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Cryopreservation solution and cryopreservation method for induced pluripotent stem cells

A pluripotent stem cell and cryopreservation technology, which is applied in the field of induced pluripotent stem cell cryopreservation and cryopreservation, can solve the problems of interference of the biological characteristics of induced pluripotent stem cells, inappropriate, bad or inconsistent animal serum, etc. The method is simple, easy to operate, and the effect of restoring activity

Pending Publication Date: 2021-08-10
郑州优倍得生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, for the clinical use of induced pluripotent stem cells, the presence of animal serum is not necessarily suitable
First, animal serum has the risk of carrying pathogens and other pathogenic factors; second, due to the complexity of serum components, differences in sources and production batches may cause adverse or inconsistent effects; third, serum is rich in various A variety of cytokines, hormones and other active ingredients are prone to interfere and affect the biological characteristics of induced pluripotent stem cells

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] An induced pluripotent stem cell cryopreservation solution, including basal medium DMEM / F12 and additives, in terms of final concentration, the concentration of each component in the additive is: raspberry alcohol 10.5 μg / mL, losartan potassium 9.8 μg / mL , Heparin sodium 17.0ng / mL, forskolin 13.2ng / mL, hyperoside 7.5μg / mL, vitamin E 8.0μg / mL, insulin 5.5ng / mL, sodium selenite 9.0ng / mL.

[0028] A cryopreservation method for induced pluripotent stem cells, comprising the following steps:

[0029] (1) Add subcultured P3 induced pluripotent stem cells into the above cryopreservation solution, the concentration of induced pluripotent stem cells is 5×10 6 cells / mL to obtain a suspension containing induced pluripotent stem cells, which was divided into 2mL cryopreservation tubes, each containing 1.5mL;

[0030] (2) Seal the aliquoted cryopreservation tubes and freeze at -80°C for 7 hours, then transfer to liquid nitrogen for storage.

Embodiment 2

[0032] An induced pluripotent stem cell cryopreservation solution, including basal medium DMEM / F12 and additives, in terms of final concentration, the concentration of each component in the additive is: raspberry alcohol 9.8 μg / mL, losartan potassium 8.5 μg / mL , Heparin sodium 15.5ng / mL, forskolin 10.8ng / mL, hyperoside 6.5μg / mL, vitamin E 5.0μg / mL, insulin 4.0ng / mL, sodium selenite 8.5ng / mL.

[0033] A cryopreservation method for induced pluripotent stem cells, comprising the following steps:

[0034] (1) Add subcultured P4 induced pluripotent stem cells into the above cryopreservation solution, the concentration of induced pluripotent stem cells is 1×10 6 / mL to obtain a suspension containing induced pluripotent stem cells, which was dispensed into 2mL cryopreservation tubes, each containing 1.5mL;

[0035] (2) Seal the aliquoted cryopreservation tubes and freeze at -80°C for 5 hours, then transfer to liquid nitrogen for storage.

Embodiment 3

[0037] An induced pluripotent stem cell cryopreservation solution, including basal medium DMEM / F12 and additives, in terms of final concentration, the concentration of each component in the additive is: raspberry alcohol 12.5 μg / mL, losartan potassium 11.2 μg / mL , Heparin sodium 18.5ng / mL, forskolin 14.5ng / mL, hyperoside 8.5μg / mL, vitamin E 9.5μg / mL, insulin 10.0ng / mL, sodium selenite 10.5ng / mL.

[0038] A cryopreservation method for induced pluripotent stem cells, comprising the following steps:

[0039] (1) Add subcultured P5 induced pluripotent stem cells into the above cryopreservation solution, the concentration of induced pluripotent stem cells is 1×10 7 cells / mL to obtain a suspension containing induced pluripotent stem cells, which was divided into 2mL cryopreservation tubes, each containing 1.5mL;

[0040] (2) Seal the aliquoted cryopreservation tubes and freeze at -80°C for 9 hours, then transfer to liquid nitrogen for storage.

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Abstract

The invention discloses a cryopreservation solution forinduced pluripotent stem cells. The cryopreservation solutioncomprises a basic culture medium and an additive, and the additive comprises the following components: raspberry alcohol, losartan potassium, heparin sodium, calomel, hyperoside, vitamin E, insulin and sodium selenite. According to the cryopreservation solution, raspberry alcohol and losartan potassium are added to replace DMSO, on one hand, ice crystals formed in induced pluripotent stem cells when the temperature is reduced to the freezing point are reduced; on the other hand, cell membranes are stabilized, osmotic pressure in the induced pluripotent stem cell cryopreservation process is adjusted, and the cells are prevented from losing activity due to excessive water loss. The heparin sodium is added to prevent the induced pluripotent stem cells from aggregating to influence the activity of the cryopreserved cells. The activity of the induced pluripotent stem cells cryopreserved by liquid nitrogen can be quickly recovered by adding the eriochin. Hyperoside and vitamin E are added, so that damage to cells caused by free radicals formed in the cryopreservation process is avoided. The invention further provides a cryopreservation method for the induced pluripotent stem cells. The method is simple, convenient and easy to operate and has good application prospects.

Description

technical field [0001] The invention relates to a cryopreservation solution, in particular to a cryopreservation solution and a cryopreservation method for induced pluripotent stem cells. Background technique [0002] Induced pluripotent stem cells (Induced Pluripotent Stem Cells, iPSCs) are cells that dedifferentiate adult cells through reprogramming technology, and have similar self-renewal and three-germ layer differentiation potentials to embryonic stem cells, because they can avoid immune transplantation to the host. immune diseases, and avoid ethical issues, has broad application prospects. [0003] In the application process of induced pluripotent stem cells, it is often necessary to freeze the cells. The cryopreservation solution is a compound buffer that must be used for the cryopreservation of induced pluripotent stem cells. Its main function includes protecting induced pluripotent stem cells during cryogenic freezing. After being destroyed and injured, induced pl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01N1/02
CPCA01N1/0226A01N1/0221
Inventor 鄂国立陈三飞陈友林
Owner 郑州优倍得生物科技有限公司
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