Stem cell

[admin]

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[miswar]

Hello Everyone,

Please help me, I need protocol for cordblood & adult stem cell processing for storage.

Thankyou verymuch.

Miswar Fattah

[bbsun]

Dear Miswar,

From whole cord blood, just dilute the blood 10ml : 20ml of 1xPBS inside a 50ml centrifuge tube, and underlay with 14ml of 1.077g/cm3 Ficol-Paque (from Amersham (now GE Biosciences I believe)). Centrifuge at 1300 RPM (400G's) at room temp for about half an hour, and then harvest the thin white ring of cells sitting at the interphase, which will be your mononuclear cells. Pool the harvest in as few tubes as you can and spin them down hard at 3000 RPM or 1000G's, and then resuspend the cell pellet in a cold buffer containing around 0.5% BSA, 1mM EDTA, in 1xPBS. Then you can use magnetic selection kits from such company as Miltenyi Biotec or Stemcell Technologies to purify the CD34+ /hematopoetic progenitor cells from the MNCs. Please refer to their websites and kits for protocols. Then you can freeze the purified cells down in fetal bovine or calf serum containing a final DMSO concentration of 5 - 10%. Adult stem cells such as from bone marrow can be processed in the same way as from cord blood. If you do not want to select the cells yet, you can also freeze the entire raw material down. You can spin the cord blood down first, collect the supernatant (autologous plasma) and use that in place of animal serum with final 5 - 10% of DMSO to freeze the whole blood cells. You can revive the whole blood later and purify the progenitor cells the same way as above. Good luck!

[admin]

thank you that is a great protocol bbsun

[nikomaster]

Nice info, thanks