Lymphocyte serum-free medium, as well as preparation method and application thereof

A serum-free medium and lymphocyte technology, applied in the field of cell biology, can solve the problems of unstable cell culture, poor cell growth, poor repeatability, etc., to improve lymphocyte differentiation, meet clinical diagnosis and scientific research, and achieve good results. Effect

Active Publication Date: 2015-09-30
广州达晖生物技术股份有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The above-mentioned various media contain L-glutamine, which is easily degraded to produce amines and has certain toxicity to cells; in addition, glutamine is the main energy source of animal cells cultured in vitro, and lack of it can lead to poor growth or even death of cells
Therefore, the above-mentioned medium is unstable to cell culture after being stored for a period of time, and the reproducibility is poor

Method used

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  • Lymphocyte serum-free medium, as well as preparation method and application thereof
  • Lymphocyte serum-free medium, as well as preparation method and application thereof
  • Lymphocyte serum-free medium, as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Embodiment 1: the culture medium effect comparison of different formulations

[0025] 1. Prepare basal medium SSF-1: prepare 1L basal medium 1640 medium, add 2g NaHCO 3 , 5g HEPES, 110mg pyruvate, 1.5mg ethanolamine, 10mg transferrin, 10mg insulin, 300mg lipid bovine serum albumin, 0.1gPHA.

[0026] 2. Preparation of SSF-2 medium: take 1 part of basal medium SSF-1, add 2mg vitamin E.

[0027] 3. Preparation of SSF-3 medium: take 1 portion of basal medium SSF-1, add 2 mg of vitamin E, and add a final concentration of 0.01 mg / L sodium selenite.

[0028] 4. Preparation of SSF-4 medium: Take 1 portion of basal medium SSF-1, add 2mg of vitamin E, add final concentration of 0.01mg / L sodium selenite, and final concentration of 0.00005mg / L manganese dichloride.

[0029] 5. Preparation of SSF-5 medium: Take 1 part of basal medium SSF-1, add 2mg of vitamin E, add final concentration of 0.01mg / L sodium selenite, final concentration of 0.00005mg / L manganese dichloride, final The...

Embodiment 2

[0059] Embodiment 2: Lymphocyte serum-free medium preparation

[0060] 1. Materials: transferrin, insulin, vitamin E, sodium selenite, manganese dichloride, provided by Sigma; zinc sulfate, ammonium metavanadate, copper sulfate, HEPES, NaHCO 3 , ethanolamine, provided by Shanghai Sangong Engineering; 1640 dry powder, lipid bovine serum albumin, alanyl glutamine, provided by GIBCO; methionine enkephalin, provided by Penta Biotech Inc.

[0061] 2. Equipment: pH meter, stirrer.

[0062] 3. Preparation method:

[0063] (1) Preparation of 100× sodium selenite mother liquor: Weigh 10 mg of sodium selenite and dissolve it in 1000 ml of ultrapure water.

[0064] (2) Preparation of ammonium metavanadate mother liquor: Weigh 50 mg of ammonium metavanadate and dissolve it in 1000 ml of ultrapure water; then measure 10 ml of the solution and add 990 ml of ultrapure water to obtain the ammonium metavanadate mother liquor.

[0065] (3) Manganese dichloride mother liquor Mother liquor pre...

Embodiment 3

[0070] Example 3: Comparison of the culture effect of lymphocyte serum-free culture and traditional lymphocyte culture medium containing bovine serum

[0071] Select the two most common brands of traditional lymphocyte culture medium containing bovine serum in the Guangzhou market, self-numbered as DH and YSJ. Get the blood sample, drip into the blood 0.5ml of the same individual in the above-mentioned two brands and the product of the present invention respectively, cell culture, chromosome preparation. The results are shown in Table 3, and Table 3 is the comparison result of the culture effect between the lymphocyte serum-free medium and the traditional lymphocyte medium containing bovine serum.

[0072] table 3

[0073]

[0074] Remark

[0075] Lymphatic transfer rate (percentage of transformed lymphocytes) = percentage of transformed cells / total number of lymphocytes × 100%;

[0076] Division index = number of dividing cells / (total number of lymphocytes - number of d...

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Abstract

The invention provides a lymphocyte serum-free medium which comprises the following materials per liter: a basic culture medium, 10 mg of transferrin, 0.01 mg of sodium selenite, 110 mg of pyroracemic acid, 1.5 mg of ethanolamine, 10 mg of insulin, 10 to 30 mg of glutamine substitute, 300 mg of lipid-type bovine serum albumin, 0.001 mg of cupper sulfate, 0.0005 mg of ammonium meta-vanadate, 0.00005 mg of manganese chloride, 0.8 mg of zinc sulfate, 2 mg of Vitamin E, 100 mg of PHA, and 10 to 25 mg of an MHEPES/NaHCO3 buffering system. The invention further provides a preparation method and application to lymphocyte culture of the lymphocyte serum-free medium. The lymphocyte serum-free medium has the advantages that the repeatability of lymphocyte culture is high; poisoning and polluting of blood serum to lymphocyte are avoided; convenience is brought for differentiation of the lymphocyte, so as to obtain various mitosis phases.

Description

technical field [0001] The invention belongs to the field of cell biology, and in particular relates to a serum-free culture medium for lymphocytes and its preparation method and application. Background technique [0002] Genetic diseases refer to diseases caused by changes in genetic material or controlled by disease-causing genes. There are congenital ones, such as congenital stupidity, polydactyly (toes), congenital deaf-mute, hemophilia, etc., and there are also acquired ones caused by gene mutations and chromosomal lesions. Genetic diseases are divided into three categories, one is chromosomal disease or chromosomal syndrome, the other is single-gene disease, and the third is polygenic disease. More than 3,000 genetic diseases have been discovered so far, and it is estimated that about There are 3 to 10 suffering from various degrees of genetic diseases. Genetic diseases involve many disciplines, including obstetrics and gynecology - infertility, prenatal and postnata...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0781C12N5/0783
Inventor 李晓辉胡可存李万红莫房德徐素茹刘强
Owner 广州达晖生物技术股份有限公司
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