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Cryopreservation of Human Embryonic Stem Cell-Derived Retinal Pigment Epithelial Cells at the Optimal Stage
Published on: November 3, 2023
An efficient and easy-to-use cryopreservation protocol for human ES and iPS cells
Hossein Baharvand1, Ghasem Hosseini Salekdeh, Adeleh Taei
1Department of Stem Cells and Developmental Biology, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran. Baharvand@RoyanInstitute.org
Nature Protocols
|March 6, 2010
Summary
This study presents a simple, efficient cryopreservation protocol for human embryonic stem (ES) and induced pluripotent stem (iPS) cells using a Rho-associated kinase (ROCK) inhibitor. The method enhances cell cloning efficiency, offering a robust platform for stem cell applications.
Area of Science:
- Stem Cell Biology
- Cryobiology
- Regenerative Medicine
Background:
- Human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) are crucial for regenerative medicine.
- Effective cryopreservation is essential for maintaining the viability and functionality of these valuable cell types.
- Existing cryopreservation protocols can be complex and may result in reduced cell survival and cloning efficiency.
Purpose of the Study:
- To develop a simple and highly efficient cryopreservation protocol for feeder-free human ESCs and iPSCs.
- To evaluate the impact of Rho-associated kinase (ROCK) inhibitor (Y-27632) on cell survival and cloning efficiency during cryopreservation.
- To establish a robust and broadly applicable method for long-term storage and expansion of human pluripotent stem cells.
Main Methods:
- Utilized a feeder-free system for cryopreservation of dissociated human ESCs and iPSCs.
- Incorporated the ROCK inhibitor Y-27632 into pre- and post-thaw culture media.
- Investigated the effect of adding Y-27632 to Matrigel on cloning efficiency.
- Assessed technical simplicity, cloning efficiency, and post-thawing recovery.
Main Results:
- The addition of ROCK inhibitor Y-27632 significantly enhanced cloning efficiency in both pre- and post-thaw media.
- Supplementing Matrigel with Y-27632 further improved cloning efficiency.
- The developed protocol demonstrated high technical simplicity and superior post-thawing passaging capabilities compared to existing methods.
- The entire process, excluding overnight incubation, takes approximately 2 hours.
Conclusions:
- This novel cryopreservation protocol offers a simple, efficient, and robust method for feeder-free human ESC and iPSC storage.
- The use of ROCK inhibitor Y-27632 is key to improving cloning efficiency and cell survival.
- This protocol provides a valuable platform for the widespread application and future expansion of human pluripotent stem cell research and therapies.
