CN110157673A - A three-dimensional model of tumor cell and its construction method and application - Google Patents

Abstract

The invention belongs to the technical field of three-dimensional culture of tumor cells, and in particular relates to a three-dimensional model of tumor cells and a construction method and application thereof. In the present invention, an alginate solution is added to a mixed cell suspension containing tumor cells and fibroblasts to obtain a mixed cell-alginate solution, and then the mixed cell-alginate solution is dropped into a calcium salt solution to obtain a mixed solution Cell microspheres, the invention adopts alginate and calcium salt to cross-link to prepare microspheres as cell scaffolds, which reduces the cost of three-dimensional culture of tumor cells. Alginate has good biocompatibility and non-immunogenicity, and can Provide a good three-dimensional growth environment for cells, and the present invention co-cultures tumor cells and fibroblasts three-dimensionally, which can more accurately simulate the living environment of tumor cells in vivo, so that the constructed three-dimensional tumor cell model can be used for anti-tumor drugs. Screening and/or evaluation is more accurate and reliable, which can reduce the cost of anti-tumor drug research and development.

Description

A three-dimensional model of tumor cell and its construction method and application

technical field

The invention belongs to the technical field of three-dimensional culture of tumor cells, and in particular relates to a three-dimensional model of tumor cells and a construction method and application thereof.

Background technique

Currently, drug detection based on two-dimensional cell culture models is still the main method for tumor research. However, this method cannot accurately simulate the complex growth environment of tumor cells, so the drug evaluation using a two-dimensional cell culture model is inaccurate, and a cost-effective model that can more accurately simulate the human tumor growth environment can be constructed to replace the two-dimensional cell culture model. Cell models are essential for drug development and evaluation screening.

To improve the accuracy and reliability of antitumor drug evaluation, it is necessary to change the traditional in vitro cell culture model. According to some current studies, three-dimensional in vitro cell culture can reproduce the physiological microenvironment and is highly consistent with the physiological response in vivo. The construction of three-dimensional tumor cell models requires special materials as cell scaffolds to support cell growth. At present, the materials used as cell scaffolds include natural extracellular matrix materials, synthetic biological materials, natural purified molecular materials, etc. These materials have good biocompatibility and physical properties. Chemical properties that allow cells to remain active in growth. However, the above-mentioned materials used as cell scaffolds have the disadvantage of being expensive.

SUMMARY OF THE INVENTION

In view of this, the present invention provides a three-dimensional tumor cell model and its construction method and application, which are used to solve the problem of high cost of using existing materials as cell scaffolds.

The concrete technical scheme of the present invention is as follows:

A method for constructing a three-dimensional model of tumor cells, comprising the following steps:

a) preparing tumor cells and fibroblasts into a mixed cell suspension, adding an alginate solution to the mixed cell suspension to obtain a mixed cell-alginate solution;

b) dropping the mixed cell-alginate solution in the calcium salt solution to obtain mixed cell microspheres;

c) placing the mixed cell microspheres in a culture medium to obtain a three-dimensional model of tumor cells.

Preferably, the number ratio of the tumor cells and the fibroblasts in the mixed cell suspension is (2-3): (1-2).

Preferably, the concentration of the tumor cells in the mixed cell suspension is 1*10 ⁵ cells/ml to 5*10 ⁶ cells/ml;

The concentration of the fibroblasts in the mixed cell suspension is 1*10 ⁵ cells/ml to 5*10 ⁶ cells/ml.

Preferably, the tumor cells are selected from prostate cancer cells, breast cancer cells, uterine fibroids or liver cancer cells.

Preferably, the alginate is sodium alginate;

The calcium salt is selected from calcium chloride or calcium sulfate.

Preferably, the mass volume concentration of the alginate solution is 2% to 4%;

The mass ratio of the mixed cell suspension to the alginate solution is (1-3): (1-3).

Preferably, the concentration of the calcium salt solution is 0.1-0.2 mol/L.

Preferably, the volume ratio of the mixed cell microspheres to the culture solution is (1-3):(1-3).

The present invention also provides the three-dimensional tumor cell model obtained by the construction method described in the above technical solution.

The present invention also provides the application of the three-dimensional tumor cell model described in the above technical solution in the screening and/or evaluation of anti-tumor drugs.

In summary, the present invention provides a method for constructing a three-dimensional tumor cell model, comprising the following steps: a) preparing a mixed cell suspension from tumor cells and fibroblasts, and adding alginic acid to the mixed cell suspension salt solution to obtain a mixed cell-alginate solution; b) dropping the mixed cell-alginate solution in a calcium salt solution to obtain a mixed cell microsphere; c) placing the mixed cell microsphere in a culture solution 3-D model of tumor cells was obtained. In the present invention, an alginate solution is added to the mixed cell suspension containing tumor cells and fibroblasts to obtain a mixed cell-alginate solution, and then the mixed cell-alginate solution is dropped into a calcium salt solution to obtain a mixed solution Cell microspheres, the invention adopts alginate and calcium salt to cross-link to prepare microspheres as cell scaffolds, which reduces the cost of three-dimensional culture of tumor cells. Alginate has good biocompatibility and non-immunogenicity, and can Provides a good three-dimensional growth environment for cells, and the present invention co-cultures tumor cells and fibroblasts three-dimensionally, which can more accurately simulate the living environment of tumor cells in vivo, so that the constructed three-dimensional tumor cell model can be used for anti-tumor drugs. Screening and/or evaluation is more accurate and reliable, which can reduce the cost of anti-tumor drug research and development.

Description of drawings

In order to illustrate the embodiments of the present invention or the technical solutions in the prior art more clearly, the following briefly introduces the accompanying drawings that are required in the description of the embodiments or the prior art.

FIG. 1 is the survival rate of cells on the 4th day, the 8th day, the 10th day and the 12th day in a three-dimensional tumor cell model provided in the embodiment of the present invention.

Detailed ways

The present invention provides a three-dimensional model of tumor cells and a construction method and application thereof, which are used to solve the problem of high cost of using existing materials as cell scaffolds.

The technical solutions in the embodiments of the present invention will be described clearly and completely below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

A method for constructing a three-dimensional model of tumor cells, comprising the following steps:

a) preparing tumor cells and fibroblasts into a mixed cell suspension, adding an alginate solution to the mixed cell suspension to obtain a mixed cell-alginate solution;

b) dropping the mixed cell-alginate solution in the calcium salt solution to obtain mixed cell microspheres;

c) placing the mixed cell microspheres in a culture medium to obtain a three-dimensional model of tumor cells.

In the present invention, an alginate solution is added to the mixed cell suspension containing tumor cells and fibroblasts to obtain a mixed cell-alginate solution, and then the mixed cell-alginate solution is dropped into a calcium salt solution to obtain a mixed solution Cell microspheres, the invention adopts alginate and calcium salt to cross-link to prepare microspheres as cell scaffolds, which reduces the cost of three-dimensional culture of tumor cells, alginate has good biocompatibility and non-immunogenicity, and seaweed The calcium alginate hydrogel obtained by the cross-linking of acid salts and calcium salts has a hydrophobic and porous structure, which is convenient for the attachment and growth of cells, as well as the transport of nutrients, and can provide a good three-dimensional growth environment for cells. In addition, using alginate and calcium salt to cross-link to prepare microspheres as cell scaffolds, the cost is low, the manufacturing process is simple, and the operation is easy.

In vivo, tumor cells co-exist with a variety of cells, and there are various interactions between them, which also leads to great differences in the physiological characteristics of tumor cells when they are cultured alone and when they grow in the human body. In the present invention, tumor cells and fibroblasts are co-cultured in three dimensions, and the three-dimensional model of tumor cells constructed has a complex three-dimensional network of interaction between cells and cells and cells and matrices, which can more accurately simulate the living environment of tumor cells in vivo. When the three-dimensional tumor cell model is used for the screening and/or evaluation of anti-tumor drugs, tumor cells are not only related to the penetration of drugs, but also the distribution and function of fibroblast-secreted factors also affect the growth, differentiation and death of tumor cells, making The constructed three-dimensional tumor cell model is more accurate and reliable when applied to the screening and/or evaluation of anti-tumor drugs, and can reduce the cost of anti-tumor drug research and development.

In the embodiment of the present invention, the number ratio of tumor cells and fibroblasts in the mixed cell suspension is (2-3):(1-2), preferably 2:1.

In the embodiment of the present invention, the concentration of tumor cells in the mixed cell suspension is 1*10 ⁵ cells/ml to 5*10 ⁶ cells/ml, preferably 1*10 ⁶ cells/ml;

The concentration of fibroblasts in the mixed cell suspension is 1*10 ⁵ cells/ml to 5*10 ⁶ cells/ml, preferably 5*10 ⁵ cells/ml.

In the embodiment of the present invention, the tumor cells are selected from prostate cancer cells, breast cancer cells, uterine fibroids or liver cancer cells.

In the embodiment of the present invention, it is necessary to select cells co-cultured with tumor cells according to the growth environment of the tumor in vivo and the composition of the tumor. base culture). The tumor cells are preferably prostate cancer cells. The growth and division cycles of prostate cancer cells are very different from those of fibroblasts. The cell culture conditions are similar and can be cultured in the same medium. In addition, prostate cancer cells grow rapidly, do not have high requirements on the growth environment, are easy to culture, and are suitable for culturing a large number of cells in a short period of time for experiments.

Prostate cancer cells are selected from PC3, LNCaP, DU145, VCaP or VLcop; fibroblasts are selected from 3T3, BMF, HPF, HFF or BJ.

In the embodiment of the present invention, the alginate is sodium alginate, and sodium alginate is a natural polysaccharide extracted from algae, which is low in cost and easy to manufacture;

The calcium salt is selected from calcium chloride or calcium sulfate.

In the embodiment of the present invention, the mass concentration of the alginate solution is 2% to 4%, preferably 3.5%. The experimental results show that the cells have a higher survival rate in the pellets with a concentration of 3.5% by mass of sodium alginate. more stable.

The mass ratio of the mixed cell suspension to the alginate solution is (1-3):(1-3), preferably 1:1.

The molecular weight of the alginate is preferably 216.12303.

In the embodiment of the present invention, the concentration of the calcium salt solution is 0.1-0.2 mol/L, preferably 0.1 mol/L.

In the embodiment of the present invention, the volume ratio of the mixed cell microspheres to the culture solution is (1-3):(1-3), preferably 1:3.

In the embodiment of the present invention, before step a), the method further includes: culturing tumor cells and fibroblasts on a plane respectively.

Step a) The tumor cells are tumor cells of the third to sixth passages, and the fibroblasts are the fibroblasts of the third to sixth passages.

The present invention also provides a three-dimensional tumor cell model obtained by the construction method of the above technical solution.

The present invention also provides the application of the three-dimensional tumor cell model of the above technical solution in the screening and/or evaluation of anti-tumor drugs.

The tumor cell three-dimensional model of the present invention is simple to use and has low requirements on the experimental environment, reduces the possibility of experimental errors, is accurate and reliable when applied to the screening and/or evaluation of anti-tumor drugs, and can reduce the cost of research and development of anti-tumor drugs .

The tumor cell three-dimensional model of the invention can be applied to the screening and detection of new anticancer drugs. Since the cells are cultured in calcium alginate microspheres, it is convenient to take out and test at any time, so as to study the effect of drugs on tumor cells at different action times. The three-dimensional tumor cell model of the present invention can also be matched with other devices such as Transwell and microfluidics to construct a new tumor cell model with more biomimetic effects.

In order to further understand the present invention, the present invention will be described in detail below with reference to specific embodiments.

Example 1

In this example, the preparation of a three-dimensional tumor cell model includes the following steps:

1) Preparation of sodium alginate solution and calcium chloride solution

Weigh the sodium alginate powder into the reagent bottle and add deionized water, then put it into a magnetic stirrer, and put it on the magnetic stirrer for 12h to 24h stirring, and the mass concentrations are 3%, 3.5% and 4% respectively. of sodium alginate solution.

Calcium chloride powder was weighed into a reagent bottle and deionized water was added to prepare a 0.1 mol/L calcium chloride solution.

Put the sodium alginate solution and the calcium chloride solution together into a high temperature and high pressure sterilizer for sterilization at 125°C for 15 minutes, and the sodium alginate solution and the calcium chloride solution are prepared.

2) Flat culture of prostate cancer cells (PC3) and fibroblasts (3T3)

The cryovial containing PC3 cells was taken out, thawed in a water bath, centrifuged, and the PC3 cells were cultured with low-glucose DMEM medium containing 1% double antibody and 10% fetal bovine serum, and the medium was changed every 2 days. When the cells are 75%-80% confluent at the bottom of the culture flask, digest with trypsin, observe the PC3 cells under the microscope until the shape of the PC3 cells is oval. Centrifuge the PC3 cell suspension, discard the supernatant, add an appropriate amount of fresh culture medium to resuspend, and prepare a PC3 cell suspension with a cell concentration of (1*10 ⁶ cells/ml).

The cryovial containing 3T3 cells was taken out, thawed in a water bath, centrifuged, and 3T3 cells were cultured in low-glucose DMEM medium containing 1% double antibody and 10% fetal bovine serum, and the medium was changed every 2 days. When the cells cover 75%-80% of the bottom of the flask, digest with trypsin, observe the 3T3 cells under a microscope until the 3T3 cells are oval in shape, and stop the digestion when they are about to detach from the wall. The 3T3 cell suspension was centrifuged, the supernatant was discarded, and an appropriate amount of fresh culture medium was added to resuspend to obtain a 3T3 cell suspension. The cell concentration of the 3T3 cell suspension was consistent with that of the PC3 cell suspension.

3) Preparation of mixed cell microspheres

Add PC3 cell suspension and 3T3 cell suspension to a centrifuge tube at a ratio of 1:1 to prepare a mixed cell suspension with a total cell concentration of 1×10 ⁶ /mL, then take three centrifuge tubes and add them to them respectively. The sodium alginate solution with the mass concentration of 3%, 3.5% and 4%, respectively, was added to the mixed cell suspension in the ratio of sodium alginate solution: mixed cell suspension to 1:1, and then the solution was pipetted evenly with a pipette gun to obtain Mixed cells-sodium alginate solution 1, mixed cells-sodium alginate solution 2, and mixed cells-sodium alginate solution 3.

Take the petri dish, add 0.1mol/L calcium chloride solution in the same amount, use a syringe to draw mixed cell-sodium alginate solution 1, mixed cell-sodium alginate solution 2, and mixed cell-sodium alginate solution 3, respectively. The needle is suspended above the calcium chloride liquid level in the petri dish, and the solution is slowly dripped at a rate of 1 drop/second, so that the mixed cell-sodium alginate solution 1, the mixed cell-sodium alginate solution 2 and the mixed cell-alginic acid The sodium solution 3 and the calcium chloride solution are respectively cross-linked to form mixed cell microspheres 1, mixed cell microspheres 2 and mixed cell microspheres 3, and then the mixed cell microspheres 1, mixed cell microspheres 2 and mixed cell microspheres 3 are cultured In fresh low-glucose DMEM medium containing 1% double antibody and 10% fetal bovine serum, three-dimensional tumor cell model 1, three-dimensional tumor cell model 2 and three-dimensional tumor cell model 3 were obtained. Among them, 5 groups of parallel experiments were set up for tumor cell 3D model 1, tumor cell 3D model 2 and tumor cell 3D model 3.

The tumor cell three-dimensional model 1, tumor cell three-dimensional model 2 and tumor cell three-dimensional model 3 were collected on the 0th day, the 4th day, the 8th day, the 10th day and the 12th day, respectively, and the cell viability was determined to determine the cell viability. Day 0 cell viability was counted as 100%. Please refer to FIG. 1, which is the survival rate of cells in a three-dimensional tumor cell model provided in the embodiment of the present invention on the 4th day, the 8th day, the 10th day and the 12th day. The results show that the mass concentration is 3.5%. The 3D model of tumor cells constructed with sodium alginate had the highest cell survival rate.

The above are only the preferred embodiments of the present invention. It should be pointed out that for those skilled in the art, without departing from the principles of the present invention, several improvements and modifications can be made. It should be regarded as the protection scope of the present invention.

Claims (10)

1. a construction method of a tumor cell three-dimensional model, is characterized in that, comprises the following steps: a) preparing tumor cells and fibroblasts into a mixed cell suspension, adding an alginate solution to the mixed cell suspension to obtain a mixed cell-alginate solution; b) dropping the mixed cell-alginate solution in the calcium salt solution to obtain mixed cell microspheres; c) placing the mixed cell microspheres in a culture medium to obtain a three-dimensional model of tumor cells. 2 . The construction method according to claim 1 , wherein the number ratio of the tumor cells and the fibroblasts in the mixed cell suspension is (2-3): (1-2). 3 . 3 . The construction method according to claim 2 , wherein the concentration of the tumor cells in the mixed cell suspension is 1*10 ⁵ cells/ml to 5*10 ⁶ cells/ml; 3 . The concentration of the fibroblasts in the mixed cell suspension is 1*10 ⁵ cells/ml to 5*10 ⁶ cells/ml. 4 . The construction method according to claim 1 , wherein the tumor cells are selected from prostate cancer cells, breast cancer cells, uterine fibroids or liver cancer cells. 5 . 5. construction method according to claim 1, is characterized in that, described alginate is sodium alginate; The calcium salt is selected from calcium chloride or calcium sulfate. 6. construction method according to claim 1 is characterized in that, the mass volume ratio concentration of described alginate solution is 2%～4%; The mass ratio of the mixed cell suspension to the alginate solution is (1-3): (1-3). 7. The construction method according to claim 1, wherein the concentration of the calcium salt solution is 0.1-0.2 mol/L. 8 . The construction method according to claim 1 , wherein the volume ratio of the mixed cell microspheres to the culture solution is (1-3): (1-3). 9 . 9. The three-dimensional model of tumor cells obtained by the construction method according to any one of claims 1 to 8. 10. The application of the three-dimensional tumor cell model of claim 9 in the screening and/or evaluation of anti-tumor drugs.