HOW to culture bone marrow monoclear cells(BMMNCs)? - (Oct/14/2005 )

hi,everyone

now i begin to culture bone marrow mononuclear cell (BMMNCs),

METHODs: Bone marrow was obtained from a population of screened normal donors . Marrow was aspirated from the posterior iliac crest and placed into sterile heparinized tubes. In the laboratory, approximately 25 ml of heparinized bone marrow was diluted with an equal volume of GIBCO BRL
HBSS without Ca++ or Mg++ and carefully layered over GIBCO BRL NYCOPREP™ 1.077 in 50-ml tubes. Samples were then centrifuged at 1500 × g for 20 min at room temperature. After
centrifugation, the band of bone marrow mononuclear cells (BMMC) at the interface was
removed using a pipette. The cells were washed once with HBSS without Ca++ or Mg++ and
counted by hemocytometer, using trypan blue dye exclusion to measure viability.


and then.....i don't know how to culture them , which medium can i chose?........

thanks,fengqiouhuang
fengqiouhuang@sina.com

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our lab also use BM cell, and isolation of MNC is quite similar although there are some differences in reagents we use.

anyway, we usually further isolate mesenchymal stem cells from MNC, and culture on culture flask.

culture condition is following

cover the entire floor of culture flask with 0.2%gelatin solution, and remove excess

set the flask aside for 10-15 min, so gelatin will dry.

and now, flask is ready to use, so simply use medium (we use MSC GM from Cambrex) to resuspend cell and put in flask.

but if you are just doing culture of MNC, you probably dont need to use MSC-GM medium. you can probably use IMDM or DMEM base media with FBS supplimentation.

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thx!
i see.but there are some other questions.

though mediums such as RPMI1640,MEM,IMDM,DMEM-LG and DMEM-HG exist more or less shortcomings, we uaually used them to culture MSCs, especially prefer to DMEM-LG,why you use medium ( MSC GM from Cambrex)?
and then you had said that if i am just doing culture of BMNCs, I don’t need to use MSC-GM medium,and IMDM or DMEM base medium with FBS supplementation is probably better ,Why?

thx,fengqiouhuang
fengqiouhuang@sina.com

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well, first of all, MSC-GM is for mesenchymal stem cell medium,
so if you are not trying to culture stem cell specifically, MSC-GM is just a too expensive medium

IMDM and DMEM with FBS can also used for MSC, but so far our lab using MSC-GM, maybe because MSC-GM has differentiation inhibitor in it (I think that I heard so) or just maybe becuase it is working so far...??

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ok!thx,now i begin to culture MSCs for transfection with recombinated adenovirus type 5,are you ?could you tell me the conditions, or convey the protocol to me (fengqiouhuang@sina.com).thx a lot!

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I dont have specific protocol for that, but I did the transduction of MSC with retrovirus before
when I did that, transduced cell grew relatively slower than the negative control, so just keep the cell concentration retatively high. then cells are always happy.

Good luck

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Thanks,the same to you !

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