CN116083363A

CN116083363A - Application of composite hydrogel in promotion of cell balling culture

Info

Publication number: CN116083363A
Application number: CN202310030299.4A
Authority: CN
Inventors: 相佳佳; 郭云舟; 陈玉平; 邵世群
Original assignee: ZJU Hangzhou Global Scientific and Technological Innovation Center
Current assignee: ZJU Hangzhou Global Scientific and Technological Innovation Center
Priority date: 2023-01-09
Filing date: 2023-01-09
Publication date: 2023-05-09

Abstract

The invention discloses an application of composite hydrogel in promoting cell balling culture. The composite hydrogel is prepared by crosslinking polyvinyl alcohol and sodium alginate, and when cells are subjected to balling culture, the polyvinyl alcohol/sodium alginate composite hydrogel is placed at the bottom of a culture solution, and the cells are cultured above the polyvinyl alcohol/sodium alginate composite hydrogel. The method of the invention can lead the cultured cells to be rapidly balled, and has obvious advantages compared with the matrix glue which is most widely applied in the current commerce. In addition, the technology uses polyvinyl alcohol and sodium alginate as main raw materials, and the polyvinyl alcohol and the sodium alginate are mature commercial polymers, so that the cost is low. And the preparation method through calcium ion crosslinking is simple and efficient.

Description

Application of composite hydrogel in promotion of cell balling culture

Technical Field

The invention belongs to the technical field of biology, and particularly relates to application of composite hydrogel in promoting cell balling culture.

Background

Cancer is one of the greatest threats to public health in today's society, however, the therapeutic effects on cancer have been difficult to progress clinically for a long time. In order to solve the difficult problem of cancer treatment, researchers culture cancer cells in vitro, establish various in vitro cancer models so as to deepen understanding of the mechanism of occurrence and development of cancer, and simultaneously facilitate screening medicines for cancer treatment. Cancer cells, fibroblasts, immune cells and the like in real tumor tissues are aggregated into compact multicomponent cell spheres, so that medicines are difficult to enter the inside of the cell spheres to kill the cancer cells, however, the traditional cell culture mode is to uniformly spread out cells and attach the cells to the inner surface of a culture dish for growth, and the difference between the traditional cell culture mode and the real tumor cell growth environment is large, so that the technology for constructing the multicomponent cell spheres in vitro is the most attractive in the current construction of in vitro cancer models, and meanwhile, the technology is also the basis for constructing large tissues and complex organs. Currently, three techniques for constructing multicomponent cell spheres are used:

(1) Liquid coating method: the method is simple to operate, however, requires a long time and is unfavorable for multi-component cell balling. In addition, the environment in which the cells grow remains in a liquid environment, similar to that of blood circulation, but different from the microenvironment of most tumor tissues.

(2) Mechanical stirring method: cell attachment is prevented by continuous mechanical agitation, thereby promoting cell aggregation to form a cell sphere. This method requires special equipment and requires external waste disposal equipment. Although the yield is large, the cost is high and the period is long.

(3) Microcarrier method: microcarriers are typically 100 to 300 nm microspheres prepared using different materials. Depending on the type of microcarrier, cells may be grown on the surface of the microsphere, or in the pore structure of the porous microsphere, where the cells, although distributed as a monolayer of cells, are able to secrete extracellular matrix in the porous structure, creating an environment resembling the internal tissues of the human body. Microcarrier methods are a good solution for cell types that cannot spontaneously form cell spheres in a three-dimensional liquid environment, however the main disadvantage of the current methods is the difficulty in collecting and analyzing the cells after culturing. In addition, cells grown in the microporous structure are also not easily observed under a microscope.

In summary, the novel cell ball construction technology preferably has the advantages of simple method, high efficiency and low cost, so that the cell ball construction technology is widely applied and is suitable for commercial large-scale popularization.

In the prior art, sodium alginate and polyvinyl alcohol are generally used for a carrier for cell immobilization, for example, the invention with publication number of CN112725327A discloses a preparation method of the carrier for cell immobilization, and the preparation method comprises the following steps: uniformly mixing a sodium alginate solution and polyvinyl alcohol to obtain a carrier solution; wherein the mass ratio of the sodium alginate to the polyvinyl alcohol is 1:2 to 10; adding diatomite into the carrier solution, uniformly mixing, gradually extruding the gel mixed solution dropwise into a crosslinking solution in a continuous stirring state for solidification to form coagulated beads, washing the coagulated beads, freezing and crosslinking at the temperature of-25 to-15 ℃, and thawing to obtain the cell immobilization carrier. In the prior art, no report that hydrogel prepared by crosslinking sodium alginate and polyvinyl alcohol can promote cell balling culture exists.

Disclosure of Invention

Aiming at the technical problems in the prior art, the invention provides an application of composite hydrogel in promoting cell balling culture. The invention provides a technology for rapidly inducing multicomponent cell sphere generation through polyvinyl alcohol/sodium alginate (PVA/Alg) composite hydrogel. The cell ball can be quickly constructed within 12 hours by culturing cells with different components on the surface of the composite hydrogel, and the method is simple and efficient and has low cost, thereby greatly overcoming the defects of the prior cell ball construction technology.

The invention firstly provides an application of composite hydrogel in promoting cell balling culture, the composite hydrogel is polyvinyl alcohol/sodium alginate composite hydrogel, when the cell balling culture is carried out, the polyvinyl alcohol/sodium alginate composite hydrogel is put on the bottom of a culture solution, the cell is cultured above the polyvinyl alcohol/sodium alginate composite hydrogel,

the preparation method of the polyvinyl alcohol/sodium alginate composite hydrogel comprises the following steps:

(1) Respectively preparing a polyvinyl alcohol solution, a sodium alginate solution and a calcium chloride solution;

(2) Mixing a polyvinyl alcohol solution and a sodium alginate solution to obtain a polyvinyl alcohol/sodium alginate mixed solution;

(3) Adding a cross-linking agent calcium chloride solution into the polyvinyl alcohol/sodium alginate mixed solution, cross-linking to obtain the polyvinyl alcohol and sodium alginate composite super-macroporous hydrogel,

wherein the concentration of the polyvinyl alcohol solution is 10wt%, the concentration of the sodium alginate solution is 4wt%, and the polyvinyl alcohol solution and the sodium alginate solution are mixed according to the volume ratio of 5:1.

Preferably, the concentration of the calcium chloride solution is 0.5M, and the volume ratio of the calcium chloride solution to the polyvinyl alcohol/sodium alginate mixed solution is 1:1, a step of;

before adding a calcium chloride solution for crosslinking, removing bubbles in the polyvinyl alcohol/sodium alginate mixed solution;

the crosslinking process is standing crosslinking for at least 12 hours;

after the crosslinking is completed, the obtained polyvinyl alcohol/sodium alginate composite hydrogel is soaked in ultrapure water to remove free calcium ions.

Preferably, the polyvinyl alcohol/sodium alginate composite hydrogel is sterilized before cell culture.

Preferably, the spheronized cells are one of the following:

(1) Tumor cells;

(2) Mixing tumor cells with tumor tissue peripheral cells;

(3) An immune cell.

Further preferably, the tumor tissue peripheral cells are at least one of: fibroblasts, macrophages.

The invention also provides a cell balling culture method, when the cell balling culture is carried out, the polyvinyl alcohol/sodium alginate composite hydrogel pad is arranged at the bottom of the culture solution, the cell is cultured above the polyvinyl alcohol/sodium alginate composite hydrogel,

Preferably, the concentration of the calcium chloride solution is 0.5M, and the volume ratio of the calcium chloride solution to the polyvinyl alcohol/sodium alginate mixed solution is 1:1;

the crosslinking process is standing crosslinking for at least 12 hours;

Preferably, the spheronized cells are one of the following:

(1) Tumor cells;

(2) Mixing tumor cells with tumor tissue peripheral cells;

(3) An immune cell.

The addition of polyvinyl alcohol to the cell culture solution reduces the adhesion between the cells and the well plate, thereby promoting the aggregation of the cells into spheres. In addition to the use of polyvinyl alcohol to reduce the adhesion between cells and the well plate, the cells are easier to spread on the rigid substrate material, and tend to aggregate on the flexible substrate material, so that the preparation of the flexible composite hydrogel by compounding sodium alginate with weak interaction force between cells and polyvinyl alcohol is expected to become a new generation of multicomponent cell sphere construction technology. Uniformly mixing sodium alginate and a polyvinyl alcohol solution, preparing the polyvinyl alcohol/sodium alginate composite hydrogel through calcium ion crosslinking of the sodium alginate, soaking the composite hydrogel into a cell culture solution, and adding cells on the surface of the composite hydrogel for culturing after swelling balance is achieved. The polyvinyl alcohol still plays a role in reducing cell adhesion, and the sodium alginate framework structure provides flexible support to induce cells to quickly aggregate into balls.

The technology of the invention can induce KPC cells, 3T3 cells, macrophages and the like to quickly ball within 12 hours, and the cell balls can be still induced to quickly generate by mixing different cells according to the proportion, so that the technology has obvious advantages compared with the matrigel which is most widely applied in the current commerce. In addition, the technology uses polyvinyl alcohol and sodium alginate as main raw materials, and the polyvinyl alcohol and the sodium alginate are mature commercial polymers, so that the cost is low. And the preparation method through calcium ion crosslinking is simple and efficient.

Drawings

FIG. 1 is a schematic diagram showing the preparation process of two PVA/Alg composite hydrogels of example 1 and example 2 (polyvinyl alcohol and sodium alginate in the ratios of 1:5, 1:1 and 5:1, respectively) and for cell culture.

FIG. 2 shows the surface of a composite hydrogel prepared by mixing a polyvinyl alcohol solution and a sodium alginate solution in a volume ratio of 1:1 in example 1.

FIG. 3 shows the KPC and 3T3 cells of example 1, each cultured individually and mixed at a ratio of 1:1, after various times on a 5-1 complex hydrogel. The scale bar is 200 microns.

FIG. 4 shows the KPC cells of example 1 after being cultured alone on a 1-5 complex hydrogel for various periods of time. The scale bar is 200 microns.

FIG. 5 shows the KPC and 3T3 cells of example 1 after being cultured individually and mixed on matrigel at a ratio of 1:1 for different periods of time. The scale bar is 200 microns.

FIG. 6 shows the KPC and 3T3 cells of example 1 after being cultured individually and mixed in a ratio of 1:1 on an orifice plate for different periods of time. The scale bar is 200 microns.

FIG. 7 shows the macrophage cell culture alone and the KPC cell culture mixed with the KPC cell culture in a 1:1 ratio on a complex hydrogel for various periods of time in example 2. The scale bar is 200 microns.

Detailed Description

FIG. 1 is a schematic diagram showing the preparation process of PVA/Alg composite hydrogels and cell culture methods according to the present application. Firstly, preparing PVA/Alg composite hydrogel by crosslinking sodium alginate and polyvinyl alcohol under the action of calcium chloride, then using the PVA/Alg composite hydrogel for cell culture, and after the PVA/Alg composite hydrogel is coated at the bottom of a culture hole of a culture device (such as a culture dish and a porous culture plate), culturing cells above the PVA/Alg composite hydrogel.

Example 1

(1) Preparing sodium Alginate (Alginate) solution: 8g of sodium alginate powder was dissolved in 192g of ultrapure water at room temperature to obtain a sodium alginate solution of 4 wt%.

(2) Preparing a polyvinyl alcohol (PVA) solution: 20g of PVA powder was dissolved in hot water (180 g) at 90℃with continuous stirring to give a 10wt% PVA solution.

(3) Preparing a polyvinyl alcohol/sodium alginate mixed solution: blending the sodium alginate solution and the PVA solution in the steps (1) and (2) according to the volume ratio of 5:1, 1:1 and 1:5, and continuously stirring at room temperature until the mixture is in a uniform steam drum-free state.

(4) Preparing a calcium chloride solution: 11.1g of calcium chloride particles were weighed, dissolved in 200ml of ultrapure water, and treated by ultrasonic treatment to obtain a 0.5M calcium chloride solution.

(5) Preparing a glue mold: a3 mm thick silica gel pad is prepared, cut into a photo frame shape (the middle part is hollowed), the silica gel pad is clamped between two PET templates, and the silica gel pad is clamped and fixed at the bottom and the edge part by a dovetail clamp, so that the leakage of liquid after the liquid is added into a die is prevented.

(6) Preparation of composite hydrogel: and (3) adding the mixed solution (the polyvinyl alcohol and sodium alginate are in the ratio of 1:5, 1:1 and 5:1) in the step (5) into the mold respectively, placing the mold vertically, adding the mixed solution into a half position in the mold, placing the mold at room temperature for 12 hours, waiting for the liquid level of the mixed solution, and eliminating bubbles. The calcium chloride solution was then added to the mold using a needle syringe to fill the space remaining in the mold. After the addition of the calcium chloride solution is completed, the mold is vertically placed under the room temperature environment, after standing and crosslinking are carried out for 12 hours, the gel is taken out, soaked in 0.5M calcium chloride solution for 12 hours, soaked in ultrapure water for 24 hours, free calcium ions are removed, and then the gel is processed into round slice-shaped samples with different sizes by a puncher. Wherein, the sample with the solution ratio of 5:1 is abbreviated as PVA/Alg-5-1, and the sample with the solution ratio of 1:5 is abbreviated as PVA/Alg-1-5. The composite hydrogel prepared by mixing the sodium alginate solution and the PVA solution according to the volume ratio of 1:1 has uneven surface and cannot be used for cell sphere construction.

(7) And (3) gel sterilization treatment: for cell culture, the prepared hydrogel in the form of a round plate is subjected to high-temperature sterilization for 20 minutes at 120 ℃.

(8) And (3) treating matrigel: the corning matrigel (354230) was removed from-20deg.C and placed in a refrigerator at 4deg.C overnight for thawing.

(9) Preparing a cell complete medium: 10% fetal bovine serum and 1% penicillin-streptomycin solution were added to high-sugar DMEM medium.

(10) Cell culture: KPC mouse pancreatic cancer cells and 3T3 mouse fibroblasts are all cultured in the complete culture medium prepared in the step (9), and all cells are placed at 37 ℃ and 5% CO ₂ The cells were cultured in a cell incubator, medium was changed every day, and passage was performed every 3d with digestion.

(11) Preparation of cell suspensions: the KPC and 3T3 cells were digested with trypsin and resuspended with the corresponding fresh medium, and the cell density was adjusted to 2X 10 after counting ⁴ And each mL.

(12) Cell culture on PVA/Alg complex hydrogel: placing the three 5-1 composite hydrogels sterilized in the step (7) into a 24-well plate for cell culture, adding the cell culture solution in the step (9), waiting for 12 hours, and adding the cell types to be cultured on the three gels respectively. The cultured cells are respectively mouse pancreatic cancer cells (KPC), mouse fibroblasts (3T 3) and cell groups obtained by blending KPC/3T3 according to the ratio of 1:1. Placing the 1-5 composite hydrogel in the step (7) into an orifice plate, adding the cell culture solution in the step (9), waiting for 12 hours, and adding KPC cells for culturing.

(13) Cell culture on matrigel: to compare the effect with the most widely used commercial matrigel at present, three matrigel in step (8) were placed in a 24-well plate for cell culture, the cell culture solution in step (9) was added, and after waiting for 12 hours, the cell types to be cultured were added to the three matrigel, respectively. The cultured cells are KPC cells, 3T3 cells and cell groups obtained by blending KPC/3T3 cells according to the ratio of 1:1.

(14) Cell culture in well plates: cell growth status in the well plate was the most blank. The cell culture solution in step (9) was added to three wells of a 24-well plate for cell culture, and then the cell type to be cultured was added. The cultured cells are KPC cells, 3T3 cells and cell groups obtained by blending KPC/3T3 according to the ratio of 1:1 respectively.

(15) Observing the cell state: cells cultured in different environments in steps (12) (13) (14) were subjected to light microscopy at different time nodes to observe cell morphology.

Analysis of results:

in FIG. 2, the composite hydrogel prepared by mixing sodium alginate solution and PVA solution in a volume ratio of 1:1 has uneven surface, and cannot be used for cell sphere construction. In FIG. 3, KPC cells and 3T3 cells grown on PVA/Alg-5-1 complex hydrogel can rapidly form cell pellets within 12 hours, and the diameter of the cell pellets can reach about 200 microns at maximum. And the cell sphere structure remained stable for 24 hours. The cell group obtained by blending the two components of the cell pellet in a ratio of 1:1 can achieve the same effect. In FIG. 4, KPC cells grown on PVA/Alg-1-5 complex hydrogels formed only slightly into spheres even after 24 hours and had a relatively loose structure. This is because the proportion of sodium alginate increases, which results in hardening of the gel, which hinders cell aggregation, and further, the decrease in PVA content results in a decrease in cell aggregation into spheres. Therefore, when the proportion of polyvinyl alcohol to sodium alginate is 5:1, the obtained composite hydrogel has the best effect on constructing cell spheres. In fig. 5, KPC cells grown on matrigel appeared aggregated cell spheres at 24 hours, which took significantly longer to pellet than the composite hydrogel, and the KPC cell spheres were approximately 100 microns in diameter, significantly smaller than those on the composite hydrogel. Whereas the 3T3 cells and KPC/3T3 mixed cell populations grown on matrigel had undergone some degree of aggregation, but were not able to form a cell sphere structure within 24 hours. As a blank, in fig. 6, all cells grown in the well plate had no tendency to sphere or aggregate, and all cells grown in the well plate were in a state of dispersion proliferation. The data fully demonstrate that the PVA/Alg-5-1 composite hydrogel can rapidly induce KPC cells and 3T3 cells to form cell spheres, and has obvious advantages compared with commercial matrigel.

Example 2

(3) Preparing a polyvinyl alcohol/sodium alginate mixed solution: blending the sodium alginate solution and the PVA solution in the steps (1) and (2) according to the volume ratio of 5:1, and continuously stirring at room temperature until the mixture is in a uniform steam drum-free state.

(6) Preparation of composite hydrogel: adding the mixed solution in the step (3) into the die in the step (5), placing the die vertically, adding the mixed solution into a half position in the die, placing the die at room temperature for 12 hours, waiting for the liquid level of the mixed solution, and eliminating bubbles. The calcium chloride solution was then added to the mold using a needle syringe to fill the space remaining in the mold. After the addition of the calcium chloride solution is completed, the mold is vertically placed under the room temperature environment, after standing and crosslinking are carried out for 12 hours, the gel is taken out, soaked in 0.5M calcium chloride solution for 12 hours, soaked in ultrapure water for 24 hours, free calcium ions are removed, and then the gel is processed into round slice-shaped samples with different sizes by a puncher.

(8) Preparing a cell complete medium: 10% fetal bovine serum and 1% penicillin-streptomycin solution were added to high-sugar DMEM medium.

(9) Cell culture: culturing KPC mouse pancreatic cancer cells in the complete medium prepared in the step (8), culturing RAW264.7 mouse macrophages by using a special culture medium, and placing at 37 ℃ and 5% CO ₂ In the cell culture box, the culture medium is replaced every day,every 3d passage was performed by digestion.

(10) Preparation of cell suspensions: cell suspension is obtained by re-suspending the cell suspension by using corresponding fresh culture medium after trypsin digestion, and the cell density is adjusted to 2 multiplied by 10 after counting ⁴ And each mL.

(11) Cell culture on PVA/Alg complex hydrogel: placing the two gels sterilized in the step (7) into a 24-well plate for cell culture, adding the cell culture solution in the step (8), waiting for 12 hours, and adding the cell types to be cultured on the two gels respectively. The cultured cells are macrophages respectively, and the KPC/macrophage is mixed according to the proportion of 1:1.

(12) Observing the cell state: the cells cultured in step (9) were subjected to an optical microscope at various time nodes to observe the cell morphology.

Analysis of results:

macrophages belong to immune cells, and there is also a large number of co-growth of macrophages and cancer cells in tumor tissue. It is difficult to aggregate them into cell spheres using conventional cell sphere construction techniques. In FIG. 7, macrophages growing on complex hydrogels aggregate to form cell spheres within 9 hours, with diameters up to about 200 microns. Macrophages and KPC cells were mixed at 1:1, and also a small amount of cell pellet formation was observed within 9 hours and a large amount of apparent cell pellet formation within 24 hours. The data fully show that the PVA/Alg-5-1 composite hydrogel can realize rapid construction of cell spheres for cells with different components, and can be fully applied to construction of in-vitro tumor tissues or organoids.

Claims

1. The application of the composite hydrogel in promoting cell balling culture is characterized in that the composite hydrogel is polyvinyl alcohol/sodium alginate composite hydrogel, when the cell balling culture is carried out, the polyvinyl alcohol/sodium alginate composite hydrogel is put on the bottom of a culture solution, the cell is cultured above the polyvinyl alcohol/sodium alginate composite hydrogel,

(3) Adding a cross-linking agent calcium chloride solution into the polyvinyl alcohol/sodium alginate mixed solution, cross-linking to obtain polyvinyl alcohol and sodium alginate composite hydrogel,

2. The use according to claim 1, wherein the concentration of the calcium chloride solution is 0.5M and the volume ratio of the calcium chloride solution to the polyvinyl alcohol/sodium alginate mixed solution is 1:1;

the crosslinking process is standing crosslinking for at least 12 hours;

3. The use according to claim 1, wherein the polyvinyl alcohol/sodium alginate composite hydrogel is sterilized prior to cell culture.

4. The use according to claim 1, wherein the spheronized cells are one of the following:

(1) Tumor cells;

(2) Mixing tumor cells with tumor tissue peripheral cells;

(3) An immune cell.

5. The use according to claim 4, wherein the tumor tissue peripheral cells are at least one of: fibroblasts, macrophages.

6. A cell balling culture method is characterized in that when the cells are balled and cultured, a polyvinyl alcohol/sodium alginate composite hydrogel pad is arranged at the bottom of a culture solution, the cells are cultured above the polyvinyl alcohol/sodium alginate composite hydrogel,

7. The method for cell pelleting culture according to claim 6, wherein the concentration of the calcium chloride solution is 0.5M, and the volume ratio of the calcium chloride solution to the polyvinyl alcohol/sodium alginate mixed solution is 1:1;

the crosslinking process is standing crosslinking for at least 12 hours;

8. The method according to claim 6, wherein the polyvinyl alcohol/sodium alginate composite hydrogel is sterilized prior to cell culture.

9. The method of claim 6, wherein the cells in the pellet culture are one of the following:

(1) Tumor cells;

(2) Mixing tumor cells with tumor tissue peripheral cells;

(3) An immune cell.

10. The cell pellet culture method of claim 9, wherein the tumor tissue peripheral cells are at least one of: fibroblasts, macrophages.