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Cryopreservation solution and cryopreservation method of DC cell

A cryopreservation method and cryopreservation solution technology, applied in the field of cell biology, can solve the problems of poor freezing effect of DC cells, exogenous viruses, allergic reactions, etc., and achieve the effect of improving the freezing effect

Inactive Publication Date: 2016-10-12
GUANGZHOU SALIAI STEMCELL SCI & TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] However, there is no clear cryopreservation solution formula that can freeze DC cells. Generally, 90% FBS + 10% DMSO is used to freeze DC cells.
However, 90% FBS + 10% DMSO cryopreservation solution is very ineffective in freezing DC cells. After recovery, the DC cell viability is low and cannot continue to proliferate.
The existing cryopreservation solution contains a large amount of bovine serum, which may cause severe allergic reactions and bring the risk of exogenous viruses, so it cannot be used clinically
Moreover, the DMSO content in the existing cryopreservation solution is too high, and the risk is greater during clinical infusion

Method used

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  • Cryopreservation solution and cryopreservation method of DC cell
  • Cryopreservation solution and cryopreservation method of DC cell
  • Cryopreservation solution and cryopreservation method of DC cell

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Freezing solution formula:

[0042]

[0043]

[0044] Freezing method:

[0045] On the 0th day, a certain amount of peripheral blood was drawn, the PBMCs were separated by the ficoll method, and the separated PBMCs were resuspended in RPMI1640 medium to a density of 5×10 5 -1×10 6 / ml, put into a culture bottle in an incubator at 37°C, 5% CO 2 Incubate for 3 hours.

[0046] The non-adherent cells were discarded, and the adherent cells were cultured with RPMI1640 medium containing 10% FBS, 500 U / ml GM-CSF and 500 U / ml IL-4 for 6 days.

[0047] Collect the DC cells, remove the supernatant after centrifugation, take a part of the cells and resuspend them in RPMI1640 medium to detect cell surface markers.

[0048] Slowly add the above freezing solution to the pellet, mix well, and adjust the cell density to 1×10 7 -3×10 7 , to detect cell viability.

[0049] Add the mixed cell suspension into a cryopreservation tube, put it in a -80°C refrigerator, and transfer...

Embodiment 2

[0054] Freezing solution formula:

[0055]

[0056]

[0057] Freezing method:

[0058] On the 0th day, a certain amount of peripheral blood was drawn, the PBMCs were separated by the ficoll method, and the separated PBMCs were resuspended in RPMI1640 medium to a density of 5×10 5 -1×10 6 / ml, put into a culture bottle in an incubator at 37°C, 5% CO 2 Incubate for 3 hours.

[0059] The non-adherent cells were discarded, and the adherent cells were cultured with RPMI1640 medium containing 10% FBS, 500 U / ml GM-CSF and 500 U / ml IL-4 for 6 days.

[0060] Collect the DC cells, remove the supernatant after centrifugation, take a part of the cells and resuspend them in RPMI1640 medium to detect cell surface markers.

[0061] Slowly add the above freezing solution to the pellet, mix well, and adjust the cell density to 1×10 7 -3×10 7 , to detect cell viability.

[0062] Add the mixed cell suspension into a cryopreservation tube, put it in a -80°C refrigerator, and transfer i...

Embodiment 3

[0067] Freezing solution formula:

[0068]

[0069]

[0070] Freezing method:

[0071] On the 0th day, a certain amount of peripheral blood was drawn, the PBMCs were separated by the ficoll method, and the separated PBMCs were resuspended in RPMI1640 medium to a density of 5×10 5 -1×10 6 / ml, put into a culture bottle in an incubator at 37°C, 5% CO 2 Incubate for 3 hours.

[0072] The non-adherent cells were discarded, and the adherent cells were cultured with RPMI1640 medium containing 10% FBS, 500 U / ml GM-CSF and 500 U / ml IL-4 for 6 days.

[0073] Collect the DC cells, remove the supernatant after centrifugation, take a part of the cells and resuspend them in RPMI1640 medium to detect cell surface markers.

[0074] Slowly add the above freezing solution to the pellet, mix well, and adjust the cell density to 1×10 7 -3×10 7 , to detect cell viability.

[0075] Add the mixed cell suspension into a cryopreservation tube, put it in a -80°C refrigerator, and transfer...

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PUM

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Abstract

The invention belongs to the field of cell biology and in particular relates to a cryopreservation solution and a cryopreservation method of a DC cell. The cryopreservation solution of the DC cell contains trehalose, bovine serum albumin, propylene glycol, DMSO, low molecular dextran, a DMEM and vitamin C. The cryopreservation method of the DC cell is characterized by adding the cryopreservation solution to the DC cell, mixing the cryopreservation solution with the DC cell uniformly and then cryopreserving the mixture. The cryopreservation solution and the cryopreservation method have the beneficial effects that the cryopreservation effects of the DC cell are obviously improved by adopting the cryopreservation solution to cryopreserve the DC cell, so the DC cell can maintain a better state in either the viability after resuscitation or proliferation.

Description

technical field [0001] The invention belongs to the field of cell biology, and in particular relates to a DC cell freezing solution and a freezing method. Background technique [0002] DC cells, also known as dendritic cells, are cells with the strongest antigen-presenting ability among known cells. Therefore, DC cells are used as research objects in many clinical treatment plans for tumor patients. However, DC cell culture takes a long time, and DC cells cannot be cultured from frozen blood. Therefore, when treating tumor patients, fresh blood must be drawn from the patient and then cultured for 14-30 days to expand the cells. Carry out reinjection. In this way, there will be some problems. First, the blood quality of tumor patients is poor, and it is difficult to amplify enough DC cells; second, the expansion time is long, and the waiting time for patients is too long. Therefore, if the peripheral blood can be drawn when the patient is in good condition, DC cells can be ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01N1/02
CPCA01N1/0221
Inventor 陈海佳王一飞葛啸虎万桦张维敏
Owner GUANGZHOU SALIAI STEMCELL SCI & TECH CO LTD
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