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T lymphocyte culture medium without any animal original or human original component and preparation method thereof

A technology of lymphocytes and culture medium, applied in the field of culture medium formulation and preparation of human T lymphocytes, can solve problems such as potential safety hazards, and achieve the effects of high growth rate, high cell density and good culture effect

Inactive Publication Date: 2015-02-25
英普乐孚生物技术(上海)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These culture media are more or less added with animal-derived or human blood-derived components. Although there are strict testing and virus inactivation treatments, there are still certain potential safety hazards.

Method used

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  • T lymphocyte culture medium without any animal original or human original component and preparation method thereof
  • T lymphocyte culture medium without any animal original or human original component and preparation method thereof
  • T lymphocyte culture medium without any animal original or human original component and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Embodiment 1: the configuration of culture medium

[0037] Culture medium configuration method

[0038] Step 1, configuring solution 1: Weigh 2 mg of cholesterol, 2 mg of oleic acid, and 2 mg of linoleic acid and dissolve them in 1 ml of absolute ethanol.

[0039] Step 2, configuring basal medium: mixing IMDM medium and DMEM / F12 medium at a ratio of 1:1.

[0040] Step 3: Add 3 g of recombinant human serum albumin, 50 mg of recombinant human transferrin, 10 mg of recombinant human insulin, 1 ml of solution one, 2 mg of ethanolamine, 0.001 mg of copper sulfate, 0.5 mg of zinc sulfate, and Manganese chloride 0.0005mg, gentamicin 10000IU, stir to fully dissolve, adjust the pH value between 6.8-7.2 with 0.1mol / L hydrochloric acid and 0.1mol / L sodium hydroxide, 0.1um filter membrane filter to sterilize, pack in aliquots , 4-8 degrees to save.

[0041] Step 4, preparation of heat-inactivated autologous plasma. Place the aseptically preserved plasma in a water bath at 56 de...

Embodiment 2

[0043] Example 2: Effect of Autologous Plasma on Lymphocyte Proliferation Activity

[0044] Heparin anticoagulation collected 50ml of peripheral blood, centrifuged at 3000 rpm for 10 minutes, and separated plasma and cells. The plasma was collected into a 15ml centrifuge tube, inactivated in a 56-degree water bath for 30 minutes, centrifuged to remove the precipitate, and the supernatant was collected for later use. The cells were diluted to the original volume with physiological saline containing 1% human albumin, added to the lymphocyte separation medium, and the peripheral blood mononuclear cells were separated by density gradient centrifugation. Collect the cells at the interface, add physiological saline containing 1% human serum albumin and centrifuge at 1000 rpm for 10 minutes, and remove the supernatant. Set as two groups, the culture medium used in the first group is the culture medium prepared in Step 2 of Example 1, the culture medium used in the second group is th...

Embodiment 3

[0046] Embodiment 3: Comparative experiment of human serum albumin and recombinant human serum albumin

[0047] Heparin anticoagulation collected 50ml of peripheral blood, centrifuged at 3000 rpm for 10 minutes, and separated plasma and cells. The plasma was collected into a 15ml centrifuge tube, inactivated in a water bath at 56°C for 30 minutes, centrifuged to remove the precipitate, and the supernatant was collected to obtain heat-inactivated autologous plasma, which was stored at 4°C for later use. The cells were diluted to the original volume with physiological saline containing 1% human albumin, added to the lymphocyte separation medium, and the peripheral blood mononuclear cells were separated by density gradient centrifugation. Collect the cells at the interface, add physiological saline containing 1% human serum albumin and centrifuge at 1000 rpm for 10 minutes, and remove the supernatant. Add 1% heat-inactivated autologous plasma to the medium to make a complete med...

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Abstract

The invention belongs to the technical field of in-vitro cell culture, and relates to a culture medium for culturing T lymphocyte. The culture medium comprises a basic culture medium and an additive for replacing human serum or cattle serum, and the additive comprises recombinant human albumin, recombinant human transferrin, recombinant human insulin, cholesterol, oleic acid, and linoleic acid. The provided culture medium has the following advantages: (1) the provided culture medium is safer, and the safety problem caused by human original and animal original components is avoided; (2) the proliferation multiple of T lymphocyte, which is cultured by the provided culture medium, is higher than that of T lymphocyte, which is cultured by a comparative culture medium.

Description

technical field [0001] The invention belongs to the technical field of cell culture, and relates to a medium formula for cultivating human T lymphocytes and a preparation method thereof. Background technique [0002] T cells are one of the main components of lymphocytes, which have a variety of biological functions, such as directly killing target cells, assisting other lymphocytes to function, responding to specific antigens or mitogens, and producing cytokines, etc. It is one of the main immune cells that the body fights against disease infection and prevents tumor formation. Enrichment, activation, and expansion of such immune cells in vitro can be used for reinfusion to treat various diseases, including malignant tumors, infections, autoimmune diseases, etc. Media for survival and expansion. [0003] The nutrients needed for the survival and expansion of T cells in vitro include: amino acids, vitamins, sugars, lipids, inorganic salts, growth factors, etc. In order to ...

Claims

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Application Information

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IPC IPC(8): C12N5/0783
Inventor 谢志明武宁
Owner 英普乐孚生物技术(上海)有限公司
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