CN110862562B

CN110862562B - A kind of 3D cell culture substrate and preparation method thereof

Info

Publication number: CN110862562B
Application number: CN201911192943.8A
Authority: CN
Inventors: 智伟; 吴月皓; 左梁蕊; 贾一凡; 吴金结; 朱秀鹏; 王振雄; 翁杰; 屈树新
Original assignee: Southwest Jiaotong University
Current assignee: Southwest Jiaotong University
Priority date: 2019-11-28
Filing date: 2019-11-28
Publication date: 2021-08-31
Anticipated expiration: 2039-11-28
Also published as: CN110862562A

Abstract

The invention discloses a 3D cell culture medium and a preparation method thereof. Oxidized alginic acid and sodium alginate are mixed in proportion, and distilled water is used to prepare a mixed sodium alginate solution; filtered in a sterile environment, and then a concentrated medium is added in sequence , serum/growth factors are mixed to obtain a culture solution; finally, mixed with sterile calcium chloride solution for cross-linking, that is, a 3D cell culture substrate is obtained. The 3D cell culture substrate prepared by the invention solves the problem that the sodium alginate solution is difficult to effectively sterilize, and the oxidized sodium alginate is too high in oxidation and too low in cross-linking. At the same time, the plasticity of the culture medium was improved by adjusting the ratio of sodium alginate and oxidized sodium alginate, which was more suitable for cell proliferation and growth. If the cells need to be cultured, the cells to be cultured are added to the culture solution according to a certain inoculation concentration, gently mixed, and finally cross-linked with sterile calcium chloride to culture the cells.

Description

3D cell culture medium and preparation method thereof

Technical Field

The invention belongs to the technical field of biological materials, and relates to a preparation method of a3D cell culture matrix and the 3D cell culture matrix prepared by the method.

Background

Sodium Alginate (SA) as a natural polymer material has good biocompatibility. The SA hydrogel has the biological characteristics similar to that of extracellular matrix, and is suitable for constructing a cell culture microenvironment. However, Sodium Alginate (SA) solution has high viscosity and is difficult to sterilize by filtration, and the sterilization means for SA in the literature at present generally adopts high-temperature high-pressure sterilization, high-temperature dry heat sterilization, ultraviolet sterilization and irradiation sterilization. However, the formability of the hydrogel is damaged by high-temperature high-pressure and high-temperature dry heat sterilization, the sterilization effect is poor by ultraviolet sterilization, and the material properties of the hydrogel are greatly influenced by irradiation sterilization. The application range of the sodium alginate hydrogel serving as a3D cell culture matrix in scientific research and clinic is greatly limited. Due to the sterilization problem of SA, no SA3D cultured cell matrix has been used for long-term cell culture.

In order to solve the problems, the invention mixes Sodium Alginate (SA) and Oxidized Sodium Alginate (OSA) according to a certain proportion to reduce the viscosity of the mixture on the basis of not influencing the crosslinking degree of gel, and then prepares the sterile hydrogel containing culture components and bioactive factors by a simple and ingenious method.

Disclosure of Invention

The technical problem to be solved by the invention is to reduce the viscosity of SA under the condition of not influencing the crosslinking of SA hydrogel so as to facilitate the filtration and sterilization of a solution, thereby facilitating the construction of a3D cell culture matrix with biological activity.

The reason why the cross-linking with calcium ions can not be affected by mixing SA and OSA is that the OSA and the long SA molecule chains form a network structure in a spatial interpenetrating mode, the hydroxyl groups on the long SA molecule chains can just make up for the shortage of the hydroxyl groups on the long OSA molecule chains, and the long SA molecule chains are cross-linked with the calcium ions in an occupying mode, so that the solution viscosity is reduced, and the formability of the hydrogel is not affected (as shown in figure 1 in particular).

A preparation method of a3D cell culture substrate comprises the following steps:

(1) preparing oxidized sodium alginate: adding sodium periodate solution into sodium alginate, stirring and reacting in the dark, adding glycol with the same molar amount as sodium periodate to terminate the reaction, dialyzing, and freeze-drying to obtain oxidized sodium alginate;

(2) preparing a mixed sodium alginate solution: mixing oxidized alginic acid and sodium alginate in proportion, and preparing a mixed sodium alginate solution by using distilled water;

(3) preparing a sterile calcium chloride solution: preparing a sterile calcium chloride solution with the mass fraction of 1.2% by using distilled water;

(4) preparing a sterile mixed sodium alginate solution: transferring the mixed sodium alginate solution and the sterile calcium chloride solution to cells in a sterile environment, and filtering by using a cell filter to obtain a sterile mixed sodium alginate solution;

(5) preparing a sodium alginate culture solution: sequentially adding a concentrated culture medium and serum/growth factors into the sterile mixed sodium alginate solution according to a proportion, and uniformly mixing to obtain a sodium alginate culture solution;

(6) preparation of 3D cell culture medium: and (3) crosslinking the mixed solution of the sterile calcium chloride solution and the sodium alginate culture solution to obtain the 3D cell culture medium.

Further, the oxidation degree of the oxidized sodium alginate in the step (1) is 50-80%.

Further, the using amount of the sodium periodate solution in the step (1) is 50-80% of the mass of the sodium alginate; the reaction time is 24 hours under the condition of light-resistant stirring; the dialysis time is not less than 24 hours, and water is changed every 4 hours.

Further, the mass ratio of the sodium alginate to the oxidized sodium alginate in the step (2) is (2-3): (3-2); the concentration of the mixed sodium alginate solution is 1-4%.

Further, the pore size of the cell filter in the step (3) is 0.22 um.

Further, the sterile calcium chloride solution in the step (4) is prepared into a sterile calcium chloride solution with the mass fraction of 1.2% by using distilled water.

Further, the concentration culture medium in the step (4) is concentrated by 10-100 times.

Further, the serum in the step (4) is added according to the proportion of 1-10%.

The invention also discloses a3D cell culture substrate prepared by any preparation method.

The invention also discloses an application of the 3D cell culture substrate in culturing 3D cells, which comprises the steps of firstly culturing the cells to be cultured according to the ratio of 10⁴～10⁷Adding into sodium alginate culture solution at a concentration of/ml, mixing, and adding sterile calcium chlorideAnd (4) crosslinking the solution to perform 3D culture of the cells.

The invention has the beneficial effects that:

(1) the problem that the OSA is difficult to effectively sterilize and the OSA is too high in oxidation degree and too low in crosslinking degree due to the fact that the SA solution is difficult to effectively sterilize is solved by mixing, filtering and then crosslinking with calcium ions.

(2) The matrix hardness of the hydrogel can be adjusted by adjusting the ratio of SA to OSA, so that the 3D cell culture matrix has good plasticity, and matrixes with different shapes can be obtained through a mould.

(3) The culture medium has good biocompatibility due to the main component of sodium alginate, contains cell culture components with appropriate concentration, and is suitable for proliferation and growth of cells.

Drawings

FIG. 1 is a schematic representation of OSA and SA through space occupation and then cross-linking with calcium ions;

FIG. 2 is a graph of matrix hardness of hydrogels obtained from different ratios of OSA to SA;

FIG. 3 is a gel made with a mold for 3D culture medium;

FIG. 4 is a fluorescent staining pattern of cells on the third day, wherein green viable cells are on the left and red dead cells are on the right;

FIG. 5 is a graph of fluorescent staining of cells at day seven with green viable cells on the left and red dead cells on the right;

FIG. 6 shows the results of the proliferation assay of cells cultured in 3D medium and cultured on a flat surface, and the OD value reflects the proliferation of the cells.

Detailed Description

The invention provides a cell culture substrate and an application and a using method thereof, and a person skilled in the art can realize the cell culture substrate by properly improving process parameters by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

Example 1

SA used in the experiment is a product of sigma company, the product number is 180947-250G, and the CAS number is 9005-38-3.

6gSA was added to a beaker containing the appropriate amount of water and stirred magnetically until dissolved. After sodium periodate with the amount of substance being 60 percent of sodium alginate, namely 3.6g of sodium periodate, is dissolved in water, the solution is added into sodium alginate solution and stirred for reaction for 24 hours in the dark. Then, ethylene glycol was added in an equimolar amount to sodium periodate to terminate the reaction. The solution was dialyzed for 24h against a dialysis bag, with water changed every 4 hours or so to remove the ethylene glycol and possibly unreacted sodium periodate. After the dialysis was completed, the dialyzed solution was lyophilized at-60 ℃ using a lyophilizer, and finally OSA having an oxidation degree of about 60% was obtained.

0.2gSA and 0.2g OSA were added to 19.6ml RO water, i.e. 1:1 to prepare a mixed SA solution with the mass fraction of 2%. Then 0.4g of calcium chloride is added into 19.6ml of RO water to prepare a calcium chloride solution with the mass fraction of 2 percent. After preparation, both solutions were transferred to the cells. The SA solution and the calcium chloride solution were mixed to be sterile by filtration sterilization using a cell filter having a diameter of 0.22 μm.

Preparation density of 2X 10⁶Individual cells/ml of cell suspension. 1ml of the sterile SA mixed solution was added to each well of a 24-well plate, followed by sequentially adding 10 Xconcentrated medium and 100. mu.l of FCS serum, and mixing. Adding 2% of CaCl₂The solution is crosslinked to form a hydrogel. The hydrogel can be used as a3D culture matrix of cells to promote cell proliferation and differentiation.

Test example 1

SA molecular chains can be crosslinked with calcium ions, but sterilization is difficult, while OSA has low viscosity, but has too few hydroxyl groups to be crosslinked with the calcium ions, so the method of mixing OSA and SA and then filtering for sterilization is selected, thereby not only reducing the solution viscosity, but also not influencing the formability of the hydrogel. The OSA and the SA molecular long chain form a network structure in a spatial interpenetrating mode, the hydroxyl on the SA molecular long chain can just make up the shortage of the hydroxyl number on the OSA molecular long chain, and the hydroxyl is crosslinked with calcium ions in an occupying mode; a schematic representation of OSA and SA through space occupation and then cross-linking with calcium ions is shown in fig. 1.

Test example 2

We put OSA and SA as 1: 1. 2: 3. 3: 2. 4: 5. 5:4, and dissolving in UP water to prepare a solution with the concentration of 2 percent. Dropwise adding 1.2% of CaCl with the same volume₂The solution was crosslinked to a hydrogel, and the matrix hardness of the hydrogel was measured using a universal tester (0.06 mm/s). The matrix hardness profile of the hydrogel obtained by different ratios of OSA to SA is shown in FIG. 2, and according to the results of FIG. 2, the stress change does not increase significantly with increasing strain when the ratio is 1:1,2:3,4:5, while the stress increases significantly with increasing strain when the ratio of OSA to SA is 3:2 and 5:4 when the ratio is 0.4. Therefore, the matrix hardness of the hydrogel can be better regulated and controlled by regulating the ratio of the OSA to the SA.

Test example 3

To verify the plasticity of the gel, the SA mixed solution is added with dye for dyeing, 2ml of the SA mixed solution is poured into a prepared mould, and the same amount of calcium ion solution is sprayed on the surface of the mould, so that the hydrogel with different shapes can be prepared. As shown in FIG. 3, the cell culture substrate was found to have high plasticity.

Test example 4

To verify the feasibility of the method of culturing cells, we prepared a density of 2X 10⁶Individual cells/ml of cell suspension. 1ml of the sterile SA cocktail was added to each well of a 24-well plate, followed by addition of 100. mu.l each of 10 Xconcentrated medium, FCS and cell suspension in that order, and mixed well. Adding 2% of CaCl₂The solution is crosslinked to form hydrogel, and cells can be cultured to serve as an experimental group.

The experimental groups were incubated in an incubator, sampled on

days

3 and 7, and stained with PI/FDA. The fluorescence staining patterns on the third and seventh days were observed (the third and seventh days correspond to fig. 4 and 5, respectively), and it was found from fig. 4 and 5 that the number of living cells was large, the number of dead cells was small, and the cells grew well. And the number of viable cells was greater on day 7 than on day 3.

To more clearly illustrate that the three-dimensional culture using the hydrogel can promote cell proliferation, working solution (Alamar blue reagent: medium: 1: 10) is added at

days

1, 3, 5 and 7 of the cell culture, the cells are incubated in an incubator at 37 ℃ for 2h for sampling, OD values are detected at a wavelength of 570nm/600nm, proliferation curves are drawn, and particularly, see FIG. 6 (a comparison graph of the OD values of the cells in a common two-dimensional culture and a three-dimensional culture in sodium alginate hydrogel) shows that, as time goes on, the OD value of the SA group is obviously higher than that of the blank group, particularly, at day 3, the two groups already show obvious difference (P < 0.05), and as time goes on, although the OD value of the SA group is also increased, the OD value of the SA group is obviously higher and far exceeds that of the blank group (P < 0.01). Therefore, the SA hydrogel three-dimensional culture substrate has good biocompatibility, contains appropriate concentrations of cell culture components, and is more suitable for the proliferation and growth of cells.

The cell 3D culture matrix provided by the invention can reduce the viscosity of SA without influencing the crosslinking degree of hydrogel and destroying the property of the SA material, and can be effectively sterilized by a filtration method, thereby obtaining a sterile 3D cell culture matrix. And the sodium alginate has good biocompatibility and contains cell culture components with proper concentration, so that the sodium alginate is beneficial to the proliferation of cells. The cell culture substrate can obtain the required substrate hardness by regulating the concentration ratio of SA and OSA, and can obtain the desired gel shape through a mold. The 3D cell culture substrate has the advantages of simple preparation method, low cost and good culture effect.

Various other modifications and adaptations of the present invention will be apparent to those skilled in the art after reading the foregoing disclosure without departing from the spirit and scope of the invention, and it is intended that all such modifications and adaptations fall within the scope of the appended claims.

Claims

1. a preparation method of 3D cell culture substrate, is characterized in that, the method comprises the steps:

(1) Preparation of oxidized sodium alginate: add sodium periodate solution with a mass of 50-80% to sodium alginate, stir the reaction in the dark for 24h, and then add ethylene glycol with an equimolar amount of sodium periodate to terminate the reaction , and then carry out dialysis treatment for no less than 24 hours, and change the water every 4 hours, and obtain oxidized sodium alginate with an oxidation degree of 50%-80% after lyophilization;

(2) Preparation of mixed sodium alginate solution: The oxidized alginic acid and sodium alginate are mixed in a mass ratio of (3-2): (2-3), and distilled water is used to configure a mixed sodium alginate solution of 1%-4% ;

(3) Preparation of sterile calcium chloride solution: using distilled water to configure a sterile calcium chloride solution with a mass fraction of 1.2%;

(4) Preparation of sterile mixed sodium alginate solution: in a sterile environment, move the mixed sodium alginate solution and sterile calcium chloride solution between cells, and then use a cell filter with a pore size of 0.22 μm to filter to obtain sterile Mix sodium alginate solution and calcium chloride solution;

(5) Preparation of sodium alginate culture solution: to the sterile mixed sodium alginate solution, add concentrated medium and serum in turn, and mix them to obtain a sodium alginate culture solution; wherein the amount of serum added is 1%-10% % ratio added;

(6) Preparation of 3D cell culture substrate: cross-linking the mixed solution of sterile calcium chloride solution and sodium alginate culture solution to obtain 3D cell culture substrate.

2. The preparation method according to claim 1, wherein the concentrated medium in step (5) is 10-100 times concentrated.

3. A 3D cell culture substrate prepared according to any one of the preparation methods of claim 1 or 2.

4. a kind of application of adopting the described 3D cell culture substrate of claim 3 in culturing 3D cells, it is characterized in that, first the cells to be cultured are added to the sodium alginate culture solution at a concentration of 10 ⁴ to 10 ⁷ cells/ml , and then add sterile calcium chloride solution for cross-linking, and then the 3D culture of cells can be carried out.