CN116462879B - Gelatin microsphere carrier and application thereof - Google Patents

Abstract

The present application discloses a gelatin microsphere carrier and its application. The gelatin is used as the main body, and an aqueous solution is prepared. Citric acid is added to adjust the pH to acidic to inhibit the reaction of the cross-linking agent genipin. Genipin is then added as a cross-linking agent, and the solution is dripped into liquid nitrogen for one-step freezing. The solution is then reacted with sodium bicarbonate to adjust the pH to alkaline to promote cross-linking and curing of genipin to obtain a gelatin microsphere carrier. The gelatin microsphere carrier of the present application has a simple preparation process, strong stability, is conducive to storage, and has a low preparation cost. It is harmless to cells, can effectively load cells and expand cells, and can be used for 3D cell culture.

Description

A gelatin microsphere carrier and its application

Technical Field

The present application relates to the technical field of biomedical materials, and in particular to a gelatin microsphere carrier and its application.

Background Art

Compared with traditional planar cell culture technology, three-dimensional cell culture technology can more realistically simulate the living environment of cells in the body, more realistically restore the interactions between cells and physiological and biochemical reactions, as well as the actual response of cells to changes in endogenous and exogenous stimuli such as temperature, pH, transport and differentiation. 3D cell culture can be used to construct organoid models or tumor models, and conduct more accurate and appropriate research such as drug metabolic dynamics, drug efficacy evaluation, and more intuitive cell biology research, making 3D cell culture an indispensable research tool.

At present, using gelatin as a cell loading matrix or making it into a spherical carrier is a hot topic in the current 3D cell culture research. Gelatin is the main product of partial hydrolysis of animal collagen. It has good biological properties such as biodegradability, biocompatibility, non-toxicity, hydrophilicity and non-immunogenicity. It is a functional biopolymer material widely used in the fields of medicine, food, tissue engineering and agriculture.

The preparation technology of gelatin microspheres is relatively mature. Currently, there are many preparation methods such as emulsification cross-linking, solvent volatilization, spray drying and phase separation. Most methods are to emulsify, stir and shear the oil phase and the water phase into balls, and then use cross-linking agents such as formaldehyde and glutaraldehyde to solidify into gelatin microspheres. There are also other cross-linking agents used for reaction, such as CN109793717A discloses a preparation method and application of gelatin microspheres, with gelatin aqueous solution as the water phase and paraffin as the oil phase emulsified and stirred into balls after freezing, and then cross-linked by genipin. Or it is a method of forming by microfluidic control chip, such as CN110200922B discloses a preparation method and application of gelatin microspheres, with gelatin aqueous solution as the water phase and oil as the oil phase through a microfluidic device into droplets into the collection phase, and then gelled at 4-12°C, and finally cross-linked with genipin. The cross-linking agent genipin used in these two preparation methods is a plant natural cross-linking agent that can be cross-linked with proteins, collagen, gelatin and chitosan to make biomaterials. Its toxicity is much lower than glutaraldehyde and other commonly used chemical cross-linking agents, but these two preparation methods require the use of an oil phase during the microsphere forming process. For example, CN112250892A first sprays or freeze-dries the gelatin solution to obtain a microsphere precursor, and then cross-links and solidifies the microsphere precursor in a poor solvent such as methanol, acetone and acetonitrile or an aqueous solution. However, CN112250892A uses an organic solvent for solidification or an organic solvent to protect the spherical shape during the fixing stage, and it must be removed after the spheroidization, which makes the cleaning step more troublesome and increases the cost. There are also microspheres that do not use crosslinking agents to solidify microspheres, such as CN112266487A, which uses chitosan as a skeleton to fix gelatin, freeze-sprays chitosan gelatin solution, and then freeze-dries under the protection of excipients such as sugar, dextran, gelatin, mannitol, etc. to obtain microspheres. However, this method requires spray drying and multiple freeze-drying processes, and the preparation process is complicated and time-consuming, involving the use of different equipment, which increases the cost. Therefore, it is necessary to provide a preparation method for microspheres that is simple and safe in preparation, green and environmentally friendly, does not harm the activity of cells, can be conducive to stem cell attachment and expansion, and saves costs and is convenient for large-scale production.

Summary of the invention

The present invention provides a gelatin microsphere carrier, which is prepared by the following method:

Step 1, dissolving gelatin in warm water and stirring evenly to obtain a gelatin aqueous solution;

Step 2: Add citric acid to the gelatin aqueous solution prepared in step 1 to adjust the pH to ≤3, dissolve and mix well to obtain a citric acid gelatin solution; using citric acid to adjust the pH of the gelatin aqueous solution to ≤3 can inhibit the reaction activity of the cross-linking agent and extend the use time of the mixed solution of gelatin, citric acid and genipin.

Step 3, adding genipin powder to the citric acid gelatin solution prepared in step 2, dissolving and mixing well to prepare a gelatin mixed solution;

Step 4, dripping the gelatin mixed solution prepared in step 3 into liquid nitrogen to solidify and form gelatin microspheres;

Step 5, place the gelatin microspheres prepared in step 4 into a sodium bicarbonate solution, react for 3 hours or until the spheres turn light blue to indicate the completion of the reaction, then wash with water to obtain a gelatin microsphere carrier.

Furthermore, the preparation method of the gelatin microsphere carrier also includes one or more of the following (1)-(7):

(1) The amount of gelatin added per liter of the gelatin aqueous solution is 50-300 g;

(2) the amount of citric acid added to each liter of the citric acid gelatin solution is 10-50 g;

(3) the amount of genipin added to each liter of the gelatin mixed solution is 3-10 g;

(4) dripping the gelatin mixed solution into liquid nitrogen through a needle;

(5) the pH value of the sodium bicarbonate solution is 8-9;

(6) The concentration of the sodium bicarbonate solution is 10-50 g/L;

(7) The temperature of the warm water is 55-60°C.

The gelatin is a mixture of 250g bloom gelatin and 100g bloom gelatin, with a mass ratio of 0.5-1:0-0.5. The source of the gelatin is one or more of fish skin, pig skin, pig bone, cow skin and cow bone.

The needle model is a No. 4 or No. 5 needle.

Alternatively, genipin powder may be first added to the gelatin aqueous solution prepared in step 1 and then citric acid may be added to adjust the pH to ≤ 3 to obtain the gelatin mixed solution.

The present invention also provides an application of the aforementioned gelatin microsphere carrier in the cell 3D culture industry chain.

Furthermore, the gelatin microsphere carrier is used for 3D culture of stem cells.

The gelatin microsphere carrier is sterilized by ethanol and/or irradiation and then used for 3D culture of stem cells. Specifically, the ethanol sterilization is to soak the gelatin microsphere carrier in anhydrous ethanol for 30 minutes; and/or the irradiation sterilization is to irradiate the gelatin microsphere carrier under ultraviolet light overnight.

The beneficial effects of the present invention include: the gelatin microsphere carrier provided by the present invention has a simple preparation process, and only requires the raw materials gelatin, citric acid and genipin to be mixed and then frozen and molded, thereby reducing the use of organic solvents such as various water-immiscible oil phases and oil phase cleaning agents; the curing process uses a natural cross-linking agent genipin, which is harmless to the human body and has no effect on the activity of cells, and does not require a removal step like the use of formaldehyde or glutaraldehyde cross-linking agents; the reaction activity of genipin can be inhibited by controlling the pH to be lower than 3, and the storage or use time of the gelatin citrate genipin mixture can be extended for several days, and the reaction activity can still be restored when the pH is adjusted to be higher than 8, and genipin has a certain antibacterial property to protect the raw material liquid from being decomposed by bacteria; the gelatin microspheres are pre-molded by freezing with liquid nitrogen, which can make the gelatin microspheres The shape of the gelatin microsphere precursor does not change in water at room temperature, so there is no need to use excipients or poor solvents for protection; the sodium bicarbonate detergent used can neutralize the citric acid in the microsphere precursor and generate bubbles to create pores on the surface of the sphere. The pH of sodium bicarbonate is milder than that of sodium hydroxide or sodium carbonate, and is more suitable for cross-linking reactions with genipin; the size of the microsphere can be adjusted by selecting different types of pinholes and flow rates; the microsphere preparation process is simple, which is more cost-effective than other preparation methods and has the potential for large-scale production; gelatin microspheres are stable and have a certain acid and alkali resistance, which is conducive to storage; gelatin microspheres can be specifically decomposed by collagenase without damaging cells, have a certain mechanical strength, can effectively load and expand cells, and are harmless to cells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a picture of the gelatin microsphere carrier prepared in Example 1 under a four-fold microscope lens;

FIG2 is a scanning electron microscope image of the surface morphology of the gelatin microsphere carrier prepared in Example 1;

FIG3 shows the attachment of hair follicle stem cells to the surface of gelatin microsphere carrier;

Figure 4 shows the viability of cells attached to the surface of gelatin microsphere carriers.

DETAILED DESCRIPTION

The present invention is further illustrated and described below in conjunction with embodiments, but the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the present invention and embodiments, all other inventions and embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

Example 1. Preparation of gelatin microsphere carrier

The specific preparation method is as follows:

(1) Weigh 0.7 g of 250 g bloom gelatin and 0.3 g of 100 g bloom gelatin, mix them, add them into 10 mL of 55°C deionized water, and stir for 10 min to completely dissolve.

(2) Weigh 0.2 g of anhydrous citric acid and add it to the prepared gelatin aqueous solution, stir and dissolve, and adjust the pH to below 3 to obtain a citric acid gelatin solution.

(3) Weigh 0.05 g of genipin and add it to the citric acid gelatin aqueous solution prepared in step (2), stir and dissolve to prepare a gelatin mixed solution.

(4) The gelatin mixed solution obtained in step (3) is aspirated and dripped into a thermos pot filled with liquid nitrogen through a No. 4 needle to solidify the solution.

(5) Weigh 5 g of sodium bicarbonate and add it to 500 mL of deionized water to completely dissolve it; soak the gelatin microspheres prepared in step (4) in the sodium bicarbonate aqueous solution for 3 h, and after the color of the spheres turns light blue, take them out and wash them with water to obtain gelatin microsphere carriers.

The obtained gelatin microsphere carriers were observed for particle size and surface morphology under a microscope and a scanning electron microscope. Figure 1 is a picture of the gelatin microsphere carrier under a four-fold microscope lens. It can be seen that the gelatin microsphere carrier is a semi-transparent particle with a size of about 500 μm and is light blue. Figure 2 is a picture of the surface morphology of the gelatin microsphere carrier under a scanning electron microscope. Among them, A in Figure 2 is a magnification of 1000 times. It can be seen that there are a large number of gully-shaped wrinkles on the surface of the gelatin microsphere carrier, which increases the specific surface area of the microspheres. B in Figure 2 is a field of view with a magnification of 2000 times. It can be observed that the width of the concave part of the gully is about 10 μm, and the width of the raised part is greater than 20 μm, which can be used for cell attachment.

Example 2 Gelatin microsphere carrier for 3D culture of hair follicle stem cells

Take 12 gelatin microsphere carriers prepared in Example 1, put them into 10 ml of anhydrous ethanol and soak for 30 minutes, repeat three times. Then place them under ultraviolet light for overnight irradiation until the alcohol is completely evaporated. Rinse the gelatin microsphere carriers with PBS buffer, take out the microsphere carriers, add culture medium and incubate in a 37°C incubator for 30 minutes. After the incubation, add 100,000 hair follicle stem cell suspensions and culture them in a 37°C incubator for 48 hours.

The gelatin microsphere carriers containing hair follicle stem cells incubated for 48 hours were fixed with glutaraldehyde and freeze-dried together with ordinary gelatin microspheres. The surface morphology of the microsphere carriers and the surface cell attachment were observed under a scanning electron microscope. The results are shown in Figure 3. A and B in Figure 3 are the cell attachment conditions on the surface of gelatin microspheres at magnifications of 1000 times and 2000 times, respectively. From Figure 3 A, it can be observed that hair follicle stem cells can attach to the grooves on the surface of the gelatin microsphere carrier. From Figure 3 B, it can be observed that the size of hair follicle stem cells is about 10μm. The hair follicle stem cells adhere to the surface of the gelatin microsphere carrier by extending synapses, and proliferate and expand in groups.

Example 3 Gelatin microsphere carrier cytotoxicity detection

Take 24 gelatin microsphere carriers prepared in Example 1, put them into 10 ml of anhydrous ethanol and soak for 30 minutes, repeat three times. Then place them under ultraviolet light for overnight irradiation until the alcohol is completely evaporated. Rinse the gelatin microsphere carriers with PBS buffer, take out the microsphere carriers, add culture medium and incubate in a 37°C incubator for 30 minutes. After the incubation, divide them into two groups and add 100,000 hair follicle stem cell suspensions and non-small cell lung cancer cell suspensions respectively, and culture them in a 37°C incubator for 48 hours.

Four microspheres were taken out from each of the two culture systems at 24h and 48h, rinsed with PBS solution, added with CCK-8 reagent and incubated in a 37°C incubator for 2h, and then taken out and the absorbance at 450nm was measured with an enzyme marker. Figure 4 shows the viability of cells attached to the surface of gelatin microspheres. The results show that the OD value at 48h is higher than that at 24h, indicating that the hair follicle stem cells and non-small cell lung cancer cells on the surface of the gelatin microsphere carrier are in a proliferative state and their cell number is increasing. Since the attachment ability of hair follicle stem cells is relatively weak and the proliferation rate of lung cancer cells is faster, the OD value of hair follicle stem cells in the two time periods of 24h and 48h is lower than that of non-small cell lung cancer cells, but overall they show an increasing trend. It shows that the gelatin microsphere carrier prepared by the present invention is harmless to cells and is conducive to cell proliferation.

Claims (7)

1. A gelatin microsphere carrier, characterized in that the gelatin microsphere carrier is prepared by the following method: Step 1, dissolving gelatin in warm water and stirring evenly to obtain a gelatin aqueous solution; Step 2, adding citric acid to the gelatin aqueous solution prepared in step 1 to adjust the pH to ≤ 3, dissolving and mixing well to obtain a citric acid gelatin solution; Step 3, adding genipin powder to the citric acid gelatin solution prepared in step 2, dissolving and mixing well to prepare a gelatin mixed solution; Step 4, dripping the gelatin mixed solution prepared in step 3 into liquid nitrogen to obtain gelatin microspheres; Step 5: Place the gelatin microspheres prepared in step 4 into a sodium bicarbonate solution, react for 3 h or until the microspheres turn light blue, then wash with water to obtain a gelatin microsphere carrier. The gelatin is a mixture of gelatin with a gel strength of 250 g bloom and gelatin with a gel strength of 100 g bloom, and the mass ratio thereof is 0.5-1:0-0.5. 2. The gelatin microsphere carrier according to claim 1, characterized in that it comprises one or more of the following (1)-(7): (1) The amount of gelatin added to each liter of the gelatin aqueous solution is 50-300 g; (2) The amount of citric acid added to each liter of the citric acid gelatin solution is 10-50 g; (3) The amount of genipin added to each liter of the gelatin mixed solution is 3-10 g; (4) Inject the gelatin mixed solution into liquid nitrogen through a needle; (5) The pH value of the sodium bicarbonate solution is 8-9; (6) The concentration of the sodium bicarbonate solution is 10-50 g/L; (7) The temperature of the warm water is 55-60°C. 3. The gelatin microsphere carrier according to claim 2, characterized in that the needle model is a No. 4 or No. 5 needle. 4. Use of the gelatin microsphere carrier according to any one of claims 1 to 3 in the field of cell 3D culture. 5. The use according to claim 4, characterized in that the gelatin microsphere carrier is used for 3D culture of stem cells. 6. The use according to claim 5, characterized in that the gelatin microsphere carrier is sterilized with ethanol and/or irradiation and then used for 3D culture of stem cells. 7. The use according to claim 6 is characterized in that the ethanol sterilization is to soak the gelatin microsphere carrier in anhydrous ethanol for 30 minutes; and/or the irradiation sterilization is to irradiate the gelatin microsphere carrier under ultraviolet light overnight.