简介：
Overview
This article demonstrates the proper techniques for thawing and freezing human embryonic stem (hES) cells. It emphasizes the importance of careful handling during these processes to ensure cell viability and successful culture.
Key Study Components
Area of Science
- Cell Biology
- Stem Cell Research
- Cell Culture Techniques
Background
- Human embryonic stem cells (hES) are sensitive to freezing and thawing stresses.
- Proper techniques are crucial for maintaining cell viability.
- Thawing and freezing procedures are routine in hES cell culture.
- These techniques allow for long-term storage and later use of hES cells.
Purpose of Study
- To provide a detailed protocol for thawing hES cells from liquid nitrogen stocks.
- To outline the freezing process for long-term storage of hES cells.
- To ensure researchers can efficiently handle hES cells without compromising their viability.
Methods Used
- Thawing hES cells in a 37°C water bath.
- Centrifugation to form a cell pellet and resuspension in culture medium.
- Plating cells on a feeder layer of irradiated mouse embryonic fibroblasts.
- Freezing cells in cryogenic vials using a controlled rate in an isopropanol container.
Main Results
- Successful thawing and plating of hES cells resulted in viable colonies.
- Proper freezing techniques preserved cell integrity for future use.
- Cells showed slow proliferation post-thaw, requiring careful monitoring.
- Detailed protocols were established for both thawing and freezing processes.
Conclusions
- Adhering to the outlined protocols ensures high viability of hES cells.
- Careful handling during thawing and freezing is essential for successful cell culture.
- These techniques are vital for researchers working with hES cells.
What temperature is used for thawing hES cells?
Thawing is performed in a 37°C water bath.
How are hES cells frozen for storage?
Cells are frozen in cryogenic vials using an isopropanol freezing container at -80°C.
What is the purpose of the feeder layer?
The feeder layer supports the growth and attachment of hES cells during culture.
How long should hES cells be incubated after thawing?
Cells should be incubated overnight at 37°C and 5% CO2.
What should be done if cells appear transparent after thawing?
This is normal; they may take a few days to establish visible colonies.
What is the typical density for freezing hES cells?
Typically, one well of cells from a six-well plate is used per cryogenic vial.
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