Animal source-free and serum-free culture medium of lymphocyte

A technology of serum-free medium and human serum albumin, which is applied in the field of serum-free medium for lymphocytes, can solve the problems of high price, unfavorable promotion, and increased cost of immune cell therapy, and achieve the effect of ensuring consistency and clear properties

Inactive Publication Date: 2013-06-12
BEIJING JING MENG STEM CELL TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the composition of these serum-free media is unclear and expensive, which greatly increases the cost of immune cell therapy, which is not conducive to the promotion of wide application

Method used

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  • Animal source-free and serum-free culture medium of lymphocyte
  • Animal source-free and serum-free culture medium of lymphocyte
  • Animal source-free and serum-free culture medium of lymphocyte

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Embodiment 1: the preparation of culture medium

[0020] Preparation of recombinant human insulin stock solution: Weigh 100mg of recombinant human insulin and dissolve it in 40mL of water for injection, add dropwise 2M HCl until the recombinant human insulin is completely dissolved, dilute to 50mL, prepare a 20mg / mL stock solution, and store at 4°C.

[0021] Preparation of human transferrin stock solution: Weigh 100 mg of human transferrin and dissolve it in 40 mL of water for injection, and dilute to 50 mL after complete dissolution, prepare a 20 mg / mL stock solution, and store at 4°C.

[0022] The medium composition of the present invention is as follows:

[0023] Component

Amount added

IMDM

17.7g / L

L-Glutamine

1-5mM

sodium bicarbonate

3.024g / L

recombinant human insulin

1-10mg / L

human transferrin

5-20mg / L

human serum albumin

1-10g / L

2-Mercaptoethanol

55uM

N-acetyl-cystein...

Embodiment 2

[0031] Example 2: Umbilical cord blood separation

[0032] Source of umbilical cord blood: The umbilical cord blood comes from the obstetrics department of Beijing Armed Police Hospital. , with an average volume of 80-130 mL. The separation process is as follows:

[0033] 1: After diluting the fresh cord blood and the buffer solution PBS at a volume ratio of 1:1, slowly add to the upper layer of the separation solution Ficoll along the wall of the test tube. Pay attention to keep the two interfaces clear and prevent blood from mixing into the separation solution.

[0034] 2: Centrifuge at 1800rpm for 15min, gently insert the white mist layer (the second layer from top to bottom) with a capillary pipette, gently suck out the mononuclear cells in this layer along the tube wall, and put it into another centrifuge tube.

[0035] 3: The resulting mononuclear cell suspension was washed with one volume of PBS, centrifuged at 1500 rpm for 10 min, and washed 3 times.

[0036] 4: Co...

Embodiment 3

[0037] Example 3: Peripheral Blood Separation

[0038] Peripheral blood source: Peripheral blood comes from Beijing Armed Police Hospital, which is qualified according to the "Blood Donation Physical Examination Standard" promulgated by the Ministry of Health. After intravenous disinfection, the peripheral blood is collected in a fully closed manner, anticoagulated with heparin, and the average volume is 50mL. The separation process is as follows:

[0039] 1: After diluting the fresh peripheral blood and the buffer solution PBS at a volume ratio of 1:1, slowly add to the upper layer of the separation solution Ficoll along the wall of the test tube. Pay attention to keep the two interfaces clear and prevent blood from mixing into the separation solution.

[0040]2: Centrifuge at 1800rpm for 15min, gently insert the white mist layer (the second layer from top to bottom) with a capillary pipette, gently suck out the mononuclear cells in this layer along the tube wall, and put it...

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Abstract

The invention relates to the biological field and discloses an animal source-free and serum-free culture medium of lymphocyte. The culture medium disclosed by the invention essentially consists of IMDM (Iscove Modified Dulbecco Medium), L-glutamine, sodium bicarbonate, recombinant human insulin, human transferrin, human serum albumin, 2-mercaptoethanol, N-acetyl-cysteine, lipid, amino acid, vitamin, microelement, ferric citrate, hydrocortisone, cholamine and non-essential amino acid. The serum-free culture medium disclosed by the invention has the advantages of clear chemical components, no animal source, no serum, safe and ideal culture cells; the instability caused by the doping of animal components and batches is avoided; the result of culturing lymphocyte shows that the total number of the cells and the cell phenotypes are normal; and the serum-free culture medium disclosed by the invention has a good industrial application prospect.

Description

technical field [0001] The invention relates to the field of biology, in particular to a serum-free culture medium for lymphocytes with clear chemical components and no animal source. Background technique [0002] Cellular immunotherapy is an emerging, brand-new anti-tumor treatment method with significant curative effect, which makes up for the disadvantages of traditional surgery, radiotherapy, and chemotherapy. A promising approach to treatment. Cellular immunotherapy is to collect human immune cells (mainly lymphocytes), culture them in vitro, increase their number by thousands of times, enhance their targeted killing function, and then reinfuse them back into the human body to kill pathogens in blood and tissues. Cancer cells and mutated cells break immune tolerance, activate and enhance the body's immune ability, and take into account the dual effects of treatment and health care. [0003] The most widely used reagent for in vitro culture and expansion of lymphocytes...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/078
Inventor 武晓云吕岩康会彦
Owner BEIJING JING MENG STEM CELL TECH
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