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Thawing method for frozen embryonic stem cell

A technology of embryonic stem cells and stem cells, which is applied in the field of biology, can solve problems such as time-consuming and labor-intensive operations, and the operation of vitrification is complicated.

Active Publication Date: 2006-10-11
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

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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

[0050] Cell cryopreservation

[0051] Freezing medium: mix knock-out serum replacement (referred to as SR for short) and dimethyl sulfoxide (DMSO) at a ratio of 9:1, place on ice, and pre-cool.

[0052] hES cells with different passage numbers (20-25 ES cell clones each) were taken for cryopreservation, and the experiment was repeated 3 times (see Table 1). Methods as below:

[0053] Take well-grown hES cells, digest with 1mg / ml type IV collagenase, transfer the clones to an eppendorf tube, add the culture medium dropwise, blow gently to form small cell clumps, centrifuge at 1000rpm / min for 3 minutes, and discard the supernatant Slowly add the freezing solution to the tube drop by drop, and shake it constantly. Quickly transfer the cell suspension to a cryopreservation tube, put it into a programmed cooling box, and lower the temperature at 1°C / min until -80°C. Overnight at -80°C, transfer it to a liquid nitrogen tank the next day, and store it in a freezer.

Embodiment 2

[0055] Cell recovery

[0056] The cryopreservation tube prepared in Example 1 was taken out from the liquid nitrogen, quickly placed in a 37°C water bath, shaken continuously until dissolved, and the tube wall was scrubbed with 75% alcohol cotton (the heating rate was about 100°C / min). Then the cells were slowly added to the resuscitation solution (the resuscitation solution was hES culture solution containing 0.2 mol / L trehalose), centrifuged at 1000 rpm / min for 3 minutes, and the supernatant was discarded. The ES cells were transferred to the pre-prepared feeder layer, cultured with hES culture medium containing 0.1mol / L trehalose, and changed to conventional hES culture medium after culturing for 1 hour.

Embodiment 3

[0058] Morphological Observation and Characteristic Determination of Human ES Cells after Thawing

[0059] 1. Method

[0060] (a) Morphological observation of human ES cells after recovery

[0061] Use a phase contrast microscope to observe the survival and differentiation of cells after recovery. The colony formation rate was calculated after 8 days of culture.

[0062] Colony formation rate = number of undifferentiated ES clones after thawing / total number of ES clones before cryopreservation × 100%

[0063] (b) Determination of characteristics of human ES cells after thawing

[0064] (1) Alkaline phosphatase (AKP) staining

[0065] According to the method of Xu et al. (Xu X, Tsung HC, Yan Yc.Establishment and differentialin of murine EG cell lines derived from primordial germcells. Acta Biologiae Expert Sinica 1999; 32:251-63), using NBT and BCIP (Sigma company) as Substrates and stains for AKP activity identification.

[0066] (2) Immunocytochemistry

[0067] SSEA-4,...

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Abstract

This invention discloses a method to thaw freezing embryonic stem cells by menas of adding a thawing solution containing carbonhydrates at a definite concentration during the thawing process. The mehod is characterized in that it can dramatically improve the survival rate(upto around 42%) of thawing stem cells and has a good repeatability.

Description

technical field [0001] The invention relates to the field of biology, and more specifically relates to a method for resuscitating cryopreserved embryonic stem cells, which can significantly improve the survival rate of the resuscitated stem cells. Background technique [0002] In 1998, scientists from the University of Wisconsin in the United States successfully cultured and expanded human embryonic stem (hES) cells in vitro, and confirmed that they had the potential to differentiate into three human germ layer cells (ThomsonJ.Itskovitg-Eldor J, Shapiro S.et .al, Embryonic stem cell lines derived from human blastocysts Science 1998; 282: 1145-1147.), drawing people's attention to the potential value of hES cells in clinical applications. [0003] Through the directional 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 disea...

Claims

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Application Information

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Patent Type & Authority Applications(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