Culture medium of amniotic fluid cells

An amniotic fluid cell and culture medium technology, applied in the field of biochemistry, can solve the problems of less active cells and prolong the cell culture period, and achieve the effect of improving the success rate, reducing the culture time, increasing the clone formation rate and the clone growth rate.

Active Publication Date: 2013-10-30
广州白云山拜迪生物医药有限公司
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Problems solved by technology

Therefore, when the condition of the amniotic fluid sample planted is not good, that is, there are few active cells and the number of clones is only 1-2, it is impossible to generate enough mitotic cells in a short period of time. cell culture cycle
Therefore, there is still a certain gap between the culture effect of H-1 medium and the needs of clinical testing units, and it is necessary to improve the medium formula

Method used

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  • Culture medium of amniotic fluid cells
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  • Culture medium of amniotic fluid cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Embodiment 1: the culture medium effect comparison of different formulations

[0027] 1. Preparation of H-1 medium: 1L of DMEM / F12 cell culture medium, dissolved in 950ml of water for injection, adding 1.2g of NaHCO3, 15mM HEPES, and adding 10 kinds of growth factors, including transferrin (transferrin) 10mg / L, sodium selenite 40nMol / L, insulin (insulin) 20mg / L, triiodothyronine (triiodothyronine) 0.2nMol / L, glucagon (glucagon) 2mg / L, basic fibroblast growth factor (bFGF) 20ug / L, hydrocortisone 2nMol / L, testosterone 2nMol / L, estradiol 2nMol / L, progesterone 2nMol / L. Then add water for injection to make up to 1000ml; prepare 8 parts of H-1 medium.

[0028] 2. Prepare DF-I1 medium: take 1 part of H-1 medium, add 20mg / L vitamin E.

[0029] 3. Prepare DF-I2 medium: take 1 part of H-1 medium, add L-ascorbic acid 60mg / L.

[0030] 4. Preparation of DF-Ja medium: take 1 portion of H-1 medium, add 5mg / L vitamin E and 25mg / L L-ascorbic acid.

[0031] 5. Preparation of DF-Jb m...

Embodiment 2

[0057] Embodiment 2: preparation of amniotic fluid cell culture medium

[0058] 1) Materials: transferrin, insulin, glucagon, triiodothyronine, hydrocortisone, cortisol, androgen, estradiol, provided by SIMGA; DM / F12 dry powder medium, provided by GIBCO; basic fibroblast growth factor (bFGF) freeze-dried powder, provided by Shanghai Gaming Company; sodium pyruvate, 1,4-butanediamine dihydrochloride, sodium selenite , vitamins, amino acids, deoxynucleosides and nucleosides, vitamin E, L-ascorbic acid, phenol red, glucose and other reagents are all domestic analytically pure, provided by Guangzhou Vega Reagent Co., Ltd.;

[0059] 2) Equipment: osmotic pressure detector, pH value detector;

[0060] 3) Preparation method

[0061] ① Preparation of 100X amino acid mother solution: Mix the L-amino acids required for the culture medium (glutamine is prepared separately and stored in a frozen state, not included), and prepare 100ml of 100X amino acid mother solution. The type of ami...

Embodiment 3

[0087] Embodiment 3: Containing the amniotic fluid cell complete medium culture effect of different proportions of fetal bovine serum (FBS)

[0088] Collect 8 amniotic fluid cell samples (amniotic fluid samples between 18-21 weeks of age, about 2-4ml each, about 22ml in total), mix them evenly, and make amniotic fluid cell collection samples for testing. Divide the amniotic fluid cell sample into three parts and centrifuge, suck off most of the supernatant, leave 1ml to mix and inoculate in 4ml of the amniotic fluid cell culture medium containing different FBS ratios prepared according to Example 2, and compare the addition of different volume percentages of fetal bovine The culture effect of serum amniocyte complete medium. The results are shown in Table 3.

[0089] Table 3 Comparison of culture effects of amniocyte complete medium containing different proportions of FBS

[0090]

[0091] It can be seen from Table 3 that there is no significant difference in the culture ...

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Abstract

The invention relates to a culture medium of amniotic fluid cells, which belongs to the field of biochemistry. The culture medium comprises the following components: basic medium, transferrin, sodium selenite, insulin, triiodothyronine, glucagons, basic fibroblast growth factor, hydrocortisone, testosterone, estradiol, progesterone and added antioxidant, wherein the antioxidant is a mixture of vitamin E and L-ascorbic acid; and the culture medium can be used in amniotic fluid cell culture after 4 to 15 volume percent of fetal calf serum is added in the culture medium, and a great amount of amniotic fluid cells which are at a division stage and meet the requirements of clinical diagnosis and scientific research can be obtained just within 7 to 8 days. The culture medium remarkably improves the culture effects of amniotic fluid cells and obviously increases cloning efficiency and cloning growth rate, reduces culture time, increases the success rate of cell culture, and fully plays the synergetic effects of vitamin E and L-ascorbic acid of different antioxidants.

Description

technical field [0001] The amniotic fluid cell culture medium of the invention belongs to the field of biochemistry. Background technique [0002] Prenatal diagnosis is an important part of eugenics. It is based on genetic counseling. Through prenatal testing of fetal health, early measures can be taken for children with serious genetic diseases and congenital deformities to reduce the accumulation of harmful genes in the population. , in order to reduce the birth of children and improve the quality of the population, so it is of great significance. At present, prenatal diagnosis mainly includes villous tissue sampling in the first trimester, amniotic fluid and umbilical cord blood in the second trimester, separation of fetal cells from peripheral blood of pregnant women, and preimplantation diagnosis, but amniotic fluid cell examination in the second trimester is still the most important method. This is because compared with other methods, amniocentesis has the advantages ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/073
Inventor 丘力功蔡小杰韦剑孙希海李素芬
Owner 广州白云山拜迪生物医药有限公司
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