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CIK cell culture solution and culture method for enhancing CD3+CD56 + of CIK cell

A culture method and cell culture technology, applied in the field of enhancing CIK cell CD3+CD56+ culture, can solve the problems of difficult to meet clinical needs, low activation efficiency, poor proliferation effect of CIK cells, etc.

Pending Publication Date: 2021-10-29
湖南格致生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, CIK cell culture is to separate peripheral blood mononuclear cells (PBMC) with CD3 monoclonal antibody, interleukin-1 (IL-1α), interleukin-2 (IL-2) and γ - Interferon (IFN-γ) and other factors are activated and further expanded by static culture or intermittent medium replacement. However, this method generally has low activation efficiency, slow cell growth, poor proliferation of CIK cells obtained in the end, and cytotoxicity. Low, difficult to meet clinical needs

Method used

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  • CIK cell culture solution and culture method for enhancing CD3+CD56 + of CIK cell
  • CIK cell culture solution and culture method for enhancing CD3+CD56 + of CIK cell
  • CIK cell culture solution and culture method for enhancing CD3+CD56 + of CIK cell

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Experimental program
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preparation example Construction

[0030] 1. Preparation of peripheral blood mononuclear cells

[0031] 1.1. Take 50-60ml of peripheral blood from healthy people, collect it in a heparin bottle, and store it at 37°C.

[0032] 1.2. Use two 50ml centrifuge tubes (marked 1 # and 2 # ) into 20ml of lymphatic separation fluid.

[0033] 1.3. Use a ground glass pipette to transfer 30ml of peripheral blood to two centrifuge tubes filled with lymphocyte separation medium (note: the peripheral blood needs to be placed on the upper layer of lymphocytes).

[0034] 1.4. Place the two centrifuge tubes in a centrifuge and centrifuge at 800g for 15min at 20°C.

[0035] 1.5. After centrifugation, use a negative pressure device to suck as much plasma as possible from the two centrifuge tubes, and transfer the resulting lymphocyte layer cells to another 50ml centrifuge tube (marked 3 # )middle.

[0036] 1.6. According to the dosage of 80,000 units of gentamicin in 500ml of normal saline, add 160IU / ml of gentamicin in normal ...

Embodiment 1

[0056] Cells were removed from the above liquid nitrogen at a concentration of 0.5 × 10 8 Peripheral blood mononuclear cells (PBMC) per ml were quickly placed in a water bath at 37°C-42°C, shaken quickly to melt within 1 minute; the outer surface of the cryotube was disinfected with alcohol, and moved to a biological safety cabinet; Transfer the cells in the cryopreservation tube to a 50ml centrifuge tube in a biological safety cabinet; centrifuge at 1500rpm for 10min, 4°C, increase speed to max, and decelerate to max; Resuspend in RPMI-1640 medium with % autologous serum, adjust the cell concentration to 1×10 after counting 6 within the range of cells / ml, and then inoculated into T175 culture flasks.

[0057] On the first day, add RPMI-1640 medium containing 10% autologous serum in a volume of 25 ml to the culture flask, then add IFN-2γ cytokine, and control the final concentration of IFN-2γ cytokine in the medium to 1000 μg / ml; then put the culture bottle into the incubat...

Embodiment 2

[0062] Cells were removed from the above liquid nitrogen at a concentration of 0.5 × 10 8 Peripheral blood mononuclear cells (PBMC) per ml were quickly placed in a water bath at 37°C-42°C, shaken quickly to melt within 1 minute; the outer surface of the cryotube was disinfected with alcohol, and moved to a biological safety cabinet; Transfer the cells in a cryopreservation tube to a 50ml centrifuge tube in a biological safety cabinet; centrifuge at 1500rpm for 10min, 4°C, increase speed to max, and decelerate to max; after centrifugation, absorb the supernatant in the centrifuge tube, and use Resuspend in RPMI-1640 medium with 10% autologous serum, adjust the cell concentration to 1×10 after counting 6 within the range of cells / ml, and then inoculated into T175 culture flasks.

[0063] Add IFN-2γ cytokines to the culture flask, and control the final concentration of IFN-2γ cytokines in the medium to 500 μg / ml, add RPMI-1640 medium containing 10% autologous serum to a volume o...

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Abstract

The invention discloses a CIK cell culture solution and a culture method for enhancing the CD3+CD56+of a CIK cell. The method comprises the following steps of: adding peripheral blood mononuclear cells into a culture medium of an IFN-2[gamma] cell factor, and culturing for about 16 days; in a culture period, adding a culture medium containing rHIL-22, rHIL-21 and CD3 cell factors for multiple times every 2-3 days; and after culture is finished, performing centrifugal treatment and normal saline solution cleaning to obtain the CIK cell. According to the culture method, IFN-2[gamma] is added into the cell to be cultured before rhIL-21, so that cytotoxicity can be improved, then the rhIL-21 cell factor is added to further enhance the cytotoxicity of the cell, and a CD3 monoclonal antibody is added during subsequent culture solution change, and rapid proliferation of the CIK cell can be promoted.

Description

technical field [0001] The invention relates to the field of cytology, in particular to a method for enhancing the CD3+CD56+ culture of CIK cells. Background technique [0002] CIK cells (Cytokine Induced Killer Cells) are a kind of killer cells induced by a variety of cytokines. They are the immune cells with the strongest cytotoxic activity, and have the high tumoricidal activity of T lymphocytes and non-mainly NK cells. Restricted tumoricidal effects of the histocompatibility complex (MHC). CIK cells have the advantages of fast proliferation and high tumoricidal activity, and have incomparable advantages over TIL and LAK cells. [0003] At present, CIK cell culture is to separate peripheral blood mononuclear cells (PBMC) with CD3 monoclonal antibody, interleukin-1 (IL-1α), interleukin-2 (IL-2) and γ - Interferon (IFN-γ) and other factors are activated and further expanded by static culture or intermittent medium replacement. However, this method generally has low activa...

Claims

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Application Information

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IPC IPC(8): C12N5/0783
CPCC12N5/0638C12N2501/24C12N2501/2321C12N2501/2322C12N2501/515C12N2501/998
Inventor 黎鸠鸠唐闪光
Owner 湖南格致生物科技有限公司
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