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Thawing method for frozen embryonic stem cell

A technology of human embryonic stem cells and resuscitation solution, which is applied in the field of resuscitation of frozen embryonic stem cells, which can solve the problems of time-consuming and labor-intensive operations, and complicated operation of vitrification cryopreservation

Active Publication Date: 2008-12-31
上海组织工程研究与开发中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] In addition, Reubinoff (2001) adopted the method of vitrification of stretched straws, and the undifferentiated ES cells reached 32% (Reubinof B.E, Pera, M.F, Vatija G and Trounson, A.O. Effective cryopreservation of human embryonic stem cells by the open pulled strawvitrification method. Hum. Reprod 2001; 2187-2194; Reubinof B.E, Pera, M.F, Fong. C.Y. et al. Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro. Nature Biotechnol., 2000, 18, 399-405. ), but the vitrification method is complicated to operate, time-consuming and labor-intensive

Method used

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  • Thawing method for frozen embryonic stem cell
  • Thawing method for frozen embryonic stem cell
  • Thawing method for frozen embryonic stem cell

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Cell cryopreservation

[0050] Cryopreservation solution: mix knock-out serum replacement (SR) and dimethyl sulfoxide (DMSO) in a ratio of 9:1, place on ice, and pre-cool.

[0051] Take hES cells of different passage numbers (20-25 ES cell clones each) for cryopreservation, and the experiment was repeated 3 times (see Table 1). Methods as below:

[0052] Take well-grown hES cells, digest with 1mg / ml type IV collagenase, transfer the clones to an eppendorf tube, add culture medium dropwise, gently pipette into small cell clumps, centrifuge at 1000rpm / min for 3 minutes, discard the supernatant Add the cryopreservation solution to the tube slowly drop by drop, and shake it up constantly. Quickly transfer the cell suspension to the cryopreservation tube, put it into the program cooling box, and cool down at 1°C / min until -80°C. -80°C overnight, transfer it to a liquid nitrogen tank the next day, and store it in the refrigerator.

Embodiment 2

[0054] Cell recovery

[0055] Take out the cryopreservation tube prepared in Example 1 from the liquid nitrogen, quickly place it in a 37°C water bath, shake it until it dissolves, and scrub the tube wall with 75% alcohol cotton (heating speed is about 100°C / min). Then slowly add the cells to the resuscitation solution (the resuscitation solution is hES culture solution containing 0.2 mol / L trehalose), centrifuge at 1000 rpm / min for 3 minutes, and discard the supernatant. The ES cells were transferred to a pre-prepared feeder layer, cultured in hES culture medium containing 0.1 mol / L trehalose, and replaced with conventional hES culture medium after 1 hour of culture.

Embodiment 3

[0057] Morphological observation and characterization of human ES cells after resuscitation

[0058] 1. Method

[0059] (a) Morphological observation of human ES cells after resuscitation

[0060] Use a phase contrast microscope to observe the survival and differentiation of cells after resuscitation. The clone formation rate was calculated after 8 days of culture.

[0061] Clone formation rate=number of undifferentiated ES clones after recovery / total number of ES clones before cryopreservation×100%

[0062] (b) Characterization of human ES cells after resuscitation

[0063] (1) Alkaline phosphatase (AKP) staining

[0064] According to the method of Xu et al. (Xu X, Tsung HC, Yan Yc. Establishment and differential of murine EG cell lines derived from primordial germcells. Acta Biologiae Exper Sinica 1999; 32: 251-63), NBT and BCIP (Sigma) were used as Substrate and staining agent for AKP activity identification.

[0065] (2) Immunocytochemistry

[0066] SSEA-4 and Oct-4 monoclonal...

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Abstract

The invention discloses a method for resuscitating cryopreserved embryonic stem cells. The method includes adding a resuscitation solution containing carbohydrates at a certain concentration during the resuscitation process. This method can significantly increase the survival rate of resuscitated stem cells (it can increase the resuscitated survival rate to about 42%), and has good repeatability.

Description

Technical field [0001] The present invention relates to the field of biology, and more specifically, to a method for resuscitating cryopreserved embryonic stem cells, which can significantly improve the survival rate of resuscitated stem cells. Background technique [0002] In 1998, scientists at the University of Wisconsin in the United States successfully cultured and expanded human embryonic stem (hES) cells in vitro, and confirmed that they have the potential to differentiate into three human germ layer cells (Thomson J. Itskovitg-Eldor J, Shapiro S. et al. .al, Embryonic stem cell lines derived from human blastocysts Science 1998; 282: 1145-1147.), drawing attention to the potential value of hES cells in clinical applications. [0003] Through the targeted differentiation of embryonic stem cells combined with cell therapy technology, it is expected to treat certain clinical diseases, including neurological diseases, diabetes, chronic heart disease, kidney disease, liver dise...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/06C12N5/08C12N5/0735
Inventor 曹谊林丛笑倩王彦唐郑雅吴春芳张文杰刘伟崔磊
Owner 上海组织工程研究与开发中心
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