WO2019128604A1

WO2019128604A1 - Aminotic fluid cell culture medium, aminotic fluid cell culture method, and application of culture medium

Info

Publication number: WO2019128604A1
Application number: PCT/CN2018/117900
Authority: WO
Inventors: 魏凤香; 万新红; 米占英; 胡亮; 罗小金; 温丽娟; 王艳春
Original assignee: 深圳市龙岗区妇幼保健院
Priority date: 2017-12-26
Filing date: 2018-11-28
Publication date: 2019-07-04
Also published as: CN108060118A

Abstract

Provided is an aminotic cell culture medium, comprising a basic culture medium, and further comprising the following components: transferrin, sodium selenite, insulin, triiodothyronine, glucagon, progesterone, hydrocortisone, testosterone, estradiol, a basic fibroblast growth factor, vitamin E, vitamin C, antibiotic, and fetal calf serum. Also provided are an application of the aminotic fluid cell culture medium in antenatal diagnosis of aminotic fluid cell growth and an amniotic fluid cell culture method.

Description

Amniocyte cell culture medium, amniotic fluid cell culture method and application of culture medium

Technical field

The invention relates to cell culture technology, in particular to an amniotic fluid cell culture medium, an amniotic fluid cell culture method and a culture medium application.

Background technique

China is a populous country in the world and a country with high birth defects and disability. Birth defects are not only a serious public health problem, but also a social problem that affects economic development and people's normal life. Birth defects and disability are not only increasingly important issues affecting the quality of the population, but also impose a heavy financial burden on families and society. Prenatal/prenatal diagnosis of karyotype analysis is the best way to effectively reduce birth defects. At present, major hospitals in China have developed karyotype analysis techniques for amniotic fluid and villus. The quality of amniotic fluid medium is the key to determining the results of the culture. At present, the amniotic fluid cell culture medium for clinical application is almost all imported from abroad, and China currently lacks the medium production technology and ability comparable in quality. This not only makes people feel regret, but also keeps the relevant medical expenses at a high level, and the cost is high, which limits the application of prenatal diagnosis in the prevention and treatment of birth defects to a certain extent. Therefore, the development of villus and amniotic fluid medium with independent intellectual property rights has positive significance for reducing the cost of prenatal diagnosis, improving the prenatal diagnosis rate and the level of prenatal and postnatal care.

Due to the small number of living cells in the amniotic fluid, the culture period is long, and the sterility requirement is high, and the slight failure to pay attention causes the culture to fail. Even if the cultivation is successful, due to the small number of divisions, the shape is not good, and the analytical requirements are not met, and further research is impossible. Therefore, in the successful amniocentesis, in addition to the rigorous experimental techniques and experience, the quality of the medium is also critical. In order to shorten the amniotic fluid cell culture cycle and improve the success rate, some research reports on improved media have been reported. At present, many domestic prenatal diagnostic institutions use imported German AMNIOPAN amniotic fluid medium and American Gibico amniotic fluid medium. There are also several companies in Hunan Xiangya Medical Gene Technology Co., Ltd., Chongqing and Guangzhou to carry out this technology research, but the technology. Not mature enough, the product is not widely used in the clinic.

technical problem

The technical problem to be solved by the present invention is to provide an amniotic fluid cell culture medium, a culture method of amniotic fluid cells and a culture medium.

Technical solution

In order to solve the above technical problems, the technical solution adopted by the present invention is:

The invention provides an amniotic fluid cell culture medium, comprising a basal medium, and further comprising the following components: transferrin, sodium selenite, insulin, triiodothyronine, glucagon, progesterone, hydrogenated Pine, testosterone, estradiol, basic fibroblast growth factor, vitamin E, vitamin C, antibiotics and fetal bovine serum.

The invention also provides the use of the above amniotic fluid cell culture medium for prenatal diagnosis of amniocytes growth.

In addition, the present invention also provides a method for culturing amniotic fluid cells, wherein 0.5-lmL of a sample of 18-22 weeks old primary amniocytes are inoculated on 5±1 mL of the above amniotic fluid cell culture medium for cell culture and harvesting. The amniocytes are obtained.

Beneficial effect

The beneficial effects of the invention are:

By changing the active ingredient in the amniotic fluid cell culture medium and configuring the amniotic fluid medium of the above components, the success rate of amniotic fluid cell culture can be improved, the culture speed can be accelerated, and the proportion of cell division phase for chromosome system can be improved, so that the amniotic fluid of the present invention can be improved. The cell culture medium achieves an excellent level of technology and quality, and can reach or exceed the imported medium in terms of technology and quality (for example, imported German AMNIOPAN amniotic fluid cell culture medium and American Gibico amniotic fluid cell culture medium).

DRAWINGS

Figure 1 is a picture showing the crystal violet staining of amniocytes in the culture example obtained in Test Example 1 under a light microscope of 4х10;

2 is a picture of cell crystal violet staining of amniocytes in cultured in Comparative Example 1 under a light microscope of 4×10;

Figure 3 is a picture showing the crystal violet staining of amniocytes in cultured in Comparative Example 2 under a light microscope of 4х10;

Figure 4 is a picture of cell crystal violet staining of amniocytes in cultured in Comparative Example 3 under a light microscope of 4х10;

Figure 5 is the expression levels of P-ERK1/2 of Test Example 1 and Comparative Examples 1-3;

Figure 6 is the expression levels of T-ERK1/2 of Test Example 1 and Comparative Examples 1-3;

Figure 7 is the expression levels of AKT of Test Example 1 and Comparative Examples 1-3;

Figure 8 is a graph showing the expression levels of P-AKT of Test Example 1 and Comparative Examples 1-3;

Fig. 9 shows the expression levels of actin of Test Example 1 and Comparative Examples 1-3.

Embodiments of the invention

In order to explain the technical contents, the objects and effects achieved by the present invention in detail, the embodiments will be described below in conjunction with the accompanying drawings.

The most critical idea of the present invention is to design the basic components of the amniotic fluid cell culture medium to improve the success rate of amniotic fluid cell culture.

Referring to Figures 1-9, the present invention provides an amniotic fluid cell culture medium comprising a basal medium, and further comprising the following components: transferrin, sodium selenite, insulin, triiodothyronine, glucagon Progesterone, hydrocortisone, testosterone, estradiol, basic fibroblast growth factor, vitamin E, vitamin C, antibiotics and fetal bovine serum.

As can be seen from the above description, the beneficial effects of the present invention are:

By changing the active ingredient in the amniotic fluid cell culture medium and configuring the amniotic fluid medium of the above components, the success rate of amniotic fluid cell culture can be improved, the culture speed can be accelerated, and the proportion of cell division phase for chromosome system can be improved, so that the amniotic fluid of the present invention can be improved. The cell culture medium meets or exceeds the imported medium in terms of technology and quality (for example, imported German AMNIOPAN amniotic fluid cell culture medium and American Gibico amniotic fluid cell culture medium).

Referring to Figures 1-9, a first embodiment of the present invention is:

1. Preparation of medium components

The basic components of amniotic fluid cell culture medium are: basic culture medium, bovine serum, cell growth factor, double antibody, etc., but the amount of cells in amniotic fluid is small, and most of them are aging cells. Therefore, it is difficult to culture amniotic fluid cells relative to peripheral blood. It is much larger, and the preparation of the medium is the key to the success or failure of amniotic fluid cell culture. The preparation process must ensure the quality of the raw materials used. The specific formulation of each component should also be verified by experiments, and the activity of each biological component during preparation and use should be ensured. The most important point is to ensure the sterility during preparation and use. Once contaminated, the entire experimental process will fail completely. After the amniotic fluid cell culture medium is prepared, its culture effectiveness and sterility must be checked by quality.

The invention combines the active components in the amniotic fluid cell culture medium, configures the amniotic fluid medium with different proportions of components, accurately adds the appropriate concentration of the cell growth factor, controls the appropriate concentration and proportion of the basic fibroblasts and the epidermal growth factor, and promotes the cells together. Diffusion and proliferation, observe the culture effect of gestational age amniocytes. By improving the composition of amniotic fluid cell culture medium, the project can improve the success rate of amniotic fluid cell culture, accelerate the culture rate, increase the proportion of cell division phase used in chromosome system, and make the domestic amniotic fluid medium meet or exceed the imported culture in terms of technology and quality. base.

Based on the above, the amniotic fluid cell culture medium of the present invention was obtained in accordance with the components and concentrations shown in Table 1 below.

Table 1

2. Optimization of culture conditions for amniotic fluid cells

The purpose of amniotic fluid cell culture is to make the amniotic fluid cells grow sufficiently and obtain a large number of chromosome samples in the middle of division. The quality of amniotic fluid cell culture is directly related to the quantity and quality of metaphase chromosomes. Because the number of amniotic cells is small, and the components include epithelial cells (3-4 days in culture, amniotic cells (about 7 days in culture) and fibroblasts (longest in growth), in order to obtain the dominant growth of amniocytes, it must be controlled. Conditions of cultivation.

Based on the above, the culture condition of the amniotic fluid cells of the present invention is specifically: taking 0.5 mL of a primary amniotic fluid cell sample of 18 weeks old, inoculated in 5 mL of optimized different medium for comparative culture, and culturing the medium in which the ratio of fetal calf serum is different. After 6 days, the fluid was changed. After 10 days, the growth of amniotic cells was observed and the cells were harvested. The colchicine was added 1.5 hours before the termination of the culture, so that as many amniocytes were stopped as in the middle of cell division.

The culture of amniotic fluid cells must be strictly controlled in the middle of the process. Before the culture is changed, the growth of the bottom of the culture flask is observed. Compared with the growth of the polyclonal cells, the cells are thinner. It is estimated that the growth will be more vigorous after the culture, and the liquid can be changed. After the completion of the culture, there are a large number of amniotic cells growing on the bottom of the bottle. There are many large and bright round cells in the division stage. Each round cell is full, the edges are clear, the nucleus is clear, the filaments are contained, and the cells are swollen like Amoeba. The protruding pseudopods, like the ones to be broken, some round cells are translucent into pairs, then the amniocytes can be harvested.

Embodiment 2 of the present invention is:

In the amniocyte cell culture medium of the present embodiment, only the "volume fraction of fetal bovine serum is 4-15%" is different from that of the first embodiment, and the others are the same as in the first embodiment;

The culture conditions of the amniocytes in this example are as follows: 1 mL of a 22-week-old primary amniotic fluid cell sample is inoculated into 5 mL of optimized different medium for comparative culture, cultured in a medium of different fetal bovine serum ratio, and changed after 7 days. After 11 days, the amniotic fluid cell growth was observed and the cells were harvested. The colchicine was added 1.5 hours before the termination of the culture, so that as many amniocytes were stopped as in the middle of cell division.

Embodiment 3 of the present invention is:

The culture conditions of the amniocytes in this example were as follows: 0.7 mL of a 20-week-old primary amniotic fluid cell sample was inoculated into 5 mL of optimized different medium for comparative culture, and cultured in a medium of different fetal bovine serum ratio, 6.5d. After changing the solution, the amniotic fluid cell growth was observed after 10.5 days and the cells were harvested. The colchicine was added 1.5 hours before the termination of the culture, so that as many amniocytes were stopped as in the middle of cell division.

Test test

The amniotic fluid cell culture medium obtained in the first to third embodiments was used as a test example, wherein the data of Example 1 was taken as a representative example, and the first example was Test Example 1; and the Gibico amniotic fluid cell culture medium of the United States was taken as Comparative Example 1, The AMNIOPAN amniotic fluid cell culture medium was used as Comparative Example 2, and the amniotic fluid cell culture medium produced by Guangzhou Dahui Biotechnology Co., Ltd. was used as Comparative Example 3; then the following steps were performed:

1, using the crystal violet method to detect the number of amniocytes

The amniotic fluid was concentrated by centrifugation, and inoculated in a 6-well plate in a 6-well plate at a rate of 5*105 cells per well. The cells were cultured in four groups of the above test examples 1 and 1-3, and cultured for 10-11 days until the cells were attached. After that, the liquid in each well was aspirated, and the residual liquid was lightly washed with PBS. Each well was fixed with 4% paraformaldehyde solution for 30 min, then washed gently with PBS for 3 times, added with 0.5% crystal violet solution for 10 min, and washed gently with PBS for 3 times. Take a photo.

2. Western blotting detection of PI3K/Akt and MAPK/ERK signal transduction pathways

The amniotic fluid was concentrated by centrifugation, and inoculated in a 6-well plate in a 6-well plate at a rate of 5*105 cells per well. The cells were cultured in four groups of the above test examples 1 and 1-3, and cultured for 10-11 days until the cells were attached. After that, the six-well plate was taken out on ice, the culture solution was aspirated, treated with ice PBS twice, and the cell lysate was added, and after cleavage on ice for 30 min, the protein was collected in an EP tube, and Coomassie Brilliant Blue method (wavelength 595 nm) was used. The protein concentration was measured, and then subjected to polyacrylamide gel electrophoresis, gel electrophoresis, immunoblotting, protein transfer to cellulose acetate membrane (NC), immersion in 5% bovine serum albumin solution for 1 h, and then primary antibody, Incubate overnight at 4 °C. After the primary antibody was incubated, TBST was washed 4 times for 10 min each time; the second antibody was incubated for 1 h at room temperature, and after washing, the membrane was washed 4 times with PBST for 10 min each time. Then, it was exposed to color with ECL luminescent solution, and β-actin was used as an internal reference protein.

Observation of cell culture effect under microscope:

100 cases of primary amniocytes samples of 18-22 weeks old were selected 0.5-l mL, inoculated in 5mL ABCD four groups of medium at the same time, amniotic cells adhered well, and were divided into three groups for evaluation.

The evaluation criteria are as follows:

Excellent: Strong growth (10-15 spherical splitting phase cells are seen in the same field of view), and the production effect is good.

Good: amniotic fluid cells can be attached to the wall, and the growth is medium (5-10 spherically splitting phase cells are seen in the same field of view), and the production effect is good.

Poor: amniotic fluid cells grow slowly (less than 3, even no cell growth in the same field of view, no cell growth), not adherent.

The experimental results are as follows:

1, crystal violet method to detect the number of amniocytes

Test Example 1. The culture medium of Comparative Example 1-3 cultured amniocytes to obtain the results of cell crystal violet staining as shown in Figures 1-4. 1 is a picture showing the crystal violet staining of the amniocytes in the culture example obtained in the test example 1 under the light microscope of 4×10, and FIG. 2 is a picture showing the crystal violet staining of the amniocytes in the culture of Comparative Example 1 under a light microscope of 4×10, FIG. 3 is a pair. The amylocytes obtained by the ratio 2 culture were stained with a cell crystal violet under a light microscope of 4×10, and FIG. 4 is a picture of the crystal violet staining of the amniotic cells obtained by the culture of Comparative Example 3 under a light microscope of 4×10. Under the light microscope of 4х10, it was found that after the crystal violet staining, the number of cells in the test example 1 and the comparative example 1-2 was significantly increased, which was superior to the medium of the domestic comparative example 3.

2. Western blotting detection of PI3K/Akt and MAPK/ERK signal transduction pathway results

Different amniocytes cell culture medium affects the expression of PI3K/Akt and MAPK/ERK signal transduction pathways as shown in Figure 5-9. (In Figure 5-9, the test example 1, the comparative example 1, the comparative example 2, and the comparative example 3 were sequentially selected from left to right. Among them, Fig. 5 shows the expression level of P-ERK1/2, Fig. 6 shows the expression level of T-ERK1/2, Fig. 7 shows the expression level of AKT, Fig. 8 shows the expression level of P-AKT, and Fig. 9 shows the expression of actin. the amount. The results showed changes in the expression levels of Phospho-AKT and Phospho-ERK1/2 in the control group. In contrast, the expression of Test Example 1 was comparable to the expression of Comparative Example 1-2, demonstrating that the medium prepared by the present invention reached an international level.

3. Observing the results of cell culture evaluation under the microscope:

The culture medium of Test Example 1 and Comparative Example 1-3 was compared for amniocytes culture.

In the amniotic fluid cell culture medium of Test Example 1, the cell culture was excellent in 89 cases (89%), good in 8 cases (8%), and poor in 3 cases (3%);

In Comparative Example 1, the cell culture was excellent in 87 cases (87%), good in ll cases (11 cases), and poor in 2 cases (2%);

In Comparative Example 2, cell culture was excellent in 91 cases (91%), good in 8 cases (8 cases), and poor in 1 case (1%);

In Comparative Example 3, cell culture was excellent in 79 cases (79%), good in 11 cases (11%), and poor in 10 cases (10%).

Please refer to Table 2 below, which is a table for evaluating the culture effect of the amniotic fluid cell culture medium of Test Example 1 and Comparative Example 1-3.

Table 2

组别/评价Group/evaluation	试验例1Test example 1	对比例1Comparative example 1	对比例2Comparative example 2	对比例3Comparative example 3
优良差Excellent difference	89 8 389 8 3	87 11 287 11 2	91 8 191 8 1	79 11 1079 11 10

After statistical analysis, there was no difference in the medium of Comparative Example 1 and Comparative Example 1-2, which were significantly better than the medium of Comparative Example 3.

In summary, the amniotic fluid cell culture medium provided by the present invention promotes the proliferation of amniocytes by regulating PI3K/Akt and MAPK/ERK signal transduction pathways, and the quality of amniotic fluid medium can reach international level, and the quality of amniotic fluid medium is improved. Excellent advantages.

The above is only the embodiment of the present invention, and is not intended to limit the scope of the invention, and equivalent transformations made by the description of the present invention and the contents of the drawings, or directly or indirectly applied in the related technical field, are included in the same. Within the scope of patent protection of the present invention.

Claims

An amniotic fluid cell culture medium, comprising a basal medium, characterized in that it further comprises the following components: transferrin, sodium selenite, insulin, triiodothyronine, glucagon, progesterone, hydrogenated Pine, testosterone, estradiol, basic fibroblast growth factor, vitamin E, vitamin C, antibiotics and fetal bovine serum.
The amniocytes cell culture medium according to claim 1, wherein the basal medium is obtained by mixing 1:1 with DMEM and F12.
The amniocytes cell culture medium according to claim 1, wherein the volume of the fetal bovine serum accounts for 4-15% of the total volume of the amniotic fluid cell culture medium.
The amniocytes cell culture medium according to any one of claims 1 to 3, wherein the concentration of each component of the amniotic fluid cell culture medium is as follows:

The error value of each component concentration in the amniotic fluid cell culture medium is within ±0.5.
Use of an amniotic fluid cell culture medium according to any one of claims 1 to 4 for prenatal diagnosis of amniocytes growth.
A method for culturing amniotic fluid cells, characterized in that 0.5 to 1 mL of a primary amniotic fluid cell sample of 18-22 weeks old is inoculated on 5 ± 1 mL of the amniotic fluid cell culture medium according to any one of claims 1 to 4. , performing cell culture, harvesting to obtain the amniotic fluid cells.
The method for culturing amniocytes according to claim 6, wherein the cells are changed after 6-7 days of cell culture, and the cells are further cultured for 5-6 days after the liquid exchange, and the amniocytes are harvested.
The method for culturing amniocytes according to claim 6, wherein colchicine is added at 1.4 to 1.6 hours before the amniocytes are harvested.