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 2 of 3.
hESC culture daily maintenance
Splitting hESCs from MEFs onto Matrigel
Usually a confluent 6-well plate of hESCs on MEFs can be split 1:2 to 1:3 onto Matrigel 6-well plates, with the wells becoming confluent again 4-5 days after splitting.
This video demonstrates how to passage hESCs from MEF plates to feeder cell-free Matrigel plates. Note that the colony density on Matrigel is higher than the density of colonies by usual splitting on MEFs. Immunofluorescence staining and microscopy or flow cytometry for hESC pluripotency markers, such as Oct-4 and SSEA-4, are needed to confirm maintenance of hESCs in an undifferentiated state in feeder-free culture conditions.
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.
Material Name | Typ | 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 |