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Controlled-rate freezing of human ES cells
Carol B Ware1, Angelique M Nelson, C Anthony Blau
1Department of Comparative Medicine, University of Washington, Seattle, WA 98195-7190, USA. cware@u.washington.edu
Biotechniques
|July 16, 2005
Summary
Improving human embryonic stem cell (hESC) cryopreservation is crucial. A novel slow controlled-rate freezing technique significantly enhances hESC survival rates from 1% to 20-80%.
Area of Science:
- Stem Cell Biology
- Cryobiology
- Reproductive Medicine
Background:
- Human embryonic stem cells (hESCs) are vital for regenerative medicine.
- Poor survival rates (around 1%) hinder hESC cryopreservation and application.
- Standard freezing methods are insufficient for effective hESC preservation.
Purpose of the Study:
- To develop and optimize a slow controlled-rate freezing technique for hESCs.
- To improve the survival rate of cryopreserved hESCs.
- To establish a reproducible method for hESC cryopreservation.
Main Methods:
- Utilized a slow controlled-rate freezing technique adapted from mammalian embryo cryopreservation.
- Employed dimethyl sulfoxide (DMSO) as a cryoprotectant.
- Investigated critical factors including ice crystal seeding, freeze rate (0.3-1.8°C/min), and rapid thawing.
Main Results:
- Achieved significant improvements in hESC survival rates, ranging from 20% to 80%.
- Survival was assessed by quantifying surviving colony number and colony diameter.
- Identified optimal conditions: ice crystal seeding at -7°C to -10°C and rapid thawing in room temperature water.
Conclusions:
- Slow controlled-rate cooling is an effective method for cryopreserving hESCs.
- This technique offers a rapid, simple, and reproducible approach to enhance hESC survival.
- Improved cryopreservation facilitates broader use of hESCs in research and therapy.