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Ch 8.Immunohistoch / immunology
Ch 10.GC/MS, NMR and Proteomics
Feeder-Free Culture of Human Embryonic Stem Cells
http://stemcells.nih.gov/research/nihresearch/scunit/feederfree.htm
MEF Conditioned
medium:
Plate MEFs (p4 or p5) onto gelatin-coated plates at a density of 1x106 cells/10cm culture dish.
The next day, wash
cells with PBS and add 10ml normal hESC medium containing 4ng/ml bFGF. This is collected the next day and each subsequent day for
7 days.
CM may be stored frozen for several months.
Before use, add 4ng/ml bFGF to the medium and filter.
Culture
of hESCs:
Coat 6-well tissue culture plates (Falcon) with 5% Matrigel in DMEM/F12 overnight at 4¡ãC using 1.5ml Matrigel suspension
per well.
Allow the Matrigel plate to warm to room temperature in the hood for approx 30min prior to use. DO NOT WARM IN INCUBATOR.
Remove hESC cells from MEFs using collagenase treatment and sediment as usual. Aspirate the Matrigel and plate triturated colonies
in conditioned medium supplemented with an additional 4ng/ml bFGF or in normal hESC medium containing 100ng/ml bFGF.
Feed cells
every day up to 7 days. Note: Colonies can grow bigger and more densely on Matrigel without losing morphology than on MEFs.
Passaging:
Wash
cells once with PBS and incubate at 37¡ãC with 1ml/well of 2mg/ml dispase in DMEM/F12.
Colonies should detach intact within
10¨C15 minutes upon tapping smartly on the side of the plate. DO NOT SCRAPE.
Remove the colonies in dispase to a 15ml tube and
rinse wells with an additional 1ml/well growth medium.
Sediment and wash twice more as usual.
Triturate VERY gently as
colonies grow flat and dissociate readily in dispase. Note: Small colonies and single cells do not survive well on Matrigel especially
in 100ng/ml bFGF only.
Plate as usual on fresh Matrigel
Feeder-free growth of undifferentiated human embryonic stem cells
...
www.nature.com/nbt/journal/v19/n10/full/nbt1001-971.html
Growing feeder-independent embryonic stem cells
http://baygenomics.ucsf.edu/protocols/comp1/feederES.html
Development
of defined medium for mouse, monkey and human ES cell ...
et al. 2005). In this simple serum-free adherent. monolayer culture condition
in the absence of feeder. cells, LIF promotes ES cell proliferation and self- ...
www.soc.nii.ac.jp/jsaae/zasshi/WC6_PC/paper601.pdf
Millipore - Feeder-free ES cell culture, Serum-free ES cell ...
Feeder-free ES cell culture, Serum-free ES cell culture and ESCRO -
Using ESCRO complete system protocol.
www.millipore.com/cellbiology/cb3/feederfreeprotocol
Protocols