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Animal origin-free culture medium capable of efficiently amplifying human lymphocytes

An animal-derived, lymphocyte technology, applied in the direction of cell culture active agent, culture process, tissue culture, etc., can solve problems such as unfavorable quality control, batch differences, etc., and achieve the effect of strong tumoricidal activity

Inactive Publication Date: 2016-05-25
谷超
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Because these components are derived from human serum, although there are various pathogen detection techniques and disinfection techniques, the risk of disease transmission is still unavoidable; because they come from different donors, differences between batches are caused, which is not conducive to quality control

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1: Animal-derived free medium composition

[0023] Add 10 mg of recombinant human serum albumin, 60 mg of recombinant human transferrin, 25 mg of recombinant human insulin, 1.5 mg of cholesterol, 0.3 mg of linoleic acid, 0.4 mg of linolenic acid, 0.3 mg of ferric nitrate, and 0.15 mg of sodium selenite into each LRPMI1640 medium. mg, copper sulfate 0.15 mg, zinc sulfate 0.5 mg, vitamin E 6 mg, compound Cephaloziellin N 10 mg, gentamycin 10 IU, stir to fully dissolve, adjust the pH value between 6.8 and 7.2, filter through a 0.1 μm filter membrane, and pack in aliquots.

Embodiment 2

[0024] Embodiment 2: the contrast of embodiment 1, does not add expansion promoter CephaloziellinN

[0025] Add 10 mg of recombinant human serum albumin, 60 mg of recombinant human transferrin, 25 mg of recombinant human insulin, 1.5 mg of cholesterol, 0.3 mg of linoleic acid, 0.4 mg of linolenic acid, 0.3 mg of ferric nitrate, and 0.15 mg of sodium selenite into each LRPMI1640 medium. mg, copper sulfate 0.15mg, zinc sulfate 0.5mg, vitamin E 6mg, gentamycin 10IU, stir to fully dissolve, adjust the pH value between 6.8 and 7.2, filter through a 0.1μm filter membrane, and pack in aliquots.

Embodiment 3

[0026] Embodiment 3: Lymphocyte proliferation experiment, measuring survival rate

[0027] Use lymphocyte separation medium density gradient centrifugation to separate peripheral blood mononuclear cells, or to revive frozen mononuclear cells. Add 300g physiological saline containing 5% heat-inactivated fetal bovine serum (FBS) and centrifuge for 10 minutes, and remove the supernatant. Count the cells and make 1×10 with the corresponding medium 6 / ml cell suspension. Divided into the following groups:

[0028] Negative control group: RPMI1640+10%FBS;

[0029] Example 1 group: the culture medium prepared in Example 1;

[0030] Embodiment 2 group: the culture medium prepared in embodiment 2;

[0031] Positive control group: the only difference in Example 1 group is that human albumin and transferrin are used instead of recombinant human albumin and recombinant human transferrin, respectively.

[0032] Add 2ml of culture medium to each well and insert into 6-well plate, with...

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Abstract

The invention discloses an animal origin-free culture medium capable of efficiently amplifying human lymphocytes. The animal origin-free culture medium comprises a base culture medium, a serum replacement and an amplification accelerant as well as lipids, trace element compounds and vitamins, wherein the serum replacement comprises recombinant human serum albumin, recombinant human transferrin, recombinant human insulin, lipids, trace element compounds and vitamins; the lipids include cholesterol, linoleic acid and linolenic acid; the trace element compounds include ferric nitrate, sodium selenite, copper sulfate and zinc sulfate; and the amplification accelerant is compound Ce phaloziellin N. The culture medium disclosed by the invention can remarkably increase the proliferation multiple of lymphocytes to promote the proliferation of lymphocytes, wherein the effect is basically consistent with that of a positive control group and better than that of a culture medium without amplification accelerant; and moreover, the culture medium can remarkably improve the tumor killing activity of lymphocytes, wherein the effect is better than that of the culture medium without amplification accelerant.

Description

technical field [0001] The invention belongs to the field of cell culture, and in particular relates to an animal-free culture medium capable of efficiently expanding human lymphocytes. Background technique [0002] Effective cellular immunotherapy is inseparable from the massive culture, expansion and activation of lymphocytes in vitro. Among them, the most used reagent is the culture medium. The medium provides the basic environment for the growth of lymphocytes, including providing suitable osmotic pressure and pH value for the survival of lymphocytes, and providing all the nutritional factors required for the growth of lymphocytes, including inorganic salts, amino acids, functional Proteins, vitamins, etc., are also the discharge sites of various metabolites of lymphocytes. In order to provide these nutritional factors, traditional culture media mostly use animal or human serum and its components as additives. The more common ones include newborn bovine serum, fetal bo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0781C12N5/0783
CPCC12N5/0635C12N5/0636C12N2500/20C12N2500/22C12N2500/24C12N2500/25C12N2500/36C12N2500/38C12N2501/998C12N2501/999
Inventor 谷超
Owner 谷超
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