Preparation process and reaction device of chitosan cell culture carrier

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to a preparation process of a chitosan cell culture carrier and a reaction device. Comprising the following steps: preparing an organic solvent solution containing an emulsifier, preparing a chitosan solution, placing the prepared chitosan solution in a membrane emulsifier, enabling the chitosan solution to enter the prepared organic solvent solution through a membrane tube under a certain air pressure condition, carrying out heat preservation for a period of time under the conditions of vacuumizing and certain temperature, and carrying out chitosan curing, the preparation method comprises the following steps: curing the chitosan microspheres, cooling and filtering after curing to obtain solid chitosan microspheres, washing the prepared solid chitosan microspheres with water to remove inorganic salt, placing the solid chitosan microspheres in an aqueous solution containing a cross-linking agent, carrying out a cross-linking reaction for a certain time, filtering after the cross-linking reaction is completed, washing with water, and drying to obtain the porous chitosan microspheres with uniform particle sizes. The chitosan microsphere prepared by the method has a porous structure and a large specific surface area, can be used as a microcarrier for bearing more cells, is used for cell culture, and is beneficial to adhesion, growth and reproduction of the cells.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to a preparation process and a reaction device of a chitosan cell culture carrier. Background Art

[0002] Cell culture carriers refer to materials used to support and grow cells during the cell culture process. Due to the particularity of their spherical three-dimensional structure, they are widely used in the preparation of tissue engineering materials, vaccine preparation, virus research, etc. in biomedical research and bioengineering. Their surfaces have been specially chemically modified to improve biological properties such as cell attachment, proliferation and differentiation. It is reported that the size of microcarriers that are more suitable for cell culture is about 100-300μm. (Tavassoli H, et al., Large-scale Production of Stem Cells Utilizing Microcarriers: A Biomaterials Engineering Perspective from Academic Research to Commercialized Products [J], Biomaterials, 2018, 181: 333-346)

[0003] At present, the commonly used cell carrier materials on the market are mainly CytodexⅠ, CytodexⅡ, CytodexⅢ, Cytopore, Cytoline, etc. Because there are more hydroxyl functional groups on the dextran molecular chain, it is hydrophilic and suitable for cell culture carriers. However, its production requires multiple processes (bioreactor solvents, polymer materials, additives, reducing agents, etc. react and neutralize each other), the process is complicated, the price is expensive, and it is not conducive to large-scale application in cell culture.

[0004] Chitosan is a natural, biodegradable polymer material. It is the only polycationic polysaccharide in the biological world. It can be degraded into glucosamine in the body and absorbed by the human body. It has excellent biocompatibility and biodegradability and is an ideal extracellular matrix material. As a result, chitosan and its derivatives are widely used in the biomedical field (Mao J, Liu H, Yin Y, et al. the properties of chitosan-gelatin membranes and scaffolds modified with hyaluronic acid by different methods. Biomaterials. 2003, 24 (9): 1621-1629). At the same time, the molecular structure of chitosan is similar to that of aminodextran, a component of the extracellular matrix of liver cells. The three-dimensional microenvironment constructed by chitosan with positive charge characteristics can better support the growth and reproduction of animal cells in vitro and maintain the function of cells in vivo, making it an ideal scaffold material for liver tissue engineering (He JK, Li DC, Yao B, et al. Preparation of chitosan-gelatin hybrid scaffolds with well-organized microstructures for hepatic tissue engineering. Acta Biomaterialia. 2009. 5 (1): 453-461)

[0005] At present, the method for preparing microcarriers using chitosan mainly includes emulsification crosslinking method, coagulation / precipitation method, spray drying method, coagulation method, reverse micelle method, emulsion droplet coagulation method and filtration method etc. (Ma Guanghui, Su Zhiguo compiled. Polymer microsphere materials, first edition. Beijing, Chemical Industry Press, 2005.). The microcarriers prepared by these technologies are mostly solid, difficult to control with particle diameter, and need to further screen the microcarriers of suitable particle size range. And the microcarrier specific surface area prepared by it is relatively limited, still limits its high density and high activity to carry out cell culture to a certain extent, is unfavorable for the adhesion and growth and reproduction of cells. Summary of the invention

[0006] The technical problem to be solved by the present invention is to overcome the deficiencies of the above-mentioned prior art and provide a preparation process and a reaction device for chitosan cell culture carriers. The process prepares chitosan microcarriers with uniform particle size and multiple pores that can be used for cell culture, which can provide a three-dimensional environment for cell adhesion, growth and reproduction, while maintaining the normal function and vitality of cells.

[0007] To achieve the above object, the technical solution adopted by the chitosan cell culture carrier of the present invention is:

[0008] A preparation process of a chitosan cell culture carrier, comprising:

[0009] S1: preparing an organic solvent solution: stirring and dissolving a surfactant in an organic solvent to form an organic solvent solution;

[0010] S2: preparing a chitosan solution: stirring and dissolving chitosan in an acidic solution containing an inorganic salt to form a chitosan solution;

[0011] S3 uses a reaction device to prepare chitosan microspheres: the chitosan solution prepared in S2 is placed in a membrane emulsifier. Under a certain air pressure condition, the chitosan solution passes through the membrane emulsifier and flows through the membrane tube, enters the organic solvent solution, and forms liquid beads with uniform particle size. After vacuuming and keeping warm for a certain time at a certain temperature, the chitosan is solidified. After the solidification is completed, the temperature is lowered and filtered to obtain chitosan microspheres. The organic solvent solution obtained by filtration is recovered and reused for the next batch;

[0012] S4 Chitosan microsphere pore making: The solid chitosan microspheres prepared in S3 are washed with water to remove inorganic salts, and the washed chitosan microspheres are placed in an aqueous solution containing a crosslinking agent, and crosslinking reaction is carried out for a period of time under certain temperature conditions. After the crosslinking reaction is completed, the microspheres are filtered, washed with water and dried to obtain porous chitosan microspheres with uniform particle size.

[0013] Preferably, the surfactant in step S1 is a surfactant soluble in an organic solvent with a boiling point higher than 110° C., specifically lauryl ether phosphate, fatty alcohol polyoxyethylene ether or disodium coconut oil monoethanolamide sulfosuccinate, preferably lauryl ether phosphate.

[0014] Preferably, the concentration of the surfactant in the organic solvent is 1wt%-10wt%, preferably 3wt%-5wt%, and the volume ratio of the organic solvent solution to the chitosan solution is (3-20):1, preferably 5:1.

[0015] Preferably, the concentration of the chitosan solution in step S2 is 0.5wt%-10wt%, preferably 1wt%-5wt%, more preferably 1wt%-4wt%, the molecular weight of the chitosan is 50,000-600,000, preferably 150,000-400,000, more preferably 300,000; the degree of deacetylation is 70%-95%, preferably 85%.

[0016] Preferably, the acid solution in step S2 is a 2wt% acetic acid or formic acid solution, preferably a formic acid solution, the soluble inorganic salt is a sodium salt or a potassium salt, preferably a food-grade sodium salt, and the mass ratio of chitosan to inorganic salt is 3:1-1:1, preferably 2:1.

[0017] Preferably, in step S3, the chitosan film emulsification pressure condition is 0.01-0.1MPa, preferably 0.02-0.05MPa, and more preferably 0.02MPa. The chitosan curing temperature is 90°C-180°C, preferably 95°C-120°C, and more preferably 105°C. The insulation time is 5-15h, preferably 10h. After curing, the temperature is lowered to room temperature.

[0018] Preferably, in step S4, the cross-linking reaction temperature is 50-80° C., preferably 60° C., the cross-linking reaction time is 0.5-5 h, and the cross-linking agent is polyphosphate, genipin or sodium glycerophosphate, preferably sodium glycerophosphate.

[0019] A reaction device for a chitosan cell culture carrier preparation process, comprising a heatable reactor, a heating jacket being provided at the outer edge of the reactor, a vacuum pump being provided at one side of the reactor connected thereto via a pipeline, a condensation recovery device being provided on the pipeline connecting the reactor and the vacuum pump, and a membrane emulsifier being provided at the other side of the reactor connected to a membrane tube via a catheter, and the membrane tube being completely immersed in an organic solvent solution containing a surfactant after being checked for qualified air tightness.

[0020] Preferably, the condensate recovery device includes a condensate recovery device connected to the pipeline connecting the vacuum pump and the reactor, and also includes a condenser installed on the pipeline connecting the vacuum pump and the reactor and located between the condensate recovery device and the vacuum pump. The condenser is located directly above the condensate recovery device. After condensation, the volatilized organic solvent flows into the condensate recovery device and is separated and recovered by an oil-water separator. After the chitosan is solidified, the organic solvent solution in the reactor is filtered and can be used in the next batch of experiments.

[0021] Preferably, the pore size of the membrane tube is 110 μm-350 μm, more preferably 145 μm.

[0022] Compared with the prior art, the present invention has at least the following beneficial effects: first, the chitosan microspheres prepared by the present invention have a porous structure and a large specific surface area, and can be used as microcarriers for carrying more cells, and can be used for high-density active cell culture, and the large specific surface area is conducive to the adhesion and growth and reproduction of cells; second, the prepared chitosan microspheres have uniform particle sizes and are more suitable for microcarriers for cell culture; third, the organic solvent and organic solvent solution used in the chitosan microsphere preparation process of the present invention are recyclable and reusable, which reduces costs and is environmentally friendly. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention, the drawings required to be used in the embodiments or the description of the prior art will be briefly described below.

[0024] Figure 1 It is a preparation process flow chart of the present invention;

[0025] Figure 2 It is a schematic diagram of the reaction device of the present invention;

[0026] Figure 3 This is a particle size distribution diagram of chitosan microspheres in Example 1 of the present invention;

[0027] Figure 4 is a scanning electron microscope image of chitosan microspheres according to Example 1 of the present invention;

[0028] Figure 5 This is a photo of the chitosan microspheres of Example 1 of the present invention under a microscope (20 times);

[0029] Figure 6 is a particle size distribution diagram of chitosan microspheres in Example 2 of the present invention;

[0030] Figure 7 is a scanning electron microscope image of chitosan microspheres of Example 2 of the present invention;

[0031] Figure 8 This is a photo of the chitosan microspheres of Example 2 of the present invention under a microscope (20 times);

[0032] Fig. 9 is a particle size distribution diagram of chitosan microspheres according to Example 3 of the present invention;

[0033] Fig.10 is a scanning electron microscope image of chitosan microspheres of Example 3 of the present invention;

[0034] Fig.11 This is a photo of the chitosan microspheres of Example 3 of the present invention under a microscope (20 times).

[0035] Figure 2 In the figure, 1-vacuum pump; 2-condensate recovery device; 3-heating jacket; 4-condenser; 5-reactor; 6-membrane tube; 7-membrane emulsifier. DETAILED DESCRIPTION

[0036] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by relevant technicians in the field without creative work are within the scope of protection of the present invention.

[0037] Embodiment 1:

[0038] (1) According to Figure 1 As shown, assemble the reaction device;

[0039] (2) Prepare 300 mL of an organic solvent solution containing a surfactant in a reaction kettle: 5 parts of lauryl ether phosphate are dissolved in 95 parts of propyl butyrate by stirring;

[0040] (3) Prepare 60 mL of chitosan solution: dissolve 1 part of food-grade sodium chloride in 97 parts of 2 wt% formic acid, then add 2 parts of chitosan (molecular weight of about 200,000, degree of deacetylation of 85%) and stir to dissolve;

[0041] (4) using a catheter to connect a membrane tube with a pore size of 145 μm to a membrane emulsifier, and after testing the air tightness to ensure that there is no leakage, completely immerse the membrane tube in the organic solvent solution of (2);

[0042] (5) Start the reactor (stirring at a speed of 160 rpm / min) and the condenser, transfer 60 mL of chitosan solution into the membrane emulsifier, start the membrane emulsifier, and adjust the emulsification pressure of the membrane emulsifier to 0.02 MPA. After the chitosan solution completely enters the reactor, start the vacuum pump and the heating jacket outside the reactor (heating to 105°C), keep warm for 10 hours, start sampling to observe whether the solidification is completed, keep warm until it is completely solidified, and at the same time, the volatilized propyl butyrate is condensed by the condenser and flows into the condensation recovery device, and is separated and recovered by the oil-water separator;

[0043] (6) After the chitosan is solidified, the system is cooled to room temperature and filtered using a 100-mesh filter to obtain chitosan solid microspheres with a relatively uniform particle size. The filtrate (propyl butyrate organic solvent solution) after filtering the microspheres is filtered again using an 800-mesh filter to recover the propyl butyrate solvent solution;

[0044] (7) Wash the chitosan solid microspheres with water for 6 times at room temperature with stirring, each time for 30 minutes, to dissolve and remove the sodium chloride. Then, soak the chitosan microspheres in 80 mL of 10 wt% sodium glycerophosphate and keep at 60°C for 1 hour;

[0045] (8) Filter, then wash the chitosan microspheres with water for 6 times at room temperature with stirring, each time for 30 minutes. Filter and freeze-dry. Porous chitosan microspheres with uniform particle size can be obtained.

[0046] (9) The specific surface area, particle size distribution and scanning electron microscopy of chitosan microspheres were respectively measured. The specific surface area test results are shown in Table 1; the particle size distribution is shown in Table 1. Figure 3 As shown in Figure 4, the Span value is 0.659 and D(4,3)(μm) is 131.53μm. The SEM image is shown in Figure 4. The results of observation under a microscope (20 times) are as follows Figure 5 As shown, there is no adhesion between the microspheres.

[0047] Embodiment 2:

[0048] (1) According to Figure 2 As shown, assemble the reaction device;

[0049] (2) Prepare 300 mL of an organic solvent solution containing a surfactant in a reaction kettle: 5 parts of lauryl ether phosphate are dissolved in 95 parts of propyl butyrate by stirring;

[0050] (3) Prepare 60 mL of chitosan solution: dissolve 1 part of food-grade sodium chloride in 97 parts of 2 wt% formic acid, then add 2 parts of chitosan (molecular weight of about 300,000, degree of deacetylation of 85%) and stir until the solution is uniform;

[0051] (4) using a catheter to connect a membrane tube with a pore size of 145 μm to a membrane emulsifier, and after testing the air tightness to ensure that there is no leakage, completely immerse the membrane tube in the organic solvent solution of (2);

[0052] (5) Start the reactor (stirring at a speed of 160 rpm / min) and the condenser, transfer 60 mL of chitosan solution into the membrane emulsifier, start the membrane emulsifier, and adjust the emulsification pressure of the membrane emulsifier to 0.02 MPA. After the chitosan solution completely enters the reactor, start the vacuum pump and the heating jacket outside the reactor (heating to 105°C), keep warm for 10 hours, start sampling to observe whether the solidification is completed, keep warm until it is completely solidified, and at the same time, the volatilized propyl butyrate is condensed by the condenser and flows into the condensation recovery device, and is separated and recovered by the oil-water separator;

[0053] (6) After the chitosan is solidified, the system is cooled to room temperature and filtered using a 100-mesh filter to obtain chitosan solid microspheres with a relatively uniform particle size. The filtrate (propyl butyrate organic solvent solution) after filtering the microspheres is filtered again using an 800-mesh filter to recover the propyl butyrate solvent solution;

[0054] (7) Wash the chitosan solid microspheres with water for 6 times at room temperature with stirring, each time for 30 minutes, to dissolve and remove the sodium chloride. Then, soak the chitosan microspheres in 80 mL of 10 wt% sodium glycerophosphate and keep at 60°C for 1 hour;

[0055] (8) Filter, then wash the chitosan microspheres with water for 6 times at room temperature with stirring, each time for 30 minutes. Filter and freeze-dry. Porous chitosan microspheres with uniform particle size can be obtained.

[0056] (9) The specific surface area, particle size distribution and scanning electron microscopy of chitosan microspheres were respectively measured. The specific surface area test results are shown in Table 1; the particle size distribution is shown in Table 1. Figure 6 As shown, its Span: 0.634, D (4,3) (μm): 129.30. Figure 7 The results of observation under a microscope (20 times) are as follows Figure 8 As shown, there is no adhesion between the microspheres.

[0057] Embodiment three:

[0058] (1) According to Figure 1 As shown, assemble the reaction device;

[0059] (2) Add 300 mL of the organic solvent solution containing the surfactant recovered in Example 1 and Example 2 into the reaction kettle;

[0060] (3) Prepare 60 mL of chitosan solution: dissolve 1 part of food-grade sodium chloride in 97 parts of 2 wt% formic acid, then add 2 parts of chitosan (molecular weight of about 300,000, degree of deacetylation of 85%) and stir until the solution is uniform;

[0061] (4) using a catheter to connect a membrane tube with a pore size of 145 μm to a membrane emulsifier, and after testing the air tightness to ensure that there is no leakage, completely immerse the membrane tube in the organic solvent solution of (2);

[0062] (5) Start the reactor (stirring at a speed of 160 rpm / min) and the condenser, transfer 60 mL of chitosan solution into the membrane emulsifier, start the membrane emulsifier, and adjust the emulsification pressure of the membrane emulsifier to 0.02 MPA. After the chitosan solution completely enters the reactor, start the vacuum pump and the heating jacket outside the reactor (heating to 105°C), keep warm for 10 hours, start sampling to observe whether the solidification is completed, keep warm until it is completely solidified, and at the same time, the volatilized propyl butyrate is condensed by the condenser and flows into the condensation recovery device, and is separated and recovered by the oil-water separator;

[0063] (6) After the chitosan is solidified, the system is cooled to room temperature and filtered using a 100-mesh filter to obtain chitosan solid microspheres with a relatively uniform particle size. The filtrate (propyl butyrate organic solvent solution) after filtering the microspheres is filtered again using an 800-mesh filter to recover the propyl butyrate solvent solution;

[0064] (7) Wash the chitosan solid microspheres with water for 6 times at room temperature with stirring, each time for 30 minutes, to dissolve and remove the sodium chloride. Then, soak the chitosan microspheres in 80 mL of 10 wt% sodium glycerophosphate and keep at 60°C for 1 hour;

[0065] (8) Filter, then wash the chitosan microspheres with water for 6 times at room temperature with stirring, each time for 30 minutes. Filter and freeze-dry. Porous chitosan microspheres with uniform particle size can be obtained.

[0066] (9) The specific surface area, particle size distribution and scanning electron microscopy of chitosan microspheres were respectively measured. The specific surface area test results are shown in Table 1; the particle size distribution is shown in Table 1. Fig. 9As shown, its Span: 0.661, D (4,3) (μm): 128.50. The scanning electron microscope image is shown in Figure 10. The results of observation under a microscope (20 times) are as follows Fig.11 As shown, there is no adhesion between the microspheres.

[0067] Table 1:

[0068] Serial number sample <![CDATA[Specific surface area / m 2 / g]]> Average pore size / μm Porosity / % 1 Embodiment 1 101 35.37 88.91 2 Embodiment 2 98 41.57 90.34 3 Embodiment 3 97 41.34 91.34

[0069] It can be intuitively concluded from Examples 1 to 3 and the above table that the chitosan microspheres prepared by the present invention have a stable specific surface area and a reasonable particle size distribution, and the chitosan microspheres prepared using the recovered organic solvent solution still have a relatively stable specific surface area, particle size distribution value and average pore size, thereby reducing costs and improving profits and efficiency during production and use. In addition, if the preparation system of the present application is mass-produced, it can be designed to be fully automatic, with automatic feeding of organic solvents and automatic filtering devices to avoid direct contact between personnel and organic solvents, and can achieve the preparation of microspheres under closed conditions, which is environmentally friendly.

[0070] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them; although the present invention has been described in detail with reference to the above embodiments, a person skilled in the art should understand that the technical solutions described in the above embodiments can still be modified, or some of the technical features can be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A preparation process of a chitosan cell culture carrier, characterized in that: include: S1: preparing an organic solvent solution: stirring and dissolving a surfactant in an organic solvent to form an organic solvent solution; S2: preparing a chitosan solution: stirring and dissolving chitosan in an acidic solution containing an inorganic salt to form a chitosan solution; S3 uses a reaction device to prepare chitosan microspheres: the chitosan solution prepared in S2 is placed in a membrane emulsifier. Under a certain air pressure condition, the chitosan solution passes through the membrane emulsifier and flows through the membrane tube, enters the organic solvent solution, and forms liquid beads with uniform particle size. The chitosan is solidified by keeping the temperature for a period of time under vacuum and certain temperature conditions. After the solidification is completed, the temperature is lowered and filtered to obtain solid chitosan microspheres. S4 Chitosan microsphere pore preparation: The solid chitosan microspheres prepared in S3 are washed with water to remove inorganic salts, and the washed chitosan microspheres are placed in an aqueous solution containing a crosslinking agent, and crosslinking reaction is carried out for a period of time under certain temperature conditions. After the crosslinking reaction is completed, the microspheres are filtered, washed with water, and dried to obtain porous chitosan microspheres with uniform particle size.

2. The preparation process of a chitosan cell culture carrier according to claim 1, characterized in that: In the step S1, the surfactant is selected to be soluble in an organic solvent with a boiling point higher than 110° C., specifically lauryl alcohol ether phosphate, fatty alcohol polyoxyethylene ether or disodium coconut oil monoethanolamide sulfosuccinate.

3. The preparation process of a chitosan cell culture carrier according to claim 1, characterized in that: The concentration of the surfactant in the organic solvent is 1wt%-10wt%, and the volume ratio of the organic solvent solution to the chitosan solution is (3-20):

1.

4. The preparation process of a chitosan cell culture carrier according to claim 1, characterized in that: In step S2, the concentration of the chitosan solution is 0.5wt%-10wt%, the molecular weight of the chitosan is 50,000-600,000, and the degree of deacetylation is 70%-95%.

5. The preparation process of a chitosan cell culture carrier according to claim 1, characterized in that: The acid solution in step S2 is a 2 wt % acetic acid or formic acid solution, the soluble inorganic salt is a sodium salt or a potassium salt, and the mass ratio of chitosan to the inorganic salt is 3:1-1:

1.

6. The preparation process of a chitosan cell culture carrier according to claim 1, characterized in that: In the step S3, the chitosan film emulsification pressure condition is 0.01-0.1 MPa, the chitosan curing temperature is 90° C.-180° C., the heat preservation time is 5-15 hours, and the temperature is lowered to room temperature after curing.

7. The preparation process of a chitosan cell culture carrier according to claim 1, characterized in that: In the step S4, the cross-linking reaction temperature is 50-80° C., the cross-linking reaction time is 0.5-5 h, and the cross-linking agent is polyphosphate, genipin or sodium glycerophosphate.

8. The reaction device according to the process for preparing a chitosan cell culture carrier according to claims 1-6, characterized in that: The reaction device comprises a heatable reactor, a heating jacket is provided on the outer edge of the reactor, a vacuum pump connected to the reactor via a pipeline is provided on one side of the reactor, a condensation recovery device is provided on the pipeline connecting the reactor and the vacuum pump, and a membrane emulsifier connected to a membrane tube via a catheter is provided on the other side of the reactor. After checking that the air tightness is qualified, the membrane tube is completely immersed in an organic solvent solution containing a surfactant.

9. The reaction device of a chitosan cell culture carrier according to claim 8, characterized in that: The condensate recovery device includes a condensate recovery device connected to the pipeline connecting the vacuum pump and the reactor, and also includes a condenser installed on the pipeline connecting the vacuum pump and the reactor and located between the condensate recovery device and the vacuum pump, and the condenser is located above the condensate recovery device.

10. The reaction device of a chitosan cell culture carrier according to claim 8, characterized in that: The pore size of the membrane tube is 110 μm-350 μm.

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