A kind of lymphocyte serum-free medium with stable pH value and its preparation method and application

A technology of serum-free medium and lymphocytes, which is applied in the field of cell biology, can solve the problems of poor repeatability and unstable cell culture, and achieve good results, improve lymphocyte differentiation, and reduce the concentration of ROS

Active Publication Date: 2018-12-18
广州达晖生物技术股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

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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  • A kind of lymphocyte serum-free medium with stable pH value and its preparation method and application
  • A kind of lymphocyte serum-free medium with stable pH value and its preparation method and application
  • A kind of lymphocyte serum-free medium with stable pH value and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Embodiment 1: the culture medium effect comparison of different formulations

[0028] 1. Prepare basal medium SSF-1: prepare 1L basal medium 1640 medium, add 2g disodium hydrogen phosphate, 1g potassium dihydrogen phosphate, 9g sodium glycerophosphate, 110mg pyruvate, 1.5mg ethanolamine, 10mg transferrin, 10mg insulin, 300mg lipid bovine serum albumin, 0.1gPHA.

[0029] 2. Preparation of SSF-2 medium: take 1 part of basal medium SSF-1, add 2mg vitamin E.

[0030] 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.

[0031] 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.

[0032] 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 selen...

Embodiment 2

[0062] Embodiment 2: the preparation method of a preferred embodiment of the lymphocyte serum-free medium of the present invention

[0063] 1. Materials: transferrin, insulin, vitamin E, sodium selenite, manganese dichloride, provided by Sigma; zinc sulfate, ammonium metavanadate, copper sulfate, disodium hydrogen phosphate, potassium dihydrogen phosphate, ethanolamine , provided by Shanghai Sangong Engineering; 1640 dry powder, lipid-type bovine serum albumin, alanyl glutamine, provided by GIBCO; methionine enkephalin, provided by Penta BiotechInc.

[0064] 2. Equipment: pH meter, stirrer.

[0065] 3. Preparation method:

[0066] (1) Preparation of 100× sodium selenite mother liquor: Weigh 10 mg of sodium selenite and dissolve it in 1000 ml of ultrapure water.

[0067] (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...

Embodiment 3

[0073] Example 3: Comparison of the culture effect of lymphocyte serum-free culture and traditional lymphocyte culture medium containing bovine serum

[0074] 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 4 parts of blood samples, breed blood respectively with above-mentioned two brands and the product of the present invention, cell culture, chromosome preparation. The results are shown in Table 3, and Table 3 is the comparison result of the culture effect between the new lymphocyte serum-free medium and the traditional lymphocyte medium containing bovine serum.

[0075] table 3

[0076]

[0077]

[0078] Remark

[0079] Lymphatic transfer rate (percentage of transformed lymphocytes) = percentage of transformed cells / total number of lymphocytes × 100%

[0080] Division index = number of dividing cells / (total number of lymphocytes - number of dividing cells)...

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Abstract

The invention provides a lymphocyte serum-free medium with a stable pH value. The lymphocyte serum-free medium comprises the following components: a basic medium, 10 mg / L of transferrin, 0.01 mg / L of sodium selenite, 110 mg / L of pyruvic acid, 1.5 mg / L of ethanolamine, 10 mg / L of insulin, 10-30 mg / L of a glutamine substitute, 300 mg / L of lipid-type bovine serum albumin, 0.001 mg / L of copper sulfate, 0.0005 mg / L of ammonium metavanadate, 0.00005 mg / L of manganese chloride, 0.8 mg / L of zinc sulfate, 2 mg / L of vitamin E, 100 mg / L of phytohemagglutinin (PHA) and 10-50 mM of a pH buffer system. The invention further discloses a preparation method and application of the lymphocyte serum-free medium. Through the adoption of the lymphocyte serum-free medium, lymphocyte differentiation can be improved, and a large number of division-phase lymphocytes are generated; moreover, the pH value is stable , so as to facilitate stable storage of the lymphocyte serum-free medium.

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 with a stable pH value and a preparation method and application thereof. 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 - infer...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/0781C12N5/0783
Inventor 李晓辉刘强
Owner 广州达晖生物技术股份有限公司
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