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Serum-free cell cryopreservation solution as well as preparation method and use method thereof

A cryopreservation liquid and serum-free technology, applied in the field of biomedicine, can solve the problems of limited protective effect, achieve the effects of reducing cell damage, reasonable density of cryopreserved cells, and protecting the activity of stem cells

Inactive Publication Date: 2020-11-03
上海雅酶生物医药科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] Although the non-permeable protective agent can play a certain protective role in the process of cell cryopreservation, the ability to protect alone is still limited. Therefore, a combination of various protective agents is used to optimize a suitable formula ratio , it is particularly necessary

Method used

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  • Serum-free cell cryopreservation solution as well as preparation method and use method thereof
  • Serum-free cell cryopreservation solution as well as preparation method and use method thereof
  • Serum-free cell cryopreservation solution as well as preparation method and use method thereof

Examples

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preparation example Construction

[0038] Such as figure 1 Shown, a kind of preparation method of serum-free cell cryopreservation liquid, concrete configuration method comprises the following steps (taking 100ml as example):

[0039] S01. Weigh 5 g of polyethylene glycol of the above concentration and dissolve it in 80 ml of basal medium;

[0040] S02, adding 2g of trehalose at the above concentration, stirring to dissolve it;

[0041] S03, add 2.5g of the above-mentioned concentration of glucose, stir to make it dissolve;

[0042] S04. Add 1 mg of all-trans retinoic acid at the above concentration, and stir to dissolve it;

[0043] S05, add 0.5mg ascorbic acid, stir to make it dissolve;

[0044] S06. Adjust the pH of the prepared cell cryopreservation solution to 7.2 with 1mol / L hydrochloric acid or NaOH, and finally dilute it to 100ml with basal medium, and store it at 2-8°C.

[0045] Such as figure 2 Shown, a kind of use method of serum-free cell cryopreservation liquid, comprises the steps:

[0046]...

Embodiment 1

[0049] Example 1: Detection of cryopreservation and resuscitation of cells (taking A549 cells as an example);

[0050] 1. Resuspend the A549 cells cultured in a 10cm culture dish by trypsinization and centrifugation, and press the density of 1x10 5 / well into six-well cell culture plates at 37 °C, 5% CO 2 The culture was continued for about 24 hours under certain conditions, when the cell confluency reached 80%-90%.

[0051] 2. Use trypsin to digest, centrifuge at 1500rpm for 5 minutes, discard the waste liquid, add 1ml of serum-containing common freezing solution and prepared cell freezing solution to resuspend, transfer to the cryopreservation tube, mark it, and determine the concentration of the frozen cells Usually in 1x10 6 / ml.

[0052] 3. Gradiently lower the temperature of the serum-containing cryopreservation solution, and place the prepared cryopreservation solution directly in a -80°C ultra-low temperature refrigerator for long-term storage or transfer it to liqu...

Embodiment 2

[0063] Example 2: Detection of cryopreservation and resuscitation of cells (taking human umbilical vein endothelial (HUVEC) cells as an example);

[0064] 1. After the HUVEC cells cultured in a 10cm culture dish were resuspended by trypsinization and centrifugation, the density was 1x10 5 / well into six-well cell culture plates at 37 °C, 5% CO 2 The culture was continued for about 24 hours under certain conditions, when the cell confluency reached 80%-90%.

[0065] 2. Use trypsin to digest, centrifuge at 900rpm for 5 minutes, discard the waste liquid, add 1ml of serum-containing common freezing solution and prepared cell freezing solution to resuspend, transfer to a cryopreservation tube, mark it, and determine the concentration of the frozen cells Usually in 1x10 6 / ml.

[0066] 3. Gradiently lower the temperature of the serum-containing cryopreservation solution, and place the prepared cryopreservation solution directly in a -80°C ultra-low temperature refrigerator for lo...

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Abstract

The invention discloses a serum-free cell cryopreservation solution, a preparation method and a use method thereof, and relates to the technical field of biological medicines. The serum-free cell cryopreservation solution is prepared from the following components: 4 to 6 g / 100mL of polyethylene glycol, 0.5 to 2.5 g / 100mL of trehalose, 2 to 3 g / 100mL of glucose, 0.5 to 1.5 mg / 100mL of all-trans retinoic acid and 0.5 mg / 100ml of a small amount of ascorbic acid, a serum-free basal culture medium is used as a solvent, and the final pH value is 6.8 to 7.4. The using method comprises the steps of digestion, cryopreservation and resuscitation. The cell cryopreservation solution is clear in components, does not contain exogenous serum or organic solvents, avoids exogenous protein pollution, and can be directly cryopreserved at -80 DEG C without programmed cooling; the activity of the stem cells can be effectively protected, and the activity of the freeze-thawed cells is maintained and prevented from being lost. According to the cryopreservation method, the risk that the cells absorb water and burst is reduced during recovery, and the cell activity is guaranteed.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a serum-free cell freezing solution, a preparation method of a serum-free cell freezing solution and a use method of a serum-free cell freezing solution. Background technique [0002] With the increase of biological and cell-based preparations, cells are used in more and more ways, but the storage of cells often limits the application of cells. During the cell culture process, with the increase of the number of passages, various biological characteristics of the cells will gradually change. Therefore, the timely preservation of primary cells or cell seeds is particularly necessary. In addition, purchasing, exchanging, and shipping certain cells also require the use of cryopreserved cells. Therefore, it is very necessary to freeze and store cells timely and conveniently in practical work. [0003] At present, the most mature and stable cryopreservation cell tech...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01N1/02
CPCA01N1/0221A01N1/0226
Inventor 吴军黄人卉于树祥
Owner 上海雅酶生物医药科技有限公司
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