This video demonstrates how to maintain the growth of human embryonic stem cells (hESCs) in feeder cell-free conditions and how to continuously passage hESCs in feeder cell-free conditions. Confirmation of hESC pluripotency grown in feeder cell-free conditions by immunofluorescence microscopy is also demonstrated. Part 3 of 3.
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Method Article
From MEFs to Matrigel 3: Passaging hESCs from Matrigel onto Matrigel
In This Article
Summary
Abstract
This video demonstrates how to maintain the growth of human embryonic stem cells (hESCs) in feeder cell-free conditions and how to continuously passage hESCs in feeder cell-free conditions. Confirmation of hESC pluripotency grown in feeder cell-free conditions by immunofluorescence microscopy is also demonstrated.
Protocol
Splitting hESCs from Matrigel to Matrigel
Usually a confluent 6-well plate of hESCs on Matrigel can be split 1:3 to 1:5 to another Matrigel plate, with the wells becoming confluent again 4-5 days after splitting.
- CM and Matrigel plates are prepared as described above before splitting.
- On the day of splitting, wash each well for splitting with 1×PBS, pH7.4, add 1ml of 1mg/ml Dispase (dilute 5mg/ml stock solution with DMEM/F12 media) to each well, and incubate at 37°C for 3 min.
- Wash each well three times with 1×PBS, pH7.4, add 1ml of ES media without bFGF, use a cell scraper to scrape colonies o....
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Discussion
This series of 3 videos demonstrates how to grow human embryonic stem cells (hESCs) on mouse embryonic fibroblast (MEF) feeder cells (video 1), how to passage them to Matrigel feeder cell-free plates (video 2), and how to maintain hESCs by passaging in Matrigel feeder cell-free conditions (video 3). Numerous prior studies showed that maintenance of viable, undifferentiated hESCs requires culture on inactivated MEF feeder cells. However, for many experiments, a pure population of hESCs free of feeder cell contamination is.......
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Acknowledgements
Human embryonic stem cell studies in the Teitell lab are supported by a California Institute for Regenerative Medicine (CIRM) seed grant RS1-00313. We thank members of the Broad Center of Regenerative Medicine and Stem Cell Research at UCLA, especially Dr. Amander Clark, Dr. Jerome Zack, and members of the UCLA Broad Institute Stem Cell Core Facility for their support of our studies.
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Materials
| Name | Company | Catalog Number | Comments | |
|---|---|---|---|---|
| Knockout Serum Replacer (KSR) | Reagent | GIBCO, by Life Technologies | 10828-028 | |
| DMEM/F12 | Reagent | GIBCO, by Life Technologies | 11330-057 | |
| Non-essential Amino Acids | Reagent | GIBCO, by Life Technologies | 11140-050 | |
| GlutaMax | Reagent | GIBCO, by Life Technologies | 35050-061 | |
| DMEM | Reagent | GIBCO, by Life Technologies | 11995-065 | |
| FBS | Reagent | Clontech Laboratories | 631107 | |
| L-glutamine | Reagent | GIBCO, by Life Technologies | 25030-081 | |
| BME | Reagent | Fisher Scientific | BP176-100 | |
| bFGF | Reagent | R&D Systems | 233-FB-025 | |
| Collagenase IV | Reagent | GIBCO, by Life Technologies | 17104-019 | |
| Dispase | Reagent | Stem Cell Technologies | 17105-041 | |
| Penicillin / Streptomycin | Reagent | GIBCO, by Life Technologies | 15140-122 | |
| Gelatin | Reagent | Chemicon International | ES-006-B | |
| Matrigel | Reagent | BD Biosciences | 354277 | |
| Oct-4 antibody | Reagent | Santa Cruz Biotechnology, Inc. | SC-9081 | |
| anti-h/mSSEA-4 Phyc–rythrin Conjugated Mouse IgG3 | Reagent | R&D Systems | FAB1435P | |
| FITCI-conjugated antirabbit IgG | Reagent | Jackson ImmunoResearch | 715-095-152 |
References
- Thomson, J. amesA., Itskovitz-Eldor, J. oseph, Shapiro, S. anderS., Waknitz, M. ichelleA., Swiergiel, J. enniferJ., Marshall, V. ivienneS., Jones, J. effreyM. Embryonic Stem Cell Lines Derived from Human Blastocysts . Science. 282, 1145-1147 (1998).
- Xu, C. hunhui, Inokuma, M. argaretS., Denham, J. errod, Golds, K. athaleen, Kundu, P. ratima, Gold, J. osephD., Carpenter, M. elissaK.
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