Cell frozen preservation solution and cell frozen preservation method

A technique for cryopreservation and cell, which is applied in the field of cell cryopreservation and cell cryopreservation, and can solve the problems of poor cryopreservation effect of cryopreservation solution and cumbersome operation of cryopreservation method.

Active Publication Date: 2017-08-11
沃昕生物科技(深圳)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In view of this, the present invention provides a cell cryopreservation solution and a cell cryopreservation method, which can effectively solve the technical defects of poor cryopreservation effect of the prior art cryopreservation solution and cumbersome operation of the existing cryopreservation method

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  • Cell frozen preservation solution and cell frozen preservation method
  • Cell frozen preservation solution and cell frozen preservation method
  • Cell frozen preservation solution and cell frozen preservation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Using DMEM / F12 as the base solution, add platelet lysate, bFGF, L-glutamine, hydroxyethyl starch, trehalose, sodium carboxymethylcellulose, glycerol, DMSO and FBS to make platelet lysate, bFGF, L -The final concentration / volume percentage of glutamine, hydroxyethyl starch, trehalose, sodium carboxymethylcellulose, glycerol, DMSO and FBS in the cell freezing solution is 5% (V / V), 50ng / ml respectively , 4mmol / L, 0%~5%(V / V), 0%~5%(V / V), 0%~0.1%(V / V), 0%~20%(V / V), 5 %(V / V) and 10%(V / V). Wherein, the specific volume percentage / concentration added to the cell cryopreservation solution is shown in Table 1.

[0033] Table 1 The formula of the cell cryopreservation solution of test groups 1-11 and comparative examples 1-2

[0034]

[0035]

Embodiment 2

[0037] Adipose stem cell cryopreservation and recovery, the steps are as follows:

[0038] Cell cryopreservation: Take the adipose stem cells cultured to the P6 generation, digest and collect them by centrifugation, and resuspend the cells with the cryopreservation solutions of test groups 1-11 and comparative examples 1-2 prepared in Example 1 respectively, and the adipose-derived stem cells are frozen. The density in the liquid is 1×10 7 pieces / ml.

[0039] Freezing method 1: first freeze the cells at 4°C for 40 minutes, then store them at -20°C for 2 hours, and then store them at -80°C for 3 hours. Finally transferred to liquid nitrogen for storage. Freeze for 1 week, 1 month, 6 months and 1 year respectively.

[0040] Freeze storage method 2: directly store the cells at -80°C for 12 hours and then transfer to liquid nitrogen for storage. Freeze for 1 week, 1 month, 6 months and 1 year respectively.

[0041] Resuscitation: Take out the cryopreservation tube from the li...

Embodiment 3

[0043]After resuscitating the adipose stem cells cryopreserved by two methods in Example 2, the cell survival rate was detected, and the steps were as follows:

[0044] Viability detection: after cell recovery, dilute with DMEM / F12 medium, centrifuge at 1200rpm / min for 3min, discard the supernatant, resuspend the cells with DMEM / F12 medium, calculate the cell survival rate of the adipose stem cells after recovery, The results are shown in Table 2 and Table 3.

[0045] Among them, cryopreservation method one is to first freeze the recovered cells at 4°C for 40 minutes, then store them at -20°C for 2 hours, and then store them at -80°C for 3 hours. Finally transferred to liquid nitrogen for storage. Freeze for 1 week, 1 month, 6 months and 1 year respectively; the second method of freezing is to directly store the cells at -80°C for 12 hours and then transfer them to liquid nitrogen for storage. Freeze for 1 week, 1 month, 6 months and 1 year respectively.

[0046] Table 2 St...

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Abstract

The invention belongs to the field of cell frozen preservation, particularly relates to a cell frozen preservation solution and a cell frozen preservation method and provides a cell frozen preservation solution. The cell frozen preservation solution comprises a basic culture medium, a blood platelet lysate, bFGF and L-glutamine. The cell frozen preservation solution does not contain animal serum or DMSO, the possibility that an exogenous virus can be introduced by the animal serum is eliminated and the bad effect of the DMSO on adipose-derived stem cells is eliminated. Furthermore, the invention further provides the cell frozen preservation method. According to the method, complicated programmed cooling is not needed and operation is simple. After the adipose-derived stem cells are frozen and preserved by adopting the cell culture medium and the cell frozen preservation method for a year, the survival rate of the cells can reach 93% and the differentiation capacity of the adipose-derived stem cells is not affected.

Description

technical field [0001] The invention belongs to the field of cell cryopreservation, in particular to a cell cryopreservation solution and a cell cryopreservation method. Background technique [0002] Adipose-derived stem cells (adipose-derived stem cells, ADSCs) are a kind of immature pluripotent cells with self-replication ability isolated from adipose tissue in recent years. Under certain conditions, it can differentiate into a variety of functional cells, with the potential to regenerate various tissues and organs. [0003] Adipose stem cells are taken out from the body's own tissues under sterile conditions, and after separation and purification, they are cultured in vitro under simulated in vivo physiological conditions through core optimization techniques, so that incompletely differentiated cells can continue to differentiate and grow. Proliferate in large quantities, keep the cells in a good growth state and vigorous proliferation vitality, and then inject them back...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01N1/02
CPCA01N1/0221
Inventor 王旭林洁璇李陶林词雄朱刚
Owner 沃昕生物科技(深圳)有限公司
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