From MEFs to Matrigel 3: Passaging hESCs from Matrigel onto Matrigel
Summary
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.
Abstract
Protocol
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…
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.
Materials
| Material Name | Type | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| Knockout Serum Replacer (KSR) | Reagent | Gibco | 10828-028 | |
| DMEM/F12 | Reagent | Gibco | 11330-057 | |
| Non-essential Amino Acids | Reagent | Gibco | 11140-050 | |
| GlutaMax | Reagent | Gibco | 35050-061 | |
| DMEM | Reagent | Gibco | 11995-065 | |
| FBS | Reagent | Clontech | 631107 | |
| L-glutamine | Reagent | Gibco | 25030-081 | |
| BME | Reagent | Fisher | BP176-100 | |
| bFGF | Reagent | R&D Systems | 233-FB-025 | |
| Collagenase IV | Reagent | Gibco | 17104-019 | |
| Dispase | Reagent | Stem Cell Technologies | 17105-041 | |
| Penicillin / Streptomycin | Reagent | Gibco | 15140-122 | |
| Gelatin | Reagent | Chemicon | ES-006-B | |
| Matrigel | Reagent | BD Biosciences | 354277 | |
| Oct-4 antibody | Reagent | Santa Cruz Biotechnology | SC-9081 | |
| anti-h/mSSEA-4 Phycoerythrin Conjugated Mouse IgG3 | Reagent | R&D Systems | FAB1435P | |
| FITCI-conjugated antirabbit IgG | Reagent | Jackson ImmunoResearch Laboratories. Inc. | 715-095-152 |
References
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