cross-linking agent to the gelatin solution is 1:10, and the cross-linking time is 1-24 h; preferably, glutaraldehyde is used as the crosslinking agent, the mass percentage concentration is 2.5%, the ratio of glutaraldehyde to gelatin solution is 1:10 by volume ratio, and the crosslinking time is 1-12 h.
Mechanically stirring the organic phase solution for 1h at a rotating speed of 700rpm to remove bubbles generated in the solution; the organic phase solution is edible oil; preferably, the organic phase solution is sunflower seed oil. Mixing the aqueous phase solution and the organic phase solution, stirring at a stirring 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. The reaction was carried out at room temperature for 24h, the emulsion was collected, the upper organic phase was filtered off and the microcarrier was collected. And cleaning the collected microcarriers by using 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 porous gelatin microcarrier, thus achieving the purpose of controlling pore diameter and porosity. The obtained porous gelatin microcarrier is subjected to stem cell micro-tissue culture test, so that the obtained stem cell micro-tissue is more compact and is closer to animal tissue properties. Thus, the invention 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, so that the reaction rate of forming gel by the gelatin solution is controlled, and further, the crosslinking degree and the crosslinking time of the gel are controlled, so that the pore diameter and the porosity of the porous gelatin microcarrier are uniformly regulated and controlled. The preparation process does not need to modify gelatin or adopt special reagents, and the preparation method is simple and feasible, controllable, low in cost, high in biological safety and easy for industrial production.
Drawings
FIG. 1 is a TEM image of a multi-Kong Mingjiao microcarrier made in example 1.
FIG. 2 is a TEM image of a multi-Kong Mingjiao microcarrier made in example 2.
FIG. 3 is a TEM image of a multi-Kong Mingjiao microcarrier made in example 3.
FIG. 4 is a TEM image of a multi-Kong Mingjiao microcarrier made in example 4.
FIG. 5 is an image of a cultured 5 day bovine umbilical cord-derived stem cell microtome HE section. FIG. 5a is an image of a section of a commercially available microcarrier cultured 5-day bovine umbilical cord-derived stem cell microtome; FIG. 5b is an image of a micro-tissue HE slice of 5-day bovine umbilical cord-derived stem cells cultured with a multi-Kong Mingjiao microcarrier prepared in example 4.
Detailed Description
The following describes the technical aspects of the present invention with reference to examples, but the present invention is not limited to the following examples.
Example 1
The present example provides a porous gelatin microcarrier preparation test using type a gelatin as the starting material.
In the test, the gelatin solution is prepared by heating in water bath to 60-80 ℃, adding gelatin A for swelling, and fully dissolving with deionized water, wherein the final concentration is 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 gelatin A to swell, and dissolving with deionized water in the final concentration of 5%. After the temperature had dropped to 37 ℃, 1/40,0.25M of the volume of the gelatin solution was added and stirred. Then adding glutaraldehyde with the volume of 1/10 of that of the gelatin solution, and carrying out crosslinking fixation for 1h to obtain an aqueous phase solution.
Mechanically stirring sunflower seed oil at 700rpm for 1h to remove bubbles generated in the solution and obtain an 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. The reaction was carried out at room temperature for 24h, the emulsion was collected, the upper organic phase was filtered off and the microcarrier was collected.
The collected microcarriers were washed. Firstly, petroleum ether is used for ultrasonic cleaning, secondly, acetone which is fully precooled is used for ultrasonic cleaning, precipitated microcarrier particles are collected, the microcarrier particles are dried at the temperature of 37 ℃, deionized water is added again for ultrasonic cleaning, and the ultrasonic cleaning is carried out for 5 times. The cleaned microcarrier is wetted in a small amount of water, and is transferred to a freezing device at the temperature of minus 20 ℃ for pre-freezing, and is transferred to a freeze dryer for freeze drying at the temperature of minus 40 ℃ for 24 hours after freezing. Sterilization with Co 60 irradiation.
The pore diameter 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
The present example provides a porous gelatin microcarrier preparation test with a 1:20 ratio of alkaline solution to gelatin solution.
In the test, the gelatin solution is prepared by heating in water bath at 60-80 ℃, adding gelatin A for swelling, and fully dissolving with deionized water, wherein the final concentration is 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 gelatin A to swell, and dissolving with deionized water to final concentration of 20%. After the temperature had dropped to 37 ℃, 1/20,0.25M of the volume of the gelatin solution was added and stirred. Then, 1/10 of the volume of the gelatin solution and 25% glutaraldehyde were added to carry out crosslinking fixation for 24 hours, thereby obtaining an aqueous phase solution.
Mechanically stirring sunflower seed oil at 700rpm for 1h to remove bubbles generated in the solution and obtain an 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. The reaction was carried out at room temperature for 24h, the emulsion was collected, the upper organic phase was filtered off and the microcarrier was collected.
The collected microcarriers were washed. Firstly, petroleum ether is used for ultrasonic cleaning, secondly, acetone which is fully precooled is used for ultrasonic cleaning, precipitated microcarrier particles are collected, the microcarrier particles are dried at the temperature of 37 ℃, deionized water is added again for ultrasonic cleaning, and the ultrasonic cleaning is carried out for 5 times. The cleaned microcarrier is wetted in a small amount of water, and is transferred to a freezing device at the temperature of minus 20 ℃ for pre-freezing, and is transferred to a freeze dryer for freeze drying at the temperature of minus 40 ℃ for 24 hours after freezing. Sterilization with Co 60 irradiation.
The pore diameter 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
The present example provides a porous gelatin microcarrier preparation test with a gelatin solution mass percentage concentration of 10%.
In the test, the gelatin solution is prepared by heating in water bath at 60-80 ℃, adding gelatin A for swelling, and fully dissolving by using deionized water, wherein the final concentration is 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 gelatin A to swell, and dissolving with deionized water in the final concentration of 10%. After the temperature had dropped to 37 ℃, 1/10,0.25M of the volume of the gelatin solution was added and stirred. Then, glutaraldehyde of 1/10 of the volume of the gelatin solution and 2.5% of the volume of the gelatin solution were added to carry out crosslinking fixation for 12 hours to obtain an aqueous phase solution.
Mechanically stirring sunflower seed oil at 700rpm for 1h to remove bubbles generated in the solution and obtain an 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. The reaction was carried out at room temperature for 24h, the emulsion was collected, the upper organic phase was filtered off and the microcarrier was collected.
The collected microcarriers were washed. Firstly, petroleum ether is used for ultrasonic cleaning, secondly, acetone which is fully precooled is used for ultrasonic cleaning, precipitated microcarrier particles are collected, the microcarrier particles are dried at the temperature of 37 ℃, deionized water is added again for ultrasonic cleaning, and the ultrasonic cleaning is carried out for 5 times. The cleaned microcarrier is wetted in a small amount of water, and is transferred to a freezing device at the temperature of minus 20 ℃ for pre-freezing, and is transferred to a freeze dryer for freeze drying at the temperature of minus 40 ℃ for 24 hours after freezing. Sterilization with Co 60 irradiation.
The pore diameter 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
The present example provides a porous gelatin microcarrier preparation test using type B gelatin as the starting material.
In the test, the gelatin solution is prepared by heating in water bath at 60-80 ℃, adding type B gelatin for swelling, and fully dissolving by using deionized water, wherein the final concentration is 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 gelatin B to swell, and dissolving with deionized water in the final concentration of 10%. After the temperature had dropped to 37 ℃, 1/10,0.25M of the volume of the gelatin solution was added and stirred. Then, glutaraldehyde of 1/10 of the volume of the gelatin solution and 2.5% of the volume of the gelatin solution were added to carry out crosslinking fixation for 12 hours to obtain an aqueous phase solution.
Mechanically stirring sunflower seed oil at 700rpm for 1h to remove bubbles generated in the solution and obtain an 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. The reaction was carried out at room temperature for 24h, the emulsion was collected, the upper organic phase was filtered off and the microcarrier was collected.
The collected microcarriers were washed. Firstly, petroleum ether is used for ultrasonic cleaning, secondly, acetone which is fully precooled is used for ultrasonic cleaning, precipitated microcarrier particles are collected, the microcarrier particles are dried at the temperature of 37 ℃, deionized water is added again for ultrasonic cleaning, and the ultrasonic cleaning is carried out for 5 times. The cleaned microcarrier is wetted in a small amount of water, and is transferred to a freezing device at the temperature of minus 20 ℃ for pre-freezing, and is transferred to a freeze dryer for freeze drying at the temperature of minus 40 ℃ for 24 hours after freezing. Sterilization with Co 60 irradiation.
The pore diameter 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 cell micro-tissue culture comparison test of the porous gelatin microcarrier obtained in example 4 with a commercially available microcarrier.
The model of the commercial microcarrier is F01-100/3D Table
Figure BDA0003543565350000101
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Figure BDA0003543565350000102
After the microcarriers prepared in example 4 were sterilized by Co 60 irradiation, 125mL cell reactors were selected and the experiment was divided into two groups:
experiment group 1:100mg of commercially available microcarrier +2.5X10 4 cells/mL bovine umbilical cord mesenchymal stem cells +50mL alpha-MEM culture medium containing 5% bovine platelet lysate; experiment group 2:100mg of microcarrier prepared in example 4 +2.5X10 4 cell culture was performed by cell/mL of bovine umbilical cord mesenchymal stem cells+50 mL of alpha-MEM culture medium containing 5% bovine platelet lysate. 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, the obtained stem cell micro-tissue was subjected to HE section staining, and the HE staining results are shown in FIG. 5.
The micro-tissue HE section shown in FIG. 5 shows that compared with the micro-carrier (FIG. 5 a) on the market, the micro-tissue HE section provided by the invention has more compact stem cell micro-tissue cultured by the micro-carrier provided by the invention and is more similar to animal tissue characters.
The present invention may be better implemented as described above, and the above examples are merely illustrative of preferred embodiments of the present invention and not intended to limit the scope of the present invention, and various changes and modifications made by those skilled in the art to the technical solution of the present invention should fall within the scope of protection defined by the present invention without departing from the spirit of the design of the present invention.

Claims (5)

1. The preparation method of the 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 gelatin is gelatin type A or gelatin type B; the mass percentage concentration of the gelatin solution is 5% ；
The alkaline solution can ionize OH － Is a solution of (a); the ratio of the alkaline solution to the gelatin solution is 1:40 by volume;
the alkaline solution is NaOH solution ； OH in the NaOH solution － The concentration of (C) is 0.25mol/L;
adding an alkaline solution into the gelatin solution, and then adding a cross-linking agent with the mass percentage concentration of 2.5-25% to carry out cross-linking fixation for 1-12 h; the cross-linking agent is glutaraldehyde; the proportion of the cross-linking agent glutaraldehyde to the gelatin solution is 1:10 by volume.
2. The method of preparing a porous gelatin microcarrier according to claim 1, wherein said method further comprises: and (3) obtaining an aqueous phase solution after the crosslinking and fixing are completed, mixing the aqueous phase solution with an organic phase solution, stirring and emulsifying to generate W/O type liquid drops, reacting for 24 hours at room temperature, collecting emulsion, cleaning, freeze drying and sterilizing.
3. The method of preparing a porous gelatin microcarrier of claim 2, wherein said organic phase solution is an edible oil comprising sunflower seed oil.
4. A microcarrier prepared by the method of any one of claims 1-3.
5. Use of the microcarrier according to claim 4 in cell culture.
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