Culture solution and culture method of DC cells

A technology of cell culture and culture medium, which is applied in the field of DC cell culture medium and its cultivation, can solve the problems of slow cell culture maturation time and low cell number purity, and achieve increased cell number and purity, good anti-tumor effect, and mature fast time effect

Inactive Publication Date: 2021-08-13
蓝莲(杭州)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

However, such a culture solution still has the problems of slow DC cell culture maturation time, it takes about 7-14 days to mature, and the number and purity of cells are low; the present invention solves such problems

Method used

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  • Culture solution and culture method of DC cells

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Step 1, processing plasma to obtain leukocytes;

[0042] 1. Confirm that the suction tube of the plasma bag is sealed. Wipe the pipette and scissors with two different sterile room wipes soaked in 70% ethanol.

[0043] 2. Hold the suction tube with tweezers, cut the front end of the tube and control the flow rate with tweezers to transfer the plasma into a 50ml test tube. Record the plasma volume transferred.

[0044] 3. Centrifuge the tube (400g, 4°C) for 30 minutes to remove platelets.

[0045] 4. Pour the supernatant liquid into a new 50ml test tube.

[0046] 5. Put the test tube into a constant temperature water tank (56°C) and incubate for 30 minutes.

[0047] 6. Centrifuge tubes (400g, 4°C) for 60 minutes to remove precipitated fibrin.

[0048] 7. Pour the upper liquid (autologous serum) into a new 50ml test tube. The tubes were then labeled with the patient's name, date of preparation, and refrigerated (4°C).

[0049] 8. Confirm that the suction tube of the ...

Embodiment 2

[0090] Step 1, processing plasma to obtain leukocytes;

[0091] Same as embodiment 1;

[0092] Step 2, using adherent cell culture medium to cultivate leukocytes, and after culturing for 2 hours, remove the suspension, leaving adherent cells;

[0093] Adherent cell culture medium includes: 20ml of ALyS505N-0 culture medium, 9.9ul of GM-SCF with a concentration of 800U / ml, 6.3ul of IL-13 with a concentration of 500U / ml, and 50ul of 200mM L-glutamine.

[0094] Except that the adherent cell culture medium is different, others are the same as in Example 1;

[0095] Step 3, first wash the adherent cells with normal saline for 1-3 times, then add the subsequent first addition of DC cell culture medium to the culture flask with adherent cells, and culture for 20 hours at 36-38°C, 3-8% CO 2 ;

[0096] The subsequent first addition of DC cell culture medium includes: 15ml of serum-free DC medium, 10.3ul of GM-CSF at a concentration of 800U / ml, 6.7ul of IL-4 at a concentration of 500...

Embodiment 3

[0104] Step 1, processing plasma to obtain leukocytes;

[0105] Same as embodiment 1;

[0106] Step 2, using adherent cell culture medium to cultivate leukocytes, and after culturing for 2 hours, remove the suspension, leaving adherent cells;

[0107] Adherent cell culture medium includes: 20ml of ALyS505N-0 culture medium, 9.7ul of GM-SCF with a concentration of 800U / ml, and 6.6ul of IL-13 with a concentration of 500U / ml.

[0108] Except that the adherent cell culture medium is different, others are the same as in Example 1;

[0109] Step 3, first wash the adherent cells with normal saline for 1-3 times, then add the subsequent first addition of DC cell culture medium to the culture flask with adherent cells, and culture for 20 hours at 36-38°C, 3-8% CO 2 ;

[0110] The subsequent first addition of DC cell culture medium includes: 15ml of serum-free DC medium, 10.1ul of GM-CSF at a concentration of 800U / ml, 6.9ul of IL-4 at a concentration of 500U / ml, and 3.1ul of a concen...

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Abstract

The invention discloses a culture solution and a culture method of DC cells. The culture solution of the DC cells is added in batches according to the growth condition of the DC cells, namely an adherent cell culture solution, a subsequent first supplemented DC cell culture solution and a subsequent second supplemented DC cell culture solution, wherein the adherent cell culture solution comprises a serum-free DC culture medium, GM-SCF and IL-13; the subsequent first supplemented DC cell culture solution comprises a serum-free DC culture medium, GM-CSF, IL-4, an anti-CD 83 antibody and autoserum with the concentration of 1%; and the subsequent second supplemented DC cell culture solution comprises TNF-a, vitamin B12 and nicotinamide. By applying the culture solution and the culture method disclosed by the invention, the DC cells are cultured to be mature in a short time, the DC cells can be mature in only 3 days, the number and purity of the cells are high, the antigen presentation ability of the DC cells is strong, and the culture solution has an excellent anti-tumor effect.

Description

technical field [0001] The invention relates to the field of cell culture, in particular to a DC cell culture medium and a culture method thereof. Background technique [0002] Dendritic cells (Dendritic Cells, hereinafter referred to as DC) are a kind of macrophages and also the most powerful antigen presenting cells (Antigen Presenting Cells, APCs). In the human body, DC cells mainly activate helper T cells and B cells by phagocytizing antigens and expressing a part of them on MHC II molecules on the surface of DC cells. It has been confirmed that DC is the only APC that can significantly stimulate the proliferation of naive T cells (T cells). There are many existing DC cell culture methods. The existing technology generally uses GM-CSF, IL-4, and TNF-a to culture DC cells. The function of GM-CSF in DC culture is to promote the differentiation of monocytes into macrophage-like cells. , the expression of MHC class II molecules on the cell surface is increased, thereby enh...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0784
CPCC12N5/0639C12N2501/25C12N2500/38C12N2500/30C12N2501/22C12N2501/2304C12N2501/599
Inventor 杨罕闻
Owner 蓝莲(杭州)生物科技有限公司
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