CN112322575A - Preparation method of three-dimensional gel scaffold for culturing cells - Google Patents
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Abstract
The invention discloses a preparation method of a three-dimensional gel scaffold for culturing cells, which comprises the following steps: (1) dissolving polycaprolactone in acetone, degassing to obtain spinning solution, preparing polycaprolactone fiber by wet spinning, and weaving into polycaprolactone fiber net; spreading the prepared polycaprolactone fiber mesh layer in a polytetrafluoroethylene mold, pouring chondroitin sulfate aqueous solution, and freeze-drying to obtain a modified polycaprolactone stent; (2) and (2) placing the prepared modified polycaprolactone scaffold into a keratin solution, stirring and mixing, and finally carrying out freeze drying at-5 to-10 ℃ for 10-20h to obtain the three-dimensional gel scaffold. The three-dimensional gel scaffold prepared by the invention has higher cell affinity and good mechanical property.
Description
The technical field is as follows:
the invention relates to the technical field of cell culture, in particular to a preparation method of a three-dimensional gel scaffold for culturing cells.
Background art:
cell culture is also called cell cloning technology, and the formal term in biology is cell culture technology. Cell culture is an indispensable process for both the whole bioengineering technology and one of the biological cloning technologies, and the cell culture technology can be used for culturing a large number of cells into simple single cells or cells with few differentiation, which is an indispensable link of the cloning technology, and the cell culture is the cloning of the cells. A large number of cells or their metabolites are obtained by cell culture. Since biological products are all derived from cells, it can be said that cell culture technology is the most core and basic technology in biotechnology. In the cell culture technique, two-dimensional culture and three-dimensional culture can be classified according to the culture environment.
When two-dimensional and three-dimensional cultures are compared to each other at a rough level, it is clear that the three-dimensional cellular structure more closely approximates the in vivo environment of tissues and tumors, where the extracellular matrix (ECM) interacts with multiple cell types in a complex manner, rather than a simple monolayer of cells or a series of stacked monolayers of cells. In a three-dimensional environment, cells respond differently to stimuli than do two-dimensional monolayers. This is due to the large influence of many variables in the environment surrounding the cells and materials that make up the scaffold on its properties. Hydrogels are widely used in tissue engineering as bio-carrier materials. The hydrogel is a three-dimensional network structure formed by mutual crosslinking between solid and liquid through covalent bonds, hydrogen bonds and other acting forces, can lock water which is many times of the dry weight of the hydrogel as a support material of an interpenetrating structure network which is physically or chemically crosslinked, and can better simulate the physical and chemical environments of extracellular matrix to provide a good microenvironment for the proliferation and differentiation of cells. Thus, the hydrogel scaffold can serve as a three-dimensional environment for culturing cells.
Patent CN202010615888.5 discloses a method for three-dimensionally culturing mesenchymal stem cells based on a Collagel gel scaffold method. According to the method, the Collagel gel scaffold is used as a three-dimensional matrix, and the mesenchymal stem cells and the Collagel gel are mixed to form a cell suspension for culture, so that the physical and spatial three-dimensional structure of a tissue-like sample required by cell growth can be better simulated, and the microenvironment for cell growth under different biomechanical effects of in-vivo physiological environments can be better simulated. Patent CN201210449128.7 discloses a natural biological cross-linked nano composite three-dimensional gel scaffold and a preparation method thereof. The hydrogel contains acrylamide monomer M1, inorganic nano clay M2, biological polymer M3 and biological cross-linking agent genipin M4, and the mass percentages of the four are as follows: 55-90: 7-25: 3-20: 0-5. The preparation method of the invention is that M2 is used as a cross-linking agent of M1 and M4 is used as a cross-linking agent of M3 in aqueous solution, and the nano composite three-dimensional gel scaffold with excellent biocompatibility and mechanical property is prepared by adopting in-situ free radical polymerization and a phase separation freeze drying method. As is known from the prior art, hydrogels for cell culture are generally prepared by cross-linking natural polymers and synthetic polymers. However, how to improve the compatibility between natural polymers and synthetic polymers and the cell adhesion of the scaffold during the preparation process becomes critical.
The invention content is as follows:
the invention aims to solve the technical problem that the defects of the prior art are overcome, and the preparation method of the three-dimensional gel scaffold for culturing cells is provided, the scaffold prepared by laminating polycaprolactone fiber nets is used as a substrate, then chondroitin sulfate is used for modification, and the chondroitin sulfate is deposited on the surface of the scaffold to form a layer of film, so that the hydrophilicity and cell adhesion of the scaffold are improved; finally, the modified polycaprolactone support is placed in a keratin solution for reaction, and keratin permeates into the support to be mutually crosslinked to form a three-dimensional gel support which has higher cell affinity and good mechanical property.
In order to solve the technical problems, the invention adopts the following technical scheme:
a method for preparing a three-dimensional gel scaffold for culturing cells, comprising the steps of:
(1) dissolving polycaprolactone in acetone, degassing to obtain spinning solution, preparing polycaprolactone fiber by wet spinning, and weaving into polycaprolactone fiber net; spreading the prepared polycaprolactone fiber mesh layer in a polytetrafluoroethylene mold, pouring chondroitin sulfate aqueous solution, and freeze-drying to obtain a modified polycaprolactone stent;
(2) and (2) placing the prepared modified polycaprolactone scaffold into a keratin solution, stirring and mixing, and finally carrying out freeze drying at-5 to-10 ℃ for 10-20h to obtain the three-dimensional gel scaffold.
Preferably, in step (1), the solution is subjected to ultrasonic treatment in an ultrasonic water bath at a power of 500W for 30-40 min.
Preferably, in the step (1), the concentration of the spinning solution is 15 to 20 w/v%.
As a preferred aspect of the above technical solution, in the step (1), the wet spinning process specifically includes: the extrusion speed of the spinning solution is 0.75-0.85ml/h, the diameter of a spinneret orifice is 200-500 mu m, and the distance between the spinneret orifice and the receiving container is 20-30 mm.
Preferably, in the step (1), the mass concentration of the chondroitin sulfate aqueous solution is 1-5%, and the mass ratio of the chondroitin sulfate aqueous solution to the polycaprolactone fiber web is 1: (2-3).
Preferably, in the step (1), the polytetrafluoroethylene mold has a height of 4mm and a diameter of 12 mm.
Preferably, in the step (1), the temperature of the freeze drying is-15 to-20 ℃, and the time of the freeze drying is 15 to 20 hours.
Preferably, in the step (2), the concentration of the keratin solution is 0.1 to 0.2 g/ml.
Preferably, in the step (2), the mass ratio of the modified polycaprolactone scaffold to the keratin solution is 10: (2-6).
As a preferable aspect of the above, in the step (2), the conditions of the stirring and mixing process are: the stirring speed is 800-1500rpm, and the stirring time is 1-2 h.
Due to the adoption of the technical scheme, the invention has the following beneficial effects:
polycaprolactone has good biocompatibility and biodegradability, but is poorly hydrophilic. In order to solve the problem, the invention firstly adopts a wet spinning process to prepare polycaprolactone fiber, then weaves the polycaprolactone fiber into a net, places the polycaprolactone fiber net in a polytetrafluoroethylene mould, adds chondroitin sulfate solution, the chondroitin sulfate permeates into the polycaprolactone fiber net, the chondroitin sulfate is mutually crosslinked due to the hydrogen bond action among molecules, thereby not only playing a role of bonding, improving the associativity between the polycaprolactone fiber nets, but also improving the hydrophilicity of polycaprolactone, and the modified polycaprolactone scaffold has better cell adhesion. The prepared modified polycaprolactone scaffold is placed in a keratin solution for reaction, keratin permeates into pores of the scaffold and is crosslinked, and a nanogel network is formed in the scaffold, so that the adhesion of cells is improved, and the migration and proliferation of the cells in the scaffold are improved.
The three-dimensional gel scaffold prepared by the invention has good hydrophilicity and mechanical property, can be used in the field of cell culture, can well adhere cells and promote cell proliferation in the process of culturing cells, and the cells can be uniformly distributed in the scaffold. The three-dimensional gel scaffold has excellent biocompatibility and biodegradability and is beneficial to environmental protection.
The specific implementation mode is as follows:
the present invention is further illustrated by the following examples, which are provided for the purpose of illustration only and are not intended to be limiting.
Example 1
(1) Dissolving polycaprolactone in acetone, placing the acetone in an ultrasonic water bath, carrying out ultrasonic treatment for 30min under the power of 500W to prepare spinning solution with the concentration of 15W/v%, and then preparing polycaprolactone fiber by adopting wet spinning, wherein the spinning process comprises the following steps: the extrusion speed of the spinning solution is 0.75ml/h, the diameter of a spinneret orifice is 200 mu m, and the distance between the spinneret orifice and a receiving container is 20 mm; then weaving a polycaprolactone fiber net; spreading the prepared polycaprolactone fiber mesh layer in a polytetrafluoroethylene mould with the height of 4mm and the diameter of 12mm, and pouring 1% chondroitin sulfate aqueous solution with the mass concentration, wherein the mass ratio of the chondroitin sulfate aqueous solution to the polycaprolactone fiber mesh layer is 1: 2; finally, freeze-drying for 15h at-15 ℃ to prepare the modified polycaprolactone stent;
(2) and (2) putting 10g of the prepared modified polycaprolactone scaffold into 2g of 0.1g/ml keratin solution, stirring and mixing for 1h at the stirring speed of 800rpm, and finally performing freeze drying for 10h at the temperature of-5 ℃ to prepare the three-dimensional gel scaffold.
Example 2
(1) Dissolving polycaprolactone in acetone, placing the acetone in an ultrasonic water bath, carrying out ultrasonic treatment for 40min under the power of 500W to prepare a spinning solution with the concentration of 20W/v%, and then preparing polycaprolactone fiber by adopting wet spinning, wherein the spinning process comprises the following steps: the extrusion speed of the spinning solution is 0.85ml/h, the diameter of a spinneret orifice is 500 mu m, and the distance between the spinneret orifice and a receiving container is 30 mm; then weaving a polycaprolactone fiber net; spreading the prepared polycaprolactone fiber mesh layer in a polytetrafluoroethylene mould with the height of 4mm and the diameter of 12mm, and pouring a chondroitin sulfate aqueous solution with the mass concentration of 5%, wherein the mass ratio of the chondroitin sulfate aqueous solution to the polycaprolactone fiber mesh layer is 1: 3; finally, freeze-drying for 20h at the temperature of minus 20 ℃ to prepare the modified polycaprolactone stent;
(2) and (2) putting 10g of the prepared modified polycaprolactone scaffold into 6g of 0.2g/ml keratin solution, stirring and mixing at the stirring speed of 1500rpm for 2 hours, and finally carrying out freeze drying at-10 ℃ for 20 hours to prepare the three-dimensional gel scaffold.
Example 3
(1) Dissolving polycaprolactone in acetone, placing the acetone in an ultrasonic water bath, carrying out ultrasonic treatment for 30min under the power of 500W to prepare a spinning solution with the concentration of 16W/v%, and then preparing polycaprolactone fiber by adopting wet spinning, wherein the spinning process comprises the following steps: the extrusion speed of the spinning solution is 0.8ml/h, the diameter of a spinneret orifice is 300 mu m, and the distance between the spinneret orifice and a receiving container is 20 mm; then weaving a polycaprolactone fiber net; spreading the prepared polycaprolactone fiber mesh layer in a polytetrafluoroethylene mould with the height of 4mm and the diameter of 12mm, and pouring a chondroitin sulfate aqueous solution with the mass concentration of 2%, wherein the mass ratio of the chondroitin sulfate aqueous solution to the polycaprolactone fiber mesh layer is 1: 2; finally, freeze-drying for 15h at-15 ℃ to prepare the modified polycaprolactone stent;
(2) and (2) putting 10g of the prepared modified polycaprolactone scaffold into 3g of 0.1g/ml keratin solution, stirring and mixing for 1h at the stirring speed of 1000rpm, and finally performing freeze drying for 10h at the temperature of-6 ℃ to prepare the three-dimensional gel scaffold.
Example 4
(1) Dissolving polycaprolactone in acetone, placing the acetone in an ultrasonic water bath, carrying out ultrasonic treatment for 40min under the power of 500W to prepare spinning solution with the concentration of 18W/v%, and then preparing polycaprolactone fiber by wet spinning, wherein the spinning process comprises the following steps: the extrusion speed of the spinning solution is 0.8ml/h, the diameter of a spinneret orifice is 400 mu m, and the distance between the spinneret orifice and a receiving container is 20 mm; then weaving a polycaprolactone fiber net; spreading the prepared polycaprolactone fiber mesh layer in a polytetrafluoroethylene mould with the height of 4mm and the diameter of 12mm, and pouring a chondroitin sulfate aqueous solution with the mass concentration of 3%, wherein the mass ratio of the chondroitin sulfate aqueous solution to the polycaprolactone fiber mesh layer is 1: 3; finally, freeze-drying for 20h at the temperature of minus 20 ℃ to prepare the modified polycaprolactone stent;
(2) and (2) putting 10g of the prepared modified polycaprolactone scaffold into 4g of 0.2/ml keratin solution, stirring and mixing at the stirring speed of 1200rpm for 1h, and finally carrying out freeze drying at the temperature of-5 ℃ for 15h to prepare the three-dimensional gel scaffold.
Example 5
(1) Dissolving polycaprolactone in acetone, placing the acetone in an ultrasonic water bath, carrying out ultrasonic treatment for 30min under the power of 500W to prepare a spinning solution with the concentration of 20W/v%, and then preparing polycaprolactone fiber by adopting wet spinning, wherein the spinning process comprises the following steps: the extrusion speed of the spinning solution is 0.8ml/h, the diameter of a spinneret orifice is 500 mu m, and the distance between the spinneret orifice and a receiving container is 20 mm; then weaving a polycaprolactone fiber net; spreading the prepared polycaprolactone fiber mesh layer in a polytetrafluoroethylene mould with the height of 4mm and the diameter of 12mm, and pouring a chondroitin sulfate aqueous solution with the mass concentration of 3%, wherein the mass ratio of the chondroitin sulfate aqueous solution to the polycaprolactone fiber mesh layer is 1: 2; finally, freeze-drying for 20h at the temperature of minus 20 ℃ to prepare the modified polycaprolactone stent;
(2) and (2) putting 10g of the prepared modified polycaprolactone scaffold into 5g of 0.15g/ml keratin solution, stirring and mixing at the stirring speed of 1500rpm for 1h, and finally performing freeze drying at-10 ℃ for 10h to prepare the three-dimensional gel scaffold.
Example 6
(1) Dissolving polycaprolactone in acetone, placing the acetone in an ultrasonic water bath, carrying out ultrasonic treatment for 30min under the power of 500W to prepare spinning solution with the concentration of 15W/v%, and then preparing polycaprolactone fiber by adopting wet spinning, wherein the spinning process comprises the following steps: the extrusion speed of the spinning solution is 0.85ml/h, the diameter of a spinneret orifice is 200 mu m, and the distance between the spinneret orifice and a receiving container is 20 mm; then weaving a polycaprolactone fiber net; spreading the prepared polycaprolactone fiber mesh layer in a polytetrafluoroethylene mould with the height of 4mm and the diameter of 12mm, and pouring 4% chondroitin sulfate aqueous solution with the mass concentration, wherein the mass ratio of the chondroitin sulfate aqueous solution to the polycaprolactone fiber mesh layer is 1: 2; finally, freeze-drying for 18h at the temperature of minus 20 ℃ to prepare the modified polycaprolactone stent;
(2) and (2) putting 10g of the prepared modified polycaprolactone scaffold into 5g of 0.15g/ml keratin solution, stirring and mixing at the stirring speed of 800rpm for 1h, and finally carrying out freeze drying at the temperature of-5 ℃ for 20h to prepare the three-dimensional gel scaffold.
Application examples
Taking human corneal cells as an example, cultured human corneal cells were suspended in DMEM medium containing 10% FBS and 1% penicillin/streptomycin to prepare a cell suspension with a cell density of 3X 104The three-dimensional gel scaffolds prepared in the above examples were placed in 48-well culture plates and sterilized, and then the cell suspension was inoculated into the 48-well culture plates, supplemented with 10% FBS and 250KIUml every two days-1DMEM for aprotinin. And a blank control group was set, and the cell viability after 5 days of culture was achieved by the MTT method.
The performance tests are shown in table 1:
TABLE 1
| Cell viability% | Tensile Strength, kPa | Tensile Strength, kPa | Water absorption percentage% | |
| Example 1 | 360 | 265 | 100.5 | 55 |
| Example 2 | 363 | 262 | 100.9 | 53 |
| Example 3 | 362 | 265 | 101.3 | 55 |
| Example 4 | 360 | 262 | 101.5 | 52 |
| Example 5 | 363 | 262 | 100.8 | 54 |
| Example 6 | 363 | 265 | 101.2 | 54 |
The test results show that the three-dimensional gel scaffold prepared by the invention has good hydrophilicity and excellent mechanical property, and can effectively improve cell activity and promote cell proliferation when being used for cell culture.
Although specific embodiments of the invention have been described, many other forms and modifications of the invention will be apparent to those skilled in the art. It is to be understood that the appended claims and this invention generally cover all such obvious forms and modifications which are within the true spirit and scope of the present invention.
Claims (10)
1. A method for preparing a three-dimensional gel scaffold for culturing cells, comprising the steps of:
(1) dissolving polycaprolactone in acetone, degassing to obtain spinning solution, preparing polycaprolactone fiber by wet spinning, and weaving into polycaprolactone fiber net; spreading the prepared polycaprolactone fiber mesh layer in a polytetrafluoroethylene mold, pouring chondroitin sulfate aqueous solution, and freeze-drying to obtain a modified polycaprolactone stent;
(2) and (2) placing the prepared modified polycaprolactone scaffold into a keratin solution, stirring and mixing, and finally carrying out freeze drying at-5 to-10 ℃ for 10-20h to obtain the three-dimensional gel scaffold.
2. The method for preparing a three-dimensional gel scaffold for culturing cells according to claim 1, wherein the degassing treatment in step (1) is ultrasonic treatment of the solution in an ultrasonic water bath at a power of 500W for 30-40 min.
3. The method for preparing a three-dimensional gel scaffold for culturing cells according to claim 1, wherein the concentration of the spinning dope in the step (1) is 15-20 w/v%.
4. The method for preparing a three-dimensional gel scaffold for culturing cells according to claim 1, wherein in the step (1), the wet spinning process comprises: the extrusion speed of the spinning solution is 0.75-0.85ml/h, the diameter of a spinneret orifice is 200-500 mu m, and the distance between the spinneret orifice and the receiving container is 20-30 mm.
5. The method for preparing a three-dimensional gel scaffold for culturing cells according to claim 1, wherein in the step (1), the mass concentration of the chondroitin sulfate aqueous solution is 1-5%, and the mass ratio of the chondroitin sulfate aqueous solution to the polycaprolactone fiber web is 1: (2-3).
6. The method for preparing a three-dimensional gel scaffold for culturing cells according to claim 1, wherein in the step (1), the polytetrafluoroethylene mold has a height of 4mm and a diameter of 12 mm.
7. The method for preparing a three-dimensional gel scaffold for culturing cells according to claim 1, wherein the temperature of the freeze-drying in the step (1) is-15 to-20 ℃, and the time of the freeze-drying is 15 to 20 hours.
8. The method of claim 1, wherein the concentration of the keratin solution in the step (2) is 0.1-0.2 g/ml.
9. The method for preparing a three-dimensional gel scaffold for culturing cells according to claim 1, wherein in the step (2), the mass ratio of the modified polycaprolactone scaffold to the keratin solution is 10: (2-6).
10. The method for preparing a three-dimensional gel scaffold for culturing cells according to claim 1, wherein the conditions of the stirring and mixing process in step (2) are as follows: the stirring speed is 800-1500rpm, and the stirring time is 1-2 h.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113249327A (en) * | 2021-05-06 | 2021-08-13 | 东华大学 | Three-dimensional cell culture scaffold for tumor cell culture and preparation method thereof |
| CN114288479A (en) * | 2021-12-30 | 2022-04-08 | 智享生物(苏州)有限公司 | Cell adhesion material based on supermolecule self-assembly and preparation method thereof |
| CN114622296A (en) * | 2022-02-28 | 2022-06-14 | 上海食未生物科技有限公司 | Method for continuously preparing edible fiber scaffold for cell culture meat |
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2020
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113249327A (en) * | 2021-05-06 | 2021-08-13 | 东华大学 | Three-dimensional cell culture scaffold for tumor cell culture and preparation method thereof |
| CN114288479A (en) * | 2021-12-30 | 2022-04-08 | 智享生物(苏州)有限公司 | Cell adhesion material based on supermolecule self-assembly and preparation method thereof |
| CN114622296A (en) * | 2022-02-28 | 2022-06-14 | 上海食未生物科技有限公司 | Method for continuously preparing edible fiber scaffold for cell culture meat |
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