CN113372580A - Preparation method of composite hydrogel and construction method of cell microenvironment bionic system - Google Patents
Preparation method of composite hydrogel and construction method of cell microenvironment bionic system Download PDFInfo
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Abstract
The invention discloses a preparation method of composite hydrogel and a construction method of a cell microenvironment bionic system, wherein the preparation method of the composite hydrogel comprises the following steps: mixing a gelatin solution, a sodium alginate solution, a glycerol solution, a glutaraldehyde solution, a phosphate buffer salt solution and a sterile aqueous solution according to a predetermined volume ratio to obtain a precursor solution with the volume concentration of 1-5% of glycerol; freezing the precursor solution to convert gelatin from sol to gel and freezing glycerin to form pores; and adding a calcium chloride solution to perform ionic crosslinking on the sodium alginate in the gel to obtain the composite hydrogel, wherein the concentration of the calcium chloride is related to the rigidity of the composite hydrogel. The composite hydrogel prepared by the embodiment of the invention has the advantages of good molding, stable mechanical property and uniform pore structure, and can be used for simulating the microenvironment of extracellular matrix in vitro.
Description
Technical Field
The invention relates to the technical field of biological materials and tissue engineering, in particular to a preparation method of composite hydrogel and a construction method of a cell microenvironment bionic system.
Background
The extracellular matrix is a three-dimensional network consisting of extracellular macromolecules, is an environment for the survival of cells and can provide structural and biochemical support for the cells. At present, in vitro cell culture mostly adopts two-dimensional culture with simple operation and low cost, which is far different from the complex three-dimensional structure of an in-vivo cell, and the three-dimensional cell culture technology can well construct and simulate the interaction between cells of cell-cell and cell-microenvironment. The cells can sense biochemical and physical stimulation of the microenvironment and perform responsive behaviors such as adhesion, migration, proliferation, differentiation, metabolism and the like on the stimulation, namely the environment has corresponding feedback guidance effect on specific cells, so that the construction of the three-dimensional cell culture microenvironment based on the specific extracellular matrix has profound significance. Some studies have adopted a three-dimensional hydrogel culture scaffold for cell culture, however, a three-dimensional scaffold capable of controlling only a single physical variable is difficult to simulate a real and complex extracellular matrix microenvironment, and even cannot comprehensively reflect behavior and function changes of cells under the influence of multiple complex factors. Therefore, the search for a proper strategy to design the three-dimensional culture scaffold and the application of the three-dimensional culture scaffold to simulate a complex microenvironment in a cell body in vitro is an urgent problem to be solved for studying cell behaviors in the three-dimensional microenvironment.
Disclosure of Invention
The invention mainly aims to provide a preparation method of composite hydrogel and a construction method of a cell microenvironment bionic system, and aims to solve the problem that a real and complex cell microenvironment is difficult to simulate by adopting a three-dimensional support with a single physical variable in the prior art.
According to one aspect of the present invention, a method for preparing a composite hydrogel is provided, which comprises: mixing a gelatin solution, a sodium alginate solution, a glycerol solution, a glutaraldehyde solution, a phosphate buffer salt solution and a sterile aqueous solution according to a predetermined volume ratio to obtain a precursor solution with the volume concentration of 1-5% of glycerol; freezing the precursor solution to convert gelatin from sol to gel and freezing glycerin to form pores; and adding a calcium chloride solution to perform ionic crosslinking on the sodium alginate in the gel to obtain the composite hydrogel, wherein the concentration of the calcium chloride is related to the rigidity of the composite hydrogel.
Wherein the method further comprises: gelatin solution with mass concentration of 3-6%, sodium alginate solution with mass concentration of 1-3%, glycerol solution with mass concentration of 40-60%, glutaraldehyde solution with mass concentration of 0.5-2%, and 1 × Phosphate Buffered Saline (PBS) with pH7.4 are respectively provided, and the precursor solutions are mixed to obtain the precursor solution.
Wherein the method further comprises: mixing gelatin solution, sodium alginate solution, glycerol solution, glutaraldehyde solution and phosphate buffer salt solution to prepare precursor solution with gelatin solution volume concentration, sodium alginate solution volume concentration, glycerol solution volume concentration, glutaraldehyde solution volume concentration and phosphate buffer salt solution volume concentration of 15-25%, 1-5%, 1-3% and 5-15%, respectively, and supplementing the rest volume with sterile aqueous solution.
Wherein the step of adding a calcium chloride solution to carry out ionic crosslinking on sodium alginate in the gel to obtain the composite hydrogel comprises the following steps: adding calcium chloride solution with mass concentration of 0.5-1.5% to make sodium alginate undergo the process of ion cross-linking; or adding 1.6-3% calcium chloride solution to make sodium alginate undergo the process of ion cross-linking; wherein the rigidity of the composite hydrogel obtained by adding the calcium chloride solution with the mass concentration of 0.5-1.5% is lower than that of the composite hydrogel obtained by adding the calcium chloride solution with the mass concentration of 1.6-3%.
Wherein the method further comprises: the composite hydrogel was soaked in HEPES buffer at pH7.0 to remove free calcium ions.
According to another aspect of the present invention, a method for constructing a cell microenvironment biomimetic system is provided, which comprises: mixing a gelatin solution, a sodium alginate solution, a glycerol solution, a glutaraldehyde solution, a phosphate buffer salt solution and a sterile aqueous solution according to a predetermined volume ratio to obtain a precursor solution with the volume concentration of 1-5% of glycerol; adding the precursor solution into a cell culture plate for freezing treatment, converting gelatin from sol to gel, and freezing glycerol for pore-forming; adding a calcium chloride solution into the cell culture plate to perform ionic crosslinking on the sodium alginate to obtain a composite hydrogel, wherein the concentration of the calcium chloride is associated with the rigidity of the hydrogel; freeze-drying the cell culture plate to obtain a three-dimensional porous hydrogel scaffold with independently regulated rigidity and aperture; adding cell suspension into the cell culture plate for cell culture.
Wherein the method further comprises: gelatin solution with mass concentration of 3-6%, sodium alginate solution with mass concentration of 1-3%, glycerol solution with mass concentration of 40-60%, glutaraldehyde solution with mass concentration of 0.5-2%, and 1 × Phosphate Buffered Saline (PBS) with pH7.4 are respectively provided, and the precursor solutions are mixed to obtain the precursor solution.
Wherein the method further comprises: mixing gelatin solution, sodium alginate solution, glycerol solution, glutaraldehyde solution and phosphate buffer salt solution to prepare precursor solution with the volume concentration of the gelatin solution, the volume concentration of the sodium alginate solution, the volume concentration of the glycerol solution, the volume concentration of the glutaraldehyde solution and the volume concentration of the phosphate buffer salt solution being 15-25%, 1-5%, 1-3% and 5-15% respectively, and quantitatively filling the rest volume with sterile aqueous solution.
Wherein the step of adding a calcium chloride solution to the cell culture plate to subject sodium alginate to ionic crosslinking comprises: adding a calcium chloride solution with the mass concentration of 0.5-1.5% into the cell culture plate to carry out ionic crosslinking on the sodium alginate; or adding 1.6-3% calcium chloride solution into the cell culture plate to make sodium alginate undergo the process of ion cross-linking; wherein the rigidity of the composite hydrogel obtained by adding the calcium chloride solution with the mass concentration of 0.5-1.5% is lower than that of the composite hydrogel obtained by adding the calcium chloride solution with the mass concentration of 1.6-3%.
Wherein the method further comprises: HEPES buffer with PH7.0 was added to the cell culture plate to soak the composite hydrogel to remove free calcium ions.
The composite hydrogel prepared according to the embodiment of the invention has the advantages of good molding, stable mechanical property and uniform pore structure, can conveniently and independently regulate and control the physical properties (rigidity and pore diameter) of the prepared hydrogel, is favorable for meeting the requirements of three-dimensional culture of different cells, and can better simulate the microenvironment of extracellular matrix.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this application, illustrate embodiment(s) of the invention and together with the description serve to explain the invention without limiting the invention. In the drawings:
FIG. 1 is a flow chart of a method of making a composite hydrogel according to an embodiment of the invention;
FIG. 2 is a flow chart of a method for constructing a bionic system of a cell microenvironment according to an embodiment of the invention;
FIG. 3 is a graph showing the statistical results of the elastic modulus of a hydrogel according to an embodiment of the present invention;
FIGS. 4A to 4D are scanning electron micrographs of hydrogels according to embodiments of the present invention;
FIG. 5 is a schematic illustration of hydrogel pore size statistics in accordance with an embodiment of the present invention;
FIG. 6 is a schematic diagram showing the results of activity measurement after 24h of three-dimensional culture of cells according to an embodiment of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be clearly and completely described below with reference to the specific embodiments of the present invention and the accompanying drawings. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Hydrogels, as a class of very hydrophilic three-dimensional network structural materials, have biophysical properties very similar to natural extracellular matrix, and have been widely used to construct tissue engineering scaffolds. Sodium alginate, as a natural polysaccharide, has good degradability and biocompatibility, and can be rapidly crosslinked with divalent cations to form hydrogel. Gelatin is a natural temperature-sensitive material, has good degradability, biocompatibility and low price, can provide adhesion sites for cells, and can be converted from sol to gel when the temperature is reduced. However, the sol-gel transformation of the gelatin is a reversible process, and after being mixed with the sodium alginate, the gelatin can obtain the three-dimensional hydrogel with stable performance through further crosslinking, and can well simulate the extracellular matrix. Glycerol, a commonly used cryoprotectant, determines the freezing point of the solution and enables the growth rate and size of ice crystals to be adjusted at low temperatures. During low temperature gel formation, growth of ice crystals can result in an increase in the degree of glycerol enrichment in the remaining precursor solution around the ice crystals, thereby altering the freezing point of the precursor solution. When the concentration of glycerin reaches a certain concentration to reduce the freezing point of the solution to-20 ℃, the ice crystal stops growing, and pores are formed after the position of the ice crystal is melted. Thus, the initial level of cryoprotectant will determine the size of the ice crystals formed, with higher cryoprotectant concentrations forming smaller ice crystals and smaller pores, and lower cryoprotectant concentrations forming larger ice crystals and larger pores. Therefore, by utilizing the principle that the cryoprotectant glycerol can regulate the formation of ice crystals at low temperature, the pore size of the hydrogel can be regulated by regulating the growth of the ice crystals after the cryoprotectant glycerol is introduced into the temperature-sensitive hydrogel. Therefore, by utilizing the cross-linking characteristic of sodium alginate and divalent cations, the temperature-control cross-linking characteristic of gelatin and the chemical cross-linking characteristic of glutaraldehyde, the cryoprotectant glycerol is introduced, the physical characteristic of the hydrogel is conveniently and quickly regulated and controlled by changing the proportion of the cross-linking agent and the cryoprotectant, the rigidity and the pore diameter of the hydrogel are independently and accurately regulated and controlled, and the three-dimensional degradable hydrogel with the rigidity and the pore diameter independently regulated and controlled is constructed.
The technical solutions provided by the embodiments of the present invention are described in detail below with reference to the accompanying drawings.
Fig. 1 is a flow chart of a method for preparing a composite hydrogel according to an embodiment of the present invention, as shown in fig. 1, the method including the steps of:
and S102, mixing the gelatin solution, the sodium alginate solution, the glycerol solution, the glutaraldehyde solution, the phosphate buffer salt solution and the sterile aqueous solution according to a preset volume ratio to obtain a precursor solution with the glycerol volume concentration of 1-5%.
Specifically, a gelatin solution with a mass concentration of 3-6%, a sodium alginate solution with a mass concentration of 1-3%, a glycerol solution with a mass concentration of 40-60%, a glutaraldehyde solution with a mass concentration of 0.5-2%, and a 1 x Phosphate Buffered Saline (PBS) solution with a pH of 7.4 are respectively provided; mixing gelatin solution, sodium alginate solution, glycerol solution, glutaraldehyde solution and phosphate buffer salt solution to prepare precursor solution with gelatin solution volume concentration, sodium alginate solution volume concentration, glycerol solution volume concentration, glutaraldehyde solution volume concentration and phosphate buffer salt solution volume concentration of 15-25%, 1-5%, 1-3% and 5-15%, respectively, and supplementing the rest volume of the precursor solution with sterile aqueous solution.
Step S104, freezing the precursor solution to convert gelatin from sol to gel and freezing glycerol to form pores;
and S106, adding a calcium chloride solution to perform ionic crosslinking on the sodium alginate in the gel to obtain the composite hydrogel, wherein the concentration of the calcium chloride is associated with the rigidity of the composite hydrogel.
Fig. 2 is a flowchart of a method for constructing a cell microenvironment biomimetic system according to an embodiment of the present invention, as shown in fig. 2, the method includes the following steps:
step S202, mixing a gelatin solution, a sodium alginate solution, a glycerol solution, a glutaraldehyde solution, a phosphate buffer salt solution and a sterile aqueous solution according to a predetermined volume ratio to obtain a precursor solution with the volume concentration of glycerol of 1-5%;
specifically, a gelatin solution with a mass concentration of 3-6%, a sodium alginate solution with a mass concentration of 1-3%, a glycerol solution with a mass concentration of 40-60%, a glutaraldehyde solution with a mass concentration of 0.5-2%, and a 1 x Phosphate Buffered Saline (PBS) solution with a pH of 7.4 are respectively provided; mixing gelatin solution, sodium alginate solution, glycerol solution, glutaraldehyde solution and phosphate buffer salt solution to prepare precursor solution with gelatin solution volume concentration, sodium alginate solution volume concentration, glycerol solution volume concentration, glutaraldehyde solution volume concentration and phosphate buffer salt solution volume concentration of 15-25%, 1-5%, 1-3% and 5-15%, respectively, and supplementing the rest volume of the precursor solution with sterile aqueous solution.
Step S204, adding the precursor solution into a cell culture plate for freezing treatment, converting gelatin from sol to gel, and freezing glycerol for pore-forming;
step S206, adding a calcium chloride solution into the cell culture plate to perform ionic crosslinking on the sodium alginate to obtain a composite hydrogel, wherein the concentration of the calcium chloride is associated with the rigidity of the hydrogel;
step S208, carrying out freeze drying treatment on the cell culture plate to obtain a three-dimensional porous hydrogel support with independently regulated rigidity and aperture;
and step S210, adding the cell suspension into the cell culture plate for cell culture.
According to the preparation method of the gelatin-sodium alginate-glycerol composite hydrogel, provided by the invention, the construction method of the cell microenvironment bionic system with independently controllable rigidity and aperture is characterized in that degradable natural polymer biomaterials with good biocompatibility, namely sodium alginate and gelatin, are used as raw materials, the rigidity of the hydrogel is adjusted by utilizing the crosslinking characteristic of the sodium alginate and divalent cations, adhesion sites are provided for cells by utilizing the temperature control crosslinking characteristic of the gelatin and the chemical crosslinking characteristic of glutaraldehyde, and then the aperture of the hydrogel is adjusted by introducing the cryoprotectant, namely glycerol, so that the degradable cell three-dimensional culture scaffold is finally prepared, and the independent and accurate regulation of the rigidity and the aperture of the hydrogel is realized. Compared with the prior art, the physical properties (rigidity and aperture) of the prepared hydrogel can be conveniently and independently regulated and controlled by changing the proportion of the cross-linking agent and the cryoprotectant, and the requirement of three-dimensional culture of different cells can be met.
The present application is described in detail below with reference to examples.
1. Weighing 0.3g of sodium alginate powder, adding 10ml of sterile ultrapure water, heating and stirring in a water bath at 50-60 ℃ until the sodium alginate powder is completely dissolved, preparing into a 3 wt% sodium alginate solution, sterilizing at high pressure, and refrigerating at 4 ℃ for later use.
2. Weighing 0.3g gelatin powder, adding 10ml sterile ultrapure water, heating in 40-50 deg.C water bath, stirring to dissolve completely, preparing into 3 wt% gelatin solution, filtering with 0.22 μm filter membrane, and sterilizing.
3. Diluting glycerol solution with purity of 99.0% or more with sterile ultrapure water to obtain glycerol solution with purity of 40%, autoclaving, and refrigerating at 4 deg.C.
4. Diluting glutaraldehyde solution with purity of 25% with sterile ultrapure water to obtain glutaraldehyde solution with purity of 2%, ultraviolet sterilizing for 2 hr, and refrigerating at 4 deg.C.
5. Adding sterile ultrapure water (about 900 ml) into dry powder of 1 XPhosphate buffered saline (PBS), stirring thoroughly to dissolve, then making volume to 1L to obtain 1 XPBS with pH of 7.4, sterilizing under high pressure, and refrigerating at 4 deg.C for use.
6. 11.915g of 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES) was weighed out and dissolved in 900ml of sterile ultrapure water, 0.5-1M aqueous sodium hydroxide solution was added to adjust the pH to neutrality, then the volume was adjusted to 1L with sterile ultrapure water to obtain HEPES buffer solution of pH7.0, which was autoclaved and then refrigerated at 4 ℃ for future use.
7. Weighing 1g and 3g of calcium chloride solid respectively, fully dissolving in 100ml of ultrapure water to prepare 1 wt% and 3 wt% calcium chloride solutions, autoclaving, and refrigerating at 4 ℃ for later use.
The following steps are obtained through the steps 1 to 7: a gelatin solution of 3 wt%, a sodium alginate solution of 3 wt%, a glycerol solution of 40% concentration, a glutaraldehyde solution of 2% concentration, a 1 x phosphate buffered saline solution of pH7.4, a HEPES buffer of pH7.0, and a calcium chloride solution of 1 wt% and a calcium chloride solution of 3 wt%. It should be noted that the above steps 1 to 7 are not performed in strict sequence.
8. Mixing the gelatin solution, the sodium alginate solution, the glycerol solution, the glutaraldehyde solution and the phosphate buffer salt solution obtained in the steps to prepare a precursor solution with the volume concentration of the gelatin solution, the volume concentration of the sodium alginate solution, the volume concentration of the glycerol solution, the volume concentration of the glutaraldehyde solution and the volume concentration of the phosphate buffer salt solution being respectively 20%, 2%, 10% or 20%, 5%, 2% and 10%, and quantitatively complementing the rest volume with a sterile aqueous solution. Wherein, the solution with the final volume concentration of 2% of glycerin corresponds to the large-aperture hydrogel precursor solution, and the solution with the final volume concentration of 5% of glycerin corresponds to the small-aperture hydrogel precursor solution. Wherein, glutaraldehyde is used as an organic cross-linking agent to chemically cross-link the gelatin, so as to avoid the decrosslinking of the gelatin after the gelatin is gelatinized at low temperature and is heated. The pore size of the prepared hydrogel can be adjusted by changing the volume of the cryoprotectant glycerol solution.
For example, 400. mu.l of sodium alginate solution, 400. mu.l of gelatin solution, 40. mu.l of glycerin solution, 40. mu.l of glutaraldehyde solution, 200. mu.l of phosphate buffer solution, and 920. mu.l of sterile ultrapure water are weighed and mixed well to obtain a precursor solution of the large-pore-diameter hydrogel, and the precursor solution is added to the first cell culture plate after ultrasonic defoaming for 15 to 30min, and 2ml of the precursor solution is added to each well.
For another example, 400. mu.l of sodium alginate solution, 400. mu.l of gelatin solution, 100. mu.l of glycerin solution, 40. mu.l of glutaraldehyde solution, 200. mu.l of phosphate buffer solution, and 860. mu.l of sterile ultrapure water were measured and mixed thoroughly to obtain a precursor solution of small-pore hydrogel, which was then added to the second cell culture plate after defoaming with ultrasound for 15 to 30min, and 2ml of the precursor solution was added to each well.
And (3) placing the first cell culture plate added with the large-aperture hydrogel precursor solution and the second cell culture plate added with the small-aperture hydrogel precursor solution in a refrigerator at-20 ℃ for freezing for 24h, carrying out the transformation from sol to gel at low temperature on gelatin, and simultaneously freezing and forming pores by using a cryoprotectant glycerol at low temperature.
9. After 24h of freezing, the first and second cell culture plates were removed and immediately added with different concentrations of calcium chloride solution, and the rigidity of the hydrogel was adjusted by changing the concentration of calcium chloride.
For example, 1ml of 1 wt% calcium chloride solution is added into each hole of the first cell culture plate for ion crosslinking, and the gelatin-sodium alginate-glycerol composite hydrogel with lower rigidity is obtained after crosslinking for 2 hours; for another example, 1ml of 3 wt% calcium chloride solution is added into each well of the second cell culture plate for ion crosslinking, and after 2 hours of crosslinking, the gelatin-sodium alginate-glycerol composite hydrogel with higher rigidity is obtained. That is, the higher the rigidity of the hydrogel with increasing concentration of calcium chloride solution. It should be noted that the concentration of calcium chloride herein is merely illustrative and does not limit the scope of the present application.
After 2h of cross-linking, the calcium chloride solution in the first and second cell culture plates was aspirated, 2ml of HEPES buffer was added to each well to soak the hydrogel, after 30min the HEPES buffer was aspirated off and fresh HEPES buffer was added, and the washing step was repeated three times to remove free calcium ions.
10. And placing the cleaned hydrogel in a refrigerator at the temperature of-20 ℃ for pre-freezing for 2h, then placing the frozen hydrogel in a freeze dryer, carrying out vacuum freeze drying for 24h at the temperature of-80 ℃ to obtain a three-dimensional hydrogel scaffold, and carrying out ultraviolet sterilization for 6h for later use. Thus, the three-dimensional porous hydrogel scaffold with independently controlled rigidity and pore size is obtained.
11. Resuspending the cells in culture medium to adjust the cell concentration to 5X 105Perml, seeded on first and second cell culture plates, 1ml of cell suspension added to each well, respectively, at 37 ℃ with 5% CO2And (4) carrying out cell culture in an incubator.
The preparation method realizes independent regulation and control of the rigidity and the aperture of the hydrogel, and the gelatin-sodium alginate-glycerol hydrogel has good molding, stable mechanical property and uniform pore structure.
Referring to FIG. 3, FIGS. 4A to 4D and FIG. 5, the ratio of 2% glycerol (Gly) and 1% calcium chloride solution (CaCl)2) Preparing hydrogel with low rigidity and large pore diameter, wherein the elastic modulus of the hydrogel is 6.4kPa, and the pore diameter is 170 mu m (figure 4A); preparing high-rigidity and large-aperture hydrogel by using 2% of glycerol and 3% of calcium chloride solution according to the proportion, wherein the elastic modulus is 11.5kPa, and the aperture is 170 mu m (figure 4B); the low-rigidity small-aperture hydrogel is prepared by 5% of glycerol and 1% of calcium chloride solution according to the proportion, the elastic modulus is 6.4kPa, and the aperture is 100 microns (figure 4C); preparation of 5% glycerol and 3% calcium chloride solutionA high stiffness-small pore size hydrogel with an elastic modulus of 11.5kPa and a pore size of 100 μm was obtained (FIG. 4D). The porosity of the hydrogel in all the proportions is over 90 percent, which is beneficial to the growth of cells and the exchange of nutrient substances. Note that in FIGS. 3 and 5, the statistical significance (P value), P<A significant difference was considered at 0.05, and the significance level was indicated as follows: p<0.05 mark @, P<0.01 marks are<0.001 is marked with an x and "NS" indicates no significant difference.
As shown in FIG. 6, the cells were cultured in hydrogel three-dimensionally for 24h and then subjected to flow detection, wherein 78.7% (region Q4) of the cells were live cells, which indicates that the hydrogel culture system has good biocompatibility and can better maintain the cell activity. Wherein:
q1: (annexin V-FITC) -/PI +, the cells in the region are necrotic cells, and a few late apoptotic cells may be contained, even mechanically damaged cells.
Q2 (annexin V + FITC) +/PI +, and the cells in this region are late apoptotic cells.
Q3: (AnnexinV-FITC) +/PI-, cells in this region are early apoptotic cells.
Q4: (annexin V-FITC) -/PI-, and the cells in the area are living cells.
The above description is only an example of the present invention, and is not intended to limit the present invention, and it is obvious to those skilled in the art that various modifications and variations can be made in the present invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the scope of the claims of the present invention.
Claims (10)
1. A method for preparing a composite hydrogel, comprising:
mixing a gelatin solution, a sodium alginate solution, a glycerol solution, a glutaraldehyde solution, a phosphate buffer salt solution and a sterile aqueous solution according to a predetermined volume ratio to obtain a precursor solution with the volume concentration of 1-5% of glycerol;
freezing the precursor solution to convert gelatin from sol to gel and freezing glycerin to form pores;
and adding a calcium chloride solution to perform ionic crosslinking on the sodium alginate in the gel to obtain the composite hydrogel, wherein the concentration of the calcium chloride is related to the rigidity of the composite hydrogel.
2. The method of claim 1, further comprising:
gelatin solution with mass concentration of 3-6%, sodium alginate solution with mass concentration of 1-3%, glycerol solution with mass concentration of 40-60%, glutaraldehyde solution with mass concentration of 0.5-2%, and 1 × Phosphate Buffered Saline (PBS) with pH7.4 are respectively provided, and the precursor solutions are mixed to obtain the precursor solution.
3. The method of claim 1 or 2, further comprising:
mixing gelatin solution, sodium alginate solution, glycerol solution, glutaraldehyde solution and phosphate buffer salt solution to prepare precursor solution with gelatin solution volume concentration, sodium alginate solution volume concentration, glycerol solution volume concentration, glutaraldehyde solution volume concentration and phosphate buffer salt solution volume concentration of 15-25%, 1-5%, 1-3% and 5-15%, respectively, and supplementing the rest volume with sterile aqueous solution.
4. The method as claimed in claim 1, wherein the step of adding calcium chloride solution to make sodium alginate in the gel undergo ionic crosslinking to obtain the composite hydrogel comprises:
adding calcium chloride solution with mass concentration of 0.5-1.5% to make sodium alginate undergo the process of ion cross-linking; or
Adding calcium chloride solution with mass concentration of 1.6-3% to make sodium alginate undergo the process of ion cross-linking;
wherein the rigidity of the composite hydrogel obtained by adding the calcium chloride solution with the mass concentration of 0.5-1.5% is lower than that of the composite hydrogel obtained by adding the calcium chloride solution with the mass concentration of 1.6-3%.
5. The method of claim 4, further comprising:
the composite hydrogel was soaked in HEPES buffer at pH7.0 to remove free calcium ions.
6. A method for constructing a bionic system of a cell microenvironment is characterized by comprising the following steps:
mixing a gelatin solution, a sodium alginate solution, a glycerol solution, a glutaraldehyde solution, a phosphate buffer salt solution and a sterile aqueous solution according to a predetermined volume ratio to obtain a precursor solution with the volume concentration of 1-5% of glycerol;
adding the precursor solution into a cell culture plate for freezing treatment, converting gelatin from sol to gel, and freezing glycerol for pore-forming;
adding a calcium chloride solution into the cell culture plate to perform ionic crosslinking on the sodium alginate to obtain a composite hydrogel, wherein the concentration of the calcium chloride is associated with the rigidity of the hydrogel;
freeze-drying the cell culture plate to obtain a three-dimensional porous hydrogel scaffold with independently regulated rigidity and aperture;
adding cell suspension into the cell culture plate for cell culture.
7. The method of claim 6, further comprising:
gelatin solution with mass concentration of 3-6%, sodium alginate solution with mass concentration of 1-3%, glycerol solution with mass concentration of 40-60%, glutaraldehyde solution with mass concentration of 0.5-2%, and 1 × Phosphate Buffered Saline (PBS) with pH7.4 are respectively provided, and the precursor solutions are mixed to obtain the precursor solution.
8. The method of claim 6 or 7, further comprising:
mixing gelatin solution, sodium alginate solution, glycerol solution, glutaraldehyde solution and phosphate buffer salt solution to prepare precursor solution with the volume concentration of the gelatin solution, the volume concentration of the sodium alginate solution, the volume concentration of the glycerol solution, the volume concentration of the glutaraldehyde solution and the volume concentration of the phosphate buffer salt solution being 15-25%, 1-5%, 1-3% and 5-15% respectively, and quantitatively filling the rest volume with sterile aqueous solution.
9. The method of claim 6, wherein the step of adding a calcium chloride solution to the cell culture plate to ionically crosslink sodium alginate comprises:
adding a calcium chloride solution with the mass concentration of 0.5-1.5% into the cell culture plate to carry out ionic crosslinking on the sodium alginate; or
Adding a calcium chloride solution with the mass concentration of 1.6-3% into the cell culture plate to perform ionic crosslinking on the sodium alginate;
wherein the rigidity of the composite hydrogel obtained by adding the calcium chloride solution with the mass concentration of 0.5-1.5% is lower than that of the composite hydrogel obtained by adding the calcium chloride solution with the mass concentration of 1.6-3%.
10. The method of claim 9, further comprising:
HEPES buffer with PH7.0 was added to the cell culture plate to soak the composite hydrogel to remove free calcium ions.
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