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Freezing storage liquid for freezing and storing mononuclear cells

A technique for nuclear cells and cryopreservation solution, which is applied to the field of cryopreservation solution for cryopreservation of mononuclear cells and its preparation field, can solve the problems of unsatisfactory cryopreservation effect, etc., achieve convenient clinical application, avoid the risk of cross-infection, and achieve cryopreservation effect. Good results

Inactive Publication Date: 2012-01-04
SHENZHEN BEIKE BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, the cryopreservation effect of the existing cryopreservation solution for MNC is often not ideal.

Method used

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  • Freezing storage liquid for freezing and storing mononuclear cells
  • Freezing storage liquid for freezing and storing mononuclear cells
  • Freezing storage liquid for freezing and storing mononuclear cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Example 1 Preparation of cryopreservation solution.

[0018] In the ultra-clean workbench, take fresh cord blood in a sterile centrifuge tube, centrifuge at 2800 rpm for 16 minutes, and collect the supernatant, which is plasma. The plasma (Blood plasma) and dimethyl sulfoxide (DMSO) are uniformly mixed at a volume ratio of 19:1 to obtain the plasma cryopreservation solution provided by the present invention.

[0019] In order to facilitate the comparison of cryopreservation effects, the prior art DMEM cryopreservation solution was prepared: DMEM basal medium and DMSO were mixed uniformly in a volume ratio of 19:1; HES cryopreservation solution: medical 6% hydroxyethyl starch solution (HES) Mix well with DMSO at a volume ratio of 19:1. In this article, DMEM refers to DMEM basal medium, and HES refers to medical 6% hydroxyethyl starch solution.

Embodiment 2

[0020] Example 2 Freezing storage of MNC.

[0021] Prepare the plasma cryopreservation fluid, DMEM cryopreservation fluid and HES cryopreservation fluid as described above, and place them in a refrigerator at 4°C. Collect the cultured MNC, centrifuge at 200 g for 10 min, discard the supernatant, suspend the cells in saline, and sample and count. According to the cell count, the cells were divided into three groups, named plasma cryopreservation group, DMEM cryopreservation group, and HES cryopreservation group. Centrifuge at 200g for 10 min, discard the supernatant, add the corresponding cryopreservation solution, and mix gently. uniform. Put the mixed cells into cryopreservation tubes, mark them, put the cryopreservation tubes in the program cooling box, and then move them into the -80℃ refrigerator, and transfer them to the liquid nitrogen tank for freezing the next day. MNC cells are frozen in liquid nitrogen for more than 2 days, and can be resuscitated as required by the ...

Embodiment 3

[0022] Example 3 Analysis of cell resuscitation rate of cryopreserved MNC.

[0023] Adjust the temperature of the constant temperature water bath to 40℃, take out the cryopreservation tubes of the plasma cryopreservation group, DMEM cryopreservation group and HES cryopreservation group from the liquid nitrogen tank, take three parallel samples from each group and put them into the water bath immediately , Shake quickly, spray 75% ethanol when the cell cryopreservation solution is completely thawed; operate in an ultra-clean workbench, quickly burn the lid of the cryotube with an alcohol lamp, open the lid of each cryotube, and remove the cells from each cryotube Transfer all the suspension to a labeled sterile centrifuge tube. Slowly add 5 times the volume of cold normal saline to the sterile centrifuge tube, mix gently, centrifuge at 200g for 10 min, and discard the supernatant. Resuspend the cells in culture medium or PBS, mix gently, take samples to calculate the viability of...

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Abstract

The invention relates to freezing storage liquid for freezing and storing mononuclear cells and a preparation method for the freezing storage liquid, and belongs to the technical field of biology. The freezing storage liquid for freezing and storing the mononuclear cells comprises blood plasma and dimethyl sulfoxide. The freezing storage liquid is mainly applied to the freezing storage of the mononuclear cells, and has the advantages of good freezing storage effect and cost conservation.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a cryopreservation solution for cryopreserving mononuclear cells and a preparation method thereof. Background technique [0002] Normally, liquids freeze to form regular or irregular crystals that can rupture cell membranes and cause direct damage to living cells. Therefore, in order to reduce cell damage during cryopreservation, it is often necessary to use a cryopreservation solution containing a cryoprotectant. Commonly used cryoprotectants include dimethyl sulfoxide (DMSO), glycerin, and dextran, among which DMSO is the most commonly used. The use of pure animal serum combined with DMSO is a commonly used cryopreservation solution, but animal serum may contain animal pathogenic microorganisms, which may cause cross-infection with frozen cells. Once infection occurs, subsequent resuscitation and washing are difficult to remove. Culture medium and hydroxyethyl starch ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01N1/02
Inventor 吴莉芳刘陈雄李陶姜舒胡祥
Owner SHENZHEN BEIKE BIOTECH
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