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1David Geffen School of Medicine, University of California, Los Angeles
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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.
Zhang, J., Khvorostov, I., Teitell, M. From MEFs to Matrigel 3: Passaging hESCs from Matrigel onto Matrigel. J. Vis. Exp. (16), e832, doi:10.3791/832 (2008).
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.
Detection of pluripotency markers by immunofluorescence microscopy
Immunofluorescence microscopy is used to examine the expression of hESC pluripotency markers Oct-4 and SSEA-4 during culturing.
Human embryonic stem cell receipts
ES media (Embryonic Stem cell media):
DMEM/F12 - 400ml
Knockout Serum Replacer - 100ml
Non-Essential Amino Acids - 5ml
200mM GlutaMax / BME Solution - 5ml
Penicillin / Streptomycin - 5ml
Mix well and store final hESC Culture Media in 500ml bottles at 4°C. Good for a maximum of two weeks.
MEF Culture Media:
DMEM - 1000ml
FBS - 100ml
Non-Essential Amino Acids - 10ml
Glutamine - 10ml
Penicllin / Streptomycin - 10ml
Mix well and store final MEF Culture Media at 4°C.
200mM Glutamax / BME Solution:
Glutamax - 100ml
β-mercaptoethanol (Stock solution at 14.3M) - 140 µ1
Mix two solutions well and then aliquot 10ml portions. Keep aliquots at -20°C.
bFGF Solution (final concentration of stock: 10 micrograms/ml):
bFGF (FGF2) - 50 µg
0.1% BSA in 1xPBS, pH 7.4 - 5ml
Dissolve 50 micrograms bFGF in 5ml 0.1% BSA to make a 10 µg/ml stock. Aliquot into 500 µl portions. Store at -80°C.
Collagenase IV Solution (1mg/ml):
Collagenase IV - 50mg
DMEM/F12 Media - 50ml
Add the collagenase IV powder to a 50ml falcon tube. In the hood, mix in 50ml of sterile DMEM/F12 media. Vortex the solution for < 1 min. to make sure the powder is dissolved. In the hood, pass through a 0.22 µm filter into a new sterile 50ml falcon tube. This solution is good for a maximum of 2 weeks stored at 4°C.
Dispase Solution (1mg/ml):
Dispase 5mg/ml - 2ml
DMEM/F12 Media - 8ml
Aliquot commercial 5mg/ml Dispase solution and store at -20°C. Dilute dispase to 1mg/ml using DMEM/F12. This solution can be kept for 1 week at 4°C.
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 required. To achieve this goal, we have demonstrated how to passage hESCs from MEF feeder plates to feeder cell-free Matrigel plates and how to maintain and continuously passage hESC colonies in feeder cell-free conditions. By following this protocol, a small amount of MEF feeder cell contamination could still exist at passage one on a Matrigel plate, but this contaminant is usually easy to visualize. At Matrigel passage two and beyond, no MEF contamination is usually found, leaving a pure population of hESCs for experiments. 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 during culture.
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.
|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|
|L-glutamine||Reagent||GIBCO, by Life Technologies||25030-081|
|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|
|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|
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