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Serum-free medium for vitro amplification culture of immune cells and application thereof

A serum-free culture medium and culture medium technology, applied in the fields of cell biology, molecular biology and medicine, can solve the problem of spreading pathogens, achieve the effect of clear chemical composition, ensure consistency, and overcome potential risks

Inactive Publication Date: 2016-06-29
GUANGDONG XTEM BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the serum-free medium supplemented with human-derived components such as human serum albumin and human transferrin has been screened for relevant pathogens in the raw plasma, and measures to remove and inactivate bacteria have been added to the production process, but in the There is still a theoretical potential risk of spreading certain known and unknown pathogens

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Embodiment 1, preparation of immune cell serum-free medium

[0058] Pack one bag of one liter of IMDM dry powder (Gibco12200036), recombinant human serum albumin (Invitria, Cellastim), recombinant human insulin (Escherichia coli recombinant expression, LIFE Company), recombinant human transferrin (yeast recombinant expression, Invitria Company) , β-mercaptoethanol (Invitrogen, 21985-023), ethanolamine (Sigma, E0135), cholesterol (Sigma, C1231), linoleic acid (Sigma, L1012), oleic acid (Sigma, O1383), copper sulfate (Sigma, C8027 ), zinc sulfate (Sigma, Z0251), ferric citrate (Sigma, F3388), sodium bicarbonate (Sigma, S5761) and ultrapure water were mixed to obtain an immune cell serum-free culture system. Stir to dissolve, adjust the pH value to 7.0, and filter with a 0.22 μm filter to sterilize. Store at 4°C.

[0059] In the process of cell culture, recombinant human γ-interferon (Shandong Quangang Pharmaceutical Co., Ltd.) and recombinant human interleukin-2 (Beijin...

Embodiment 2

[0061] Example 2, Application of Immune Cell Serum-Free Medium in Culturing Immune Cells

[0062] 1. Isolation and purification of peripheral blood mononuclear cells

[0063] Peripheral blood mononuclear cells were directly separated and purified by Ficoll density gradient centrifugation, and the specific steps were as follows:

[0064] 1. Transfer the peripheral blood (city hospital) to a 50ml centrifuge tube, centrifuge at 500g for 5min, and transfer the upper layer plasma into a new centrifuge tube with a pipette.

[0065] 2. Dilute the blood cells with normal saline, mix them evenly with a pipette, slowly pour into the centrifuge tube that has been added with lymphatic separation solution, and centrifuge at 2000r / min for 20 minutes. At this time, the centrifuge tube is divided into four layers from top to bottom. The first layer: plasma or cell tissue homogeneous serum layer; the second layer: milky white lymphocyte layer; the third layer: clear cell sorting liquid layer...

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PUM

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Abstract

The invention discloses a serum-free medium suitable for immune cells and a preparation method thereof. According to experimental proof, the immune cells cultured by the serum-free medium for immune cells have more stable characters, and the cell growth and proliferation effects are better. The serum-free medium for immune cells has the advantages of clear chemical components, little batch-to-batch difference and stable quality and can guarantee the consistency among the medium batches; without animal origin or human origin, the serum-free medium has high safety, overcomes potential risk caused by human or animal serum and is conducive to studying the action mechanisms of various immune cells, promoting the safety standardization of domestic cell therapy and reducing the risk of accidental infection of patients and is of great significance in the promotion and application of immunotherapy.

Description

technical field [0001] The invention belongs to the research fields of cell biology, molecular biology and medicine, and relates to a serum-free culture medium for in vitro expansion and culture of immune cells, more specifically, to a serum-free culture for in vitro expansion and culture of immune cells base and its applications. Background technique [0002] Cellular immunotherapy technology is a brand-new anti-tumor treatment method with remarkable curative effect. By collecting human immune cells, enriching, activating, and amplifying them in vitro, their targeted lethality is enhanced, and then infused back into the human body to achieve the purpose of treating various immune diseases. However, in vitro culture expansion requires a medium suitable for the growth and expansion of immune cells, including suitable osmotic pressure, to provide the cells with the nutrients necessary for growth, and to protect the cells from the damage of toxic substances produced during the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/078
CPCC12N5/0634C12N2500/12C12N2500/20C12N2500/22C12N2500/25C12N2500/30C12N2500/36C12N2500/44C12N2500/46C12N2500/90C12N2501/2302C12N2501/24C12N2501/998
Inventor 乔志平单磊
Owner GUANGDONG XTEM BIOTECH CO LTD
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