Passaging HuES Human Embryonic Stem Cell-lines with Trypsin.
October 12th, 2006
259.2K views
In this video we demonstrate how our lab routinely passages HuES human embryonic stem cell lines with trypsin. Brought to you by JoVE.
Overview
This article demonstrates the routine passaging technique for HuES human embryonic stem cell lines using trypsin. Proper passaging is critical for maintaining healthy, undifferentiated, and karyotypically normal human embryonic stem cell cultures. The protocol emphasizes careful handling to minimize the risk of karyotypic abnormalities.
Key Study Components
Area of Science
- Stem cell biology
- Cell culture techniques
- Human embryonic stem cells
Background
- Human embryonic stem cells (hESCs) are prone to acquiring karyotypic abnormalities during extended culture.
- Maintaining undifferentiated and genetically stable hESC cultures is essential for research and therapeutic applications.
- Passaging methods can influence cell health and genetic stability.
- Trypsinization is a common method for dissociating and passaging stem cells.
Purpose of Study
- To demonstrate a standardized protocol for passaging HuES human embryonic stem cell lines with trypsin.
- To highlight steps that help maintain healthy and karyotypically normal stem cell cultures.
- To provide guidance on minimizing the risk of introducing genetic abnormalities during passaging.
Methods Used
- Washing expanding hESC cultures with PBS to remove residual media and debris.
- Overlaying cells with warm 0.05% Trypsin-EDTA for up to five minutes.
- Gently dislodging cells using a serological pipette.
- Collecting and mixing the cell suspension with HuES media, followed by gentle centrifugation.
- Removing the inactivated trypsin/media mixture and resuspending cells in pre-warmed HuES media.
- Calculating an appropriate split ratio (typically 1:10 to 1:20) and replating onto feeder cell monolayers.
- Leaving newly seeded plates undisturbed for 48 hours, then changing media daily.
Main Results
- Proper passaging supports the maintenance of undifferentiated and healthy HuES cell cultures.
- Minimizing single-cell suspensions reduces the risk of karyotypic abnormalities.
- Routine use of feeder layers and careful handling are essential for optimal culture conditions.
- Consistent media changes and undisturbed incubation promote cell recovery and growth.
Conclusions
- Careful passaging with trypsin is vital for preserving the quality of HuES human embryonic stem cell lines.
- Avoiding over-trypsinization and single-cell suspensions helps maintain genetic stability.
- Following standardized protocols ensures reproducibility and reliability in stem cell research.
Frequently Asked Questions
Why is it important to avoid creating a single-cell suspension during passaging?
Creating a single-cell suspension increases the risk of introducing karyotypic abnormalities in human embryonic stem cell cultures.
What is the recommended split ratio for passaging HuES cells?
The recommended split ratio is generally between 1:10 and 1:20, depending on culture conditions and cell density.
How long should trypsin be left on the cells during passaging?
Trypsin should be left on the cells for up to five minutes to gently dissociate them without over-digesting.
What type of feeder cells are used for replating HuES cells?
A monolayer of irradiated mouse embryonic fibroblast feeder cells is used to support the growth of HuES cells.
How often should the media be changed after passaging?
After the initial 48-hour undisturbed period, the media should be changed every day to maintain optimal culture conditions.
Why is gentle centrifugation used during the protocol?
Gentle centrifugation helps collect the cells without causing mechanical stress or damage, preserving cell viability and quality.
