CN110747161A - Culture medium for amniotic fluid cells - Google Patents
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Abstract
The invention relates to an amniotic fluid cell culture medium, and belongs to the technical field of cell culture media. The amniotic fluid cell culture medium comprises the following components: a basal culture medium, an amniotic fluid cell culture additive and a human embryo trophoblast cell culture solution; the dosage of the human embryo trophoblast cell culture solution is 30-70 mL/L. The culture medium of the amniotic fluid cells is added with a human embryo trophoblast cell culture solution which is a natural composition obtained by cell culture, is an efficient multi-factor mixture, and has an effect of stimulating the growth of the amniotic fluid cells which is obviously superior to that of a common culture medium. The amniotic fluid cell culture in the invention can effectively stimulate the growth of amniotic fluid cells and obtain more cell clusters.
Description
Technical Field
The invention relates to an amniotic fluid cell culture medium, and belongs to the technical field of cell culture media.
Background
Amniocentesis is the most commonly used invasive prenatal diagnostic technique. The doctor can obtain free cells of the skin, gastrointestinal tract, urinary tract and the like of the fetus by extracting amniotic fluid, and further analyze whether the chromosome of the fetus is abnormal or not by using the free cells. The operation is generally carried out in 16-22 weeks, and the living cells in the amniotic fluid are higher in proportion. The extraction of amniotic fluid is mainly to analyze the chromosome composition of the fetus, and the most important and common is Down syndrome. Some monogenic diseases, such as type B marine anemia, hemophilia, etc., can also be diagnosed by examining the genes (DNA composition) in amniotic cells.
The free cells of skin, gastrointestinal tract, urinary tract and the like of a fetus can be obtained by a doctor in the amniocentesis through extracting amniotic fluid, the number of fetal cells in the amniotic fluid is small, the diagnosis requirement cannot be met, and therefore the cells in the amniotic fluid need to be cultured, and after the fetal cells are subjected to amplification culture, various examinations are performed. The culture medium for culturing the fetal cells in the amniotic fluid is the amniotic fluid culture medium, and the amniotic fluid culture medium generally consists of a basic culture medium and various additives, wherein the additives are used for stimulating the rapid proliferation of the amniotic fluid cells. At present, the common amniotic fluid culture medium on the market has poor culture effect on amniotic fluid cells, the cells are slowly proliferated, and the cell clusters obtained after culture are few.
Disclosure of Invention
The invention aims to provide an amniotic fluid cell culture medium, which can promote the proliferation of amniotic fluid cells and obtain a large number of cell clusters after culture.
In order to achieve the purpose, the invention adopts the technical scheme that:
an amniotic fluid cell culture medium comprises a basic culture medium, an amniotic fluid cell culture additive and a human embryo trophoblast cell culture solution; the dosage of the human embryo trophoblast cell culture solution is 30-70 mL/L.
30-70mL/L of human embryo trophoblast cell culture solution is added into the amniotic fluid cell culture medium, and the obtained amniotic fluid culture medium can effectively stimulate the growth of amniotic fluid cells to obtain more cell masses.
Preferably, the amniotic fluid cell culture additive is one or more of insulin, glucagon, hydrocortisone, sodium selenite, colony stimulating factor, estradiol, ferric nitrate, ferric citrate, fibroblast factor, nerve growth factor, epidermal growth factor, albumin and vitamins. More preferably, the dosage of each component of the amniotic fluid cell culture additive is as follows: 1-5mg/L of insulin; 1-5mg/L glucagon; hydrocortisone 1-5 nmol/L; 10-30nmol/L of sodium selenite; 1-10 mug/L of colony stimulating factor; 1-5nmol/L of estradiol; 1-5mg/L of ferric nitrate; 1-5mg/L ferric citrate; fibroblast factor 0.1-20 μ g/L; nerve growth factor 0.1-20 mug/L; 0.1-20 mug/L of epidermal growth factor; 1-5mg/L of albumin; vitamin 1-5 mg/L.
The basic medium in the invention can be a cell culture medium commonly used in the field, and preferably, the basic medium is F12/DMEM medium.
Specifically, the human embryo trophoblast cell culture solution is prepared by a method comprising the following steps:
1) culturing human embryo trophoblast cells with complete culture medium;
2) removing the complete culture medium, and culturing human embryo trophoblast cells by using a serum-free culture medium;
3) filtering the upper layer culture solution to obtain the final product.
The human embryo trophoblast cell culture solution obtained in the invention is a natural composition obtained by cell culture, is an efficient multifactorial mixture, has good effect on the culture of amniotic fluid cells, and has an effect of stimulating the growth of the amniotic fluid cells which is obviously superior to that of a common culture medium. Wherein the human embryo trophoblast cells are purchased from professional primary cell isolation culture companies (Shanghai Bohu, Jiangsu Qi's, Nanjing Sihong Rui, etc.).
Preferably, the human embryonic trophoblast cells are cultured in step 1) for 36-60 h. Preferably, the human embryonic trophoblast cells are cultured in step 1) for 24-48 h.
Preferably, the complete medium in step 1) is: the complete culture medium of human embryo trophoblast cells is purchased together with human embryo trophoblast cells.
Preferably, the serum-free medium in the step 2) is F12/DMEM.
Preferably, the filtration in step 3) is performed using a 0.22 μm filter membrane. Filtration can remove cells and other impurities that can affect the culture of amniotic fluid.
Preferably, the culture solution of the human embryo trophoblast cells in the amniotic fluid cell culture medium is 50 mL/L. Too much or too little culture solution can cause poor culture effect and influence the culture effect.
The albumin in the present invention may be albumin commonly used in the art, and is preferably human serum albumin.
Preferably, the vitamin is vitamin B1.
Detailed Description
The following examples are intended to illustrate the invention in further detail, but are not to be construed as limiting the invention in any way.
The sources of materials used in the following examples are as follows: colony stimulating factors were purchased from solibao; fibroblast factors were purchased from solibao; nerve growth factor was purchased from sigma; epidermal growth factor was purchased from sigma; the vitamin used was vitamin B1, available from Shanghai-derived leaf Biotech Co.
The human embryo trophoblast cell kit is purchased from Qie's biotechnology, Jiangsu, and contains human embryo trophoblast cells and complete culture medium for the growth of the human embryo trophoblast cells.
Example 1
The amniotic fluid cell culture medium in the embodiment comprises the following components: 2mg/L of insulin; glucagon 2 mg/L; hydrocortisone at 1 nmol/L; 10nmol/L of sodium selenite; colony stimulating factor 2 mug/L; estradiol is 1 nmol/L; 2mg/L of ferric nitrate; 5mg/L ferric citrate; fibroblast factor 0.5 mug/L; nerve growth factor 1 mug/L; 2 mug/L of epidermal growth factor; human serum albumin 5 mg/L; 2mg/L of vitamin; 50mL/L of human embryo trophoblast cell culture solution, and the balance of basal culture medium F12/DMEM.
The culture solution of the human embryo trophoblast cells is prepared by the method comprising the following steps: human embryonic trophoblasts were purchased from professional primary cell isolation and culture;
1) human embryo trophoblast cell count reaches 5X 105At individual cells/mL, transfer to T25 flask (expanded ratio at the same density)) Medium culture, at 37 deg.C, in carbon dioxide incubator, using complete culture liquid to culture for 48 h.
2) After culturing for 48h, replacing the complete culture medium in the supernatant by using a serum-free culture medium, and washing for multiple times during replacement to fully remove the complete culture medium; serum-free culture (F12/DMEM) medium was used for further 36 h.
3) After culturing, centrifuging to remove human embryo trophoblast cells, and filtering the supernatant culture solution with 0.22 μm filter membrane.
The passage of the human embryo trophoblast cells can reach about 10 generations, and after 10 generations, new human embryo trophoblast cells are needed to prepare a filtrate.
Example 2
The amniotic fluid cell culture medium in the embodiment comprises the following components:
1mg/L of insulin; glucagon 5 mg/L; hydrocortisone 5 nmol/L; 30nmol/L of sodium selenite; colony stimulating factor 10. mu.g/L; estradiol 5 nmol/L; 1mg/L of ferric nitrate; 1mg/L ferric citrate; fibroblast factor 0.1 mug/L; nerve growth factor 20 mug/L; 20 mu g/L of epidermal growth factor; 1mg/L of human serum albumin; 5mg/L of vitamin; 70mL/L of human embryo trophoblast cell culture solution, and the balance of basal culture medium F12/DMEM.
The human embryonic trophoblast cell culture medium used was the same as in example 1.
Example 3
The amniotic fluid cell culture medium in the embodiment comprises the following components:
5mg/L of insulin; glucagon 1 mg/L; hydrocortisone 3 nmol/L; sodium selenite 20 nmol/L; colony stimulating factor 1 μ g/L; estradiol is 3 nmol/L; 5mg/L of ferric nitrate; 3mg/L ferric citrate; fibroblast factor 20 mug/L; nerve growth factor 0.1 μ g/L; 0.1 mu g/L of epidermal cell growth factor; human serum albumin 3 mg/L; 1mg/L of vitamin; 30mL/L of human embryo trophoblast cell culture solution, and the balance of basal culture medium F12/DMEM.
The human embryonic trophoblast cell culture medium used was the same as in example 1.
Comparative example 1
The amniotic fluid cell culture medium used in this comparative example was Gibco medium (model No. 11269-016. sup. f. amniotic fluid medium).
Comparative example 2
The amniotic fluid cell culture medium in the comparative example comprises the following components: 2mg/L of insulin; glucagon 2 mg/L; hydrocortisone at 1 nmol/L; 10nmol/L of sodium selenite; colony stimulating factor 2 mug/L; estradiol is 1 nmol/L; 2mg/L of ferric nitrate; 5mg/L ferric citrate; fibroblast factor 0.5 mug/L; nerve growth factor 1 mug/L; 2 mug/L of epidermal growth factor; human serum albumin 5 mg/L; 2mg/L of vitamin; the balance is basal medium F12/DMEM.
Test examples
The culture media of example 1 and comparative examples 1-2 were used simultaneously for the same batch of amniotic fluid cells, specifically:
1. collecting amniotic fluid in clean environment, centrifuging, discarding supernatant, respectively supplementing culture medium with the same volume as that of the above examples 1 and 2,
2. placing in CO at 37 DEG C2Culturing in an incubator.
And counting the number of cell clone groups according to the cell morphology and the growth condition. The diameter of the cell colony was measured using a ruler in the microscope eye piece.
The statistical results are shown in tables 1 and 2, where table 1 shows the total number of cell colonies obtained in different media, and table 2 shows the diameters of the cell colonies obtained in different media.
TABLE 1 Total cell colony counts obtained on two media
| Example 1 | Comparative example 1 | Comparative example 2 | |
| First bottle | 17 | 16 | 10 |
| Second bottle | 18 | 15 | 9 |
| Third bottle | 19 | 17 | 11 |
| The fourth bottle | 22 | 20 | 13 |
| Fifth bottle | 25 | 22 | 15 |
TABLE 2 diameters of cell clones obtained in two media
It can be seen from Table 1 that the number of cell colonies obtained by the culture medium of amniotic fluid cells of the present invention is about 10% higher than that of comparative example 1; compared with comparative example 2, the number of cell colonies was more than 70%. As can be seen from Table 2, the mean diameter of the cell colony obtained from the culture medium of amniotic fluid cells of the present invention is substantially the same as that of comparative example 1, and is larger than that of comparative example 2.
Claims (9)
1. An amniotic fluid cell culture medium comprises a basic culture medium and an amniotic fluid cell culture additive, and is characterized by also comprising a human embryo trophoblast cell culture solution; the dosage of the human embryo trophoblast cell culture solution is 30-70 mL/L.
2. The amniotic fluid cell culture medium according to claim 1, wherein the amniotic fluid cell culture supplement is one or more of insulin, glucagon, hydrocortisone, sodium selenite, colony stimulating factor, estradiol, ferric nitrate, ferric citrate, fibroblast factor, nerve growth factor, epidermal growth factor, albumin, vitamins.
3. The amniotic fluid cell culture medium according to claim 1, wherein the dosage of each component of the amniotic fluid cell culture additive is as follows: 1-5mg/L of insulin; 1-5mg/L glucagon; hydrocortisone 1-5 nmol/L; 10-30nmol/L of sodium selenite; 1-10 mug/L of colony stimulating factor; 1-5nmol/L of estradiol; 1-5mg/L of ferric nitrate; 1-5mg/L ferric citrate; fibroblast factor 0.1-20 μ g/L; nerve growth factor 0.1-20 mug/L; 0.1-20 mug/L of epidermal growth factor; 1-5mg/L of albumin; vitamin 1-5 mg/L.
4. The amniotic fluid cell culture medium according to claim 1, 2 or 3, wherein the basal medium is F12/DMEM medium.
5. The amniotic fluid cell culture medium according to claim 1, 2 or 3, wherein the human embryo trophoblast cell culture fluid is prepared by a method comprising the steps of:
1) culturing human embryo trophoblast cells with complete culture medium;
2) removing the complete culture medium, and culturing human embryo trophoblast cells by using a serum-free culture medium;
3) filtering the upper layer culture solution to obtain the final product.
6. The culture medium for amniotic fluid cells according to claim 5, wherein the human embryonic trophoblast cells are cultured in step 1) for 36-60 h.
7. The culture medium for amniotic fluid cells according to claim 5, wherein the human embryonic trophoblast cells are cultured in step 1) for 24-48 h.
8. The amniotic fluid cell culture medium according to claim 1, 2 or 3, wherein the culture solution of human embryo trophoblast cells in the amniotic fluid cell culture medium is 50 mL/L.
9. The amniotic fluid cell culture medium according to claim 2 or 3, wherein the albumin is human blood albumin.
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| CN1626651A (en) * | 2003-12-10 | 2005-06-15 | 财团法人工业技术研究院 | System for culturing human embryonic stem cell and culturing method |
| WO2010047824A1 (en) * | 2008-10-24 | 2010-04-29 | Childrens Hospital Los Angeles | Amniotic fluid cells and uses thereof |
| CN101705207A (en) * | 2009-11-10 | 2010-05-12 | 广州拜迪生物医药有限公司 | Culture medium of amniotic fluid cells |
| WO2017048097A1 (en) * | 2015-09-15 | 2017-03-23 | 주식회사 강스템바이오텍 | Composition for preventing or treating inflammatory diseases, containing, as active ingredient, stem cells overexpressing sod3 |
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| CN106834215A (en) * | 2016-12-24 | 2017-06-13 | 严志海 | A kind of culture medium of amniotic fluid cells |
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