need help for cord blood mesenchymal stem cell culture - (Apr/18/2007 )

hi everyone
i m now starting my work on cord blood stem cell culture
eventually the culture seem like worikng
no growth at all
i harvested mononuclear cells from cord blood by ficoll paque separation
then i seeded the cells(50x10⁶ and 100 X 10⁶) in DMEM high glucose with 10% of preselected MSC ( stemcell technologies) and let it to adhere for 4-5 day.
can any one help in this
thanks

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well, first of all, is that human MSC or some other animal?
I work with human MSC from CB as well as BM and other organs, so I only know human MSC.
and as far as I concern, if you have good population of MSC, you dont need 4-5 days to let cell to adhere
MSC will adhere within 24 hrs usually (I usually consider the cells which do not adhere within 24 hr are dead)
so you might not have enough MSC population in your isolated mononuclear cells
Also, I am not familier to the preselected MSC from stem cell technologies, but DMEM+ 10% FBS should be sufficient as medum.
one more thing is you can coat the bottom of culture flask with 0.2% gelatin or fibronectin for better adherence of cell.

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thanks
that is a human cord blood serum
actually i do have quick a number of cells adhere
but all are monocyte( round big cell)
i am now planning to do preselection/ enrichment method for isolation
do u have any idea?
i mean other than magnetic bead and FACs sorting?
and do u think icreasing the FCS concentration to 20% will be working?
hope to hear u soon

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well, I guess there should be some way to preselect MSC, but we normally use magnetic beads with STRO-1 antibody.
( we do magnetic sorting right after the ficol)
and then we calacterize the isolated cell population with FACS.

I heard that there are some people who just culture mononuclear cells in adherent condition and remove suspension cells (lymphocyte and HSC etc) to enrich, but I am not sure the reliablity of this methods.

do you have any particular reason that you dont want to use beads separation?

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em.....
well we dun have the particular instrument here
and i would like to try different method for the project
well u do help me in some way
i will try enrichment method
if anything will contact u for help
is that ok for u?
thanks alot

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Hi,

One common problem with growing mesenchyml cells from cord blood is the age of the cord blood. Many people have great difficulty in growing mesenchymal cells if the cord blood is more than 6 hours old. How fresh is your sample?

Feel free to contact me if you would like to discuss this further.

Diane Miller
Product Manager
StemCell Technologies

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thanks for the info
some of the cord blood are really fresh(1-4)
but some are bit old(more then 6)
i was using the ACD-B completement inactivation blood collection tube
and almost all the samples had some dark clumping tissue in it
the cells do grow in the culture
but no sign of MSC
almost al R monocyte
and if there was some Fibroblast like cell
they jz din proliferate
do u have any gd solution for that..?
hear u soon

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