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Animal protein-free immune cell serum-free medium and using method thereof

A serum-free culture medium, immune cell technology, applied in the field of in vitro culture of immune cells, can solve the problems of poor cell proliferation efficiency and biological efficacy, pollution, serum quality differences, etc., to improve cell proliferation efficiency and biological efficacy, The effect of good tumor killing rate and controllable quality

Active Publication Date: 2015-03-25
上海安库生医生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The existing media on the market are not formulated for the conditions required for immune cell culture, resulting in poor cell proliferation efficiency and biological efficacy, and many media are supplemented with human or animal serum and components to fully supplement
However, human serum is expensive and not suitable for large-scale amplification, and due to the heterogeneity of donors, there are differences in the quality of serum between batches, especially the risk of contamination by hepatitis virus or HIV
The serum-free medium added with human serum albumin, human transferrin, human insulin and other human-derived components, although the source of the disease has been detected and sterilized, it is still unavoidable that there are differences between batches due to different donors, which is not conducive to quality control
Animal serum such as fetal bovine serum, newborn bovine serum and other serum components from bovine materials, although there is sufficient supply and low price, there is a risk of spreading infectious diseases, such as mycoplasma, virus (such as the risk of BSE pathogenic factor) contamination risk

Method used

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  • Animal protein-free immune cell serum-free medium and using method thereof
  • Animal protein-free immune cell serum-free medium and using method thereof
  • Animal protein-free immune cell serum-free medium and using method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Use triple-distilled water as a solvent, and dissolve amino acids, vitamins, salts, organic matter and protein to prepare a medium. To increase the solubility of organic matter, add co-solvent Tween 80, use 0.1mol / L hydrochloric acid and 0.1mol / L Sodium hydroxide is used to adjust the pH value so that the pH value of the medium is 6.8-7.2. The culture medium was sterilized twice through a 0.22um filter membrane, and stored in a refrigerator at 4°C in the dark.

[0031] Based on 1L of the prepared medium, its components contain the following concentrations of components:

[0032] Amino acid composition: glycine concentration 18mg / L, L-arginine concentration 190mg / L, L-asparagine 45mg / L, L-aspartic acid 20mg / L, L-cystine hydrochloride 60mg / L, L-glutamic acid 15mg / L, L-histidine 25mg / L, L-hydroxyproline 15mg / L, L-isoleucine 130mg / L, L-leucine 80mg / L, L-hydrochloric acid Lysine 100mg / L, L-methionine 25mg / L, L-phenylalanine 35mg / L, L-proline 25mg / L, L-serine 45mg / L, L-thre...

Embodiment 2

[0039] The culture medium prepared in embodiment 1 is carried out quality test

[0040] 1) Sterility test

[0041] Using the plate method for detection, take 1 sheep blood agar plate and 1 Saburo agar plate, and equilibrate to room temperature. Take the culture medium prepared in Example 1 as the sample, centrifuge at 4000rpm for 15min, draw the sample from the bottom of the sample tube, 100 μl per plate, and streak with an inoculation needle. Put the plate into a 37°C incubator, and after culturing for 48 hours, the observation result was negative, and the sample was qualified.

[0042] 2) Endotoxin detection

[0043] Use the gel method to detect, take the culture medium prepared in Example 1 as the sample after dilution, add the same negative control, positive control, and test sample positive control respectively in the Limulus reagent after sterility testing water reconstitution, each Parallel two tubes. The result is that the negative control tube is negative, the pos...

Embodiment 3

[0047] Use lymphocyte separation medium density gradient centrifugation to separate peripheral blood mononuclear cells, or to revive frozen mononuclear cells. Centrifuge at 500 g of normal saline for 5 minutes, and remove the supernatant. Count the cells, and use the culture medium prepared in Example 1 to make a concentration of 1 × 10 6 cells / mL of cell suspension. Add 2 mL of culture medium cell suspension to each well of 6-well plate, with 3 parallel wells in each group. On the first day, 1000IU / mL INF-r was added and cultured in a carbon dioxide incubator. After 24 hours, 50ng / mL CD3 monoclonal antibody and 500IU / mL IL-2 were added. Every 48 hours thereafter, supplement the culture medium and 500IU / mL IL-2, and count to 1×10 6 cells / mL concentration. Cell morphology was observed after co-cultivation for 18 days. The culture result is: the shape of the cells is polygonal, the shape is plump, and the growth is good.

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Abstract

The invention discloses the field of culture in vitro of immune cells and particularly relates to an animal protein-free immune cell serum-free medium and a culture method thereof, wherein the culture medium is a liquid culture medium. The culture medium per liter comprises the following components: 1400-1500mg of amino acids, 70-75mg of vitamins, 9800-10000mg of salts, 9500-9700mg of organic matters and 76-80mg of proteins. The culture medium prepared by the invention satisfies the special condition required by growth of immune cells well, so that the culture medium is more suitable for growth of immune cells, thereby remarkably improving the cell proliferation efficiency and the biological potency. The culture medium overcomes the defect that in the prior art, batches are different in quality caused by diversity of human or animal serum and component limited donors added into the culture medium, the quality is easy to control, the culture medium is not polluted due to the quality of serum, and the tumor-killing rate effect is good and the cost is low.

Description

technical field [0001] The invention relates to the field of culturing immune cells in vitro, in particular to a serum-free culture medium for immune cells without animal protein sources and a use method thereof. Background technique [0002] Adoptive cellular immunotherapy (ACI or AIT) of tumors refers to the transfer of immune cells (specific and relatively specific) with anti-tumor activity to tumor patients to directly kill tumors or stimulate the body's immune response to kill tumor cells , to achieve the purpose of treating tumors. The cell immunotherapy is an emerging and brand-new anti-tumor treatment method with significant curative effect in tumor rehabilitation medicine. It is known as the most active and promising treatment method in the comprehensive tumor treatment model in the 21st century. [0003] Effective cellular immunotherapy is inseparable from the large-scale cultivation, expansion and activation of immune cells in vitro. The medium suitable for the ...

Claims

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Application Information

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IPC IPC(8): C12N5/078C12N5/0783C12N5/0781
Inventor 王晓明苗嘉奕金宜强
Owner 上海安库生医生物科技有限公司
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