Preclinical assessment of cord blood hematopoietic stem cell expansion

by Alexey Bersenev on January 27, 2010 · 1 comment

in clinical lab, cord blood, hematopoietic, protocols

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A recent study, published in Nature Medicine a week ago, proved the principal that hematopoietic stem/progenitor cells from cord blood (CB) unit could be successfully expanded for clinical use. Besides general excitement about these clinical results, I’d like to highlight that this study provided us a clinically-grade algorithm for validation of any new CB hematopoietic stem cell (HSC) expansion protocol.

I picked the most important points from the study and made up step-by-step protocol that I’m sharing them with you here. So, If you work on a new protocol for HSC expansion for potential clinical use, you should follow all steps, described below:

    1. Optimize of in vitro expansion cell culture parameters: plastic, cytokines, serum-free expansion media, cell density, re-feeding…
    2. Evaluate of HSC (CD34+/CD38-) or progenitors (CD34+ total) purity by flow cytometry and total cell number – before and after expansion.
    3. Count fold expansion in vitro based on expression HSC surface markers by flow cytometry (at least CD34+/CD38- cells or even further) and determine optimal duration of expansion.
    Engraftment of human HSC in the study was evaluated as more than 0.5% positive bone marrow cells for human CD45 at least 9 weeks after transplantation. Reconstitution should be multilineage (at least myeloid and lymphoid).
    4. Do functionality test in vitro – colony assay (not shown in the study)
    5. Assess of in vivo repopulation ability in xenotransplant models (NOD/SCID; NOG).
    Groups – expanded, noncultured, fresh cells.
    6. Calculate frequency of expanded HSC in vivo – limiting dilution transplantation assay.
    7. Evaluate of self-renewal function of long-term expanded HSC in serial xenotransplant models.
    8. Validate the reproducibility of protocols in cGMP conditions. Toxicology tests.
    9. Apply for clinical trial.

{ 1 comment… read it below or add one }

Graham Hobba September 23, 2011 at 4:11 am

Hi Alexey,

Really good list of steps!

If people are looking for cGMP recombinant xeno-free human albumin, transferrin and growth factors to augment their serum-free medium, then please contact me to discuss (or follow the above URL).

Graham – GHOB@novozymes.com

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