A Simple and Efficient Method of Slow Freezing for Human Embryonic Stem Cells and Induced Pluripotent Stem Cells

• Published in: Methods in molecular biology (Clifton, N.J.) Vol. 1341; p. 15
• Main Authors: Imaizumi, Keitaro, Iha, Momoe, Nishishita, Naoki, Kawamata, Shin, Nishikawa, Shinichi, Akuta, Teruo
• Format: Journal Article
• Language: English
• Published: United States 01.01.2016
• Subjects: Cell Culture Techniques - methods; Cell Proliferation; Cryopreservation - economics; Cryopreservation - methods; Cryoprotective Agents - pharmacology; Dimethyl Sulfoxide - pharmacology; Ethylene Glycol - pharmacology; Freezing; Human Embryonic Stem Cells - cytology; Human Embryonic Stem Cells - drug effects; Humans; Hydroxyethyl Starch Derivatives - pharmacology; Induced Pluripotent Stem Cells - cytology; Induced Pluripotent Stem Cells - drug effects; Pronase - pharmacology; Staining and Labeling - methods; Streptomyces griseus - enzymology; Human embryonic stem cells; Ethylene glycol; Slow freezing; Cryopreservation; Dimethyl sulfoxide; Human induced pluripotent stem cells; Pronase; Hydroxyethyl starch
• ISSN: 1940-6029
• DOI: 10.1007/7651_2015_211

Protocols available for the cryopreservation of human embryonic stem (ES) and induced pluripotent stem (iPS) cells are very inefficient and laborious compared to those for the cryopreservation of murine ES/iPS cells or other general cell lines. While the vitrification method may be adequate when working with small numbers of human ES/iPS cells, it requires special skills and is unsuitable when working with large cell numbers. Here, we describe a simple and efficient method for the cryopreservation of hES/hiPS cells that is based on a conventional slow freezing method that uses a combination of Pronase/EDTA for Stem™ and CP-5E™ [final concentrations: 6 % hydroxyethyl starch, 5 % DMSO, and 5 % ethylene glycol in saline]. CP-5E™ is highly effective for the cryopreservation of small cell clumps produced by hES/hiPS colony detachment in the presence of Pronase and EDTA (Pronase/EDTA for Stem™, a formulation containing multiple digestive enzymes from Streptomyces griseus). This novel method would be quite useful for large-scale hES/iPS cell banking for use in clinical applications.