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Culture method of umbilical cord blood lymphocyte CIK

A technology of lymphocyte and culture method, applied in the field of culturing umbilical cord blood lymphocyte CIK, can solve the problems of cumbersome steps, loss of killing, reduction, etc., and achieve the effects of high multiplication multiple, strong tumoricidal activity, and low immunogenicity

Active Publication Date: 2020-07-10
GUANGDONG XIANKANGDA BIOTECH CO LTD
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AI Technical Summary

Problems solved by technology

[0007] (1) The general CIK preparation method is to treat isolated umbilical cord blood mononuclear cells (PBMC) with IFN-γ for 24 hours, then add CD3 monoclonal antibody, IL-1α and IL-2 factors for stimulation and induction, and finally obtain a certain number of CIK cells, but the multiplier and effective cell content of the finally obtained CIK cells are not ideal
[0008] (2) Existing CIK cells need to remove adherent monocytes before culturing, which is cumbersome and increases the risk of bacterial contamination during cell transfer and culture
[0009] (3) Autologous immune cells CIK easily cause immune escape of cancer cells or target cells and reduce or lose the ability to kill cancer cells, cancer cells or target cells

Method used

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  • Culture method of umbilical cord blood lymphocyte CIK
  • Culture method of umbilical cord blood lymphocyte CIK

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Experimental program
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Embodiment

[0044]The method for expanding umbilical cord blood immune cells CIK adopted in this embodiment:

[0045] 1. Isolation and Culture of Cord Blood Mononuclei

[0046] (1) Coarse separation:

[0047] Aliquot the blood into 50mL centrifuge tubes, up to 35mL per tube, and calculate the blood volume. Draw the blood sample with a Pasteur pipette, drop 2 drops of it into the EP tube, and use a hematology analyzer to detect the number of cells. Centrifuge the blood in the 50mL centrifuge tube at 650g, 10min, up to 8 gears, down to 8 gears. Prepare the same number of 50mL centrifuge tubes as the whole blood, and add 15mL of lymphocyte separation medium to each tube.

[0048] (2) Plasma preparation:

[0049] After centrifugation, transfer the centrifuge tube to the countertop of the safety cabinet, and use a 25mL pipette to draw the upper layer of plasma into a new centrifuge tube. Take 1 mL of plasma as a reserved sample, mark it and store it in a -20°C refrigerator. The remaining...

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Abstract

The invention belongs to the technical field of immune cell in-vitro culture, and particularly discloses a culture method of umbilical cord blood lymphocyte CIK. The method comprises the following steps: by using umbilical cord blood as a treatment object, separating the umbilical cord blood mononuclear cells, purifying and inducing the CIK cells, culturing and amplifying the CIK cells, and finally, centrifuging to collect the mature umbilical cord blood CIK cells, thereby obtaining the product. The IFN-gamma and alpha-galactose ceramide are added to activate the CIK cells, and CD3 monoclonalantibodies, IL-1 alpha, IL-2 and IL-1 beta are added in the early culture stage to perform continuous stimulation, thereby saving the coating time and enhancing the activation efficiency and amplification efficiency of effector cell groups.

Description

technical field [0001] The invention relates to the technical field of in vitro culture of immune cells, in particular to a culture method for umbilical cord blood lymphocyte CIK. Background technique [0002] Adoptive immune cell therapy is to infuse lymphocytes with specific immunity or their products to cellular immune dysfunction (such as tumor patients and other diseases caused by high or low immunity), and adjust the anti-tumor immunity of patients to achieve The purpose of treatment and prevention of recurrence. [0003] Cytokine induced killer cells (CIK) are human peripheral blood or umbilical cord blood mononuclear cells treated in vitro with various cytokines (such as anti-CD3 monoclonal antibody, IL-2, α-galactosylceramide , IL-1β and IFN-γ, etc.) to obtain a group of heterogeneous cells after induction of proliferation. Due to the simultaneous expression of two membrane protein molecules, CD3+ and CD56+, they are also called NK cell-like T lymphocytes, which h...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783
CPCC12N5/0638C12N2500/90C12N2501/2301C12N2501/2302C12N2501/24C12N2501/515C12N2501/999
Inventor 谢海涛薛卫巍钟家炜谢天仲王斌王乃会
Owner GUANGDONG XIANKANGDA BIOTECH CO LTD
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