Bioreactor expansion of human bone marrow: comparison of unprocessed, density-separated, and CD34-enriched cells.
Scale-up of human hematopoietic cultures was previously described in continuously perfused systems with bone marrow mononuclear cells (BMMNC), yielding expansion of both progenitors and long-term culture-initiating cells (LTC-IC). We report here on the use of these systems for expansion of unprocessed whole BM cells (WBMC) and CD34-enriched cells. Density separation recovered 84% of CFU-GM and 65% of LTC-IC from WBMC. Subsequent CD34 selection recovered 17% of CFU-GM and 48% of LTC-IC from the MNC fraction. The unabsorbed (CD34-depleted) fraction contained 37% of CFU-GM and 38% of LTC-IC, accounting for most of the lost cells. WBMC, BMMNC, and CD34-depleted cells were each placed directly in bioreactors, whereas CD34-enriched cells were placed in bioreactors containing preformed irradiated stroma. After 14 days, an average of 3.82 x 10(7) (12.7-fold expansion), 3.54 x 10(7) (11.8-fold), 2.85 x 10(7) (9.5-fold), and 3.65 x 10(7) (1298-fold) total cells were obtained from bioreactors inoculated with WBMC, BMMNC, CD34-depleted, and CD34-enriched cells on stroma, respectively. These cultures yielded 1.64 x 10(5) (27.9-fold expansion), 1.69 x 10(5) (14.3-fold), 8.36 x 10(4) (13.0-fold), and 1.91 x 10(5) (41.4-fold) CFU-GM each, respectively. Cell recovery and expansion data were combined to determine the number of expanded CFU-GM obtained per ml of BM aspirate, allowing direct comparison of performance between the four culture inocula. WBMC generated 3.76 x 10(6) CFU-GM per ml BM aspirate, whereas MNC resulted in 1.42 x 10(6) CFU-GM. CD34-enriched cells (on irradiated stroma) gave 7.00 x 10(5) CFU-GM per ml BM aspirate, whereas CD34-depleted cells generated 4.97 x 10(5) CFU-GM. The high productivity from WBMC cultures was studied further and was found to be reproducible at different inoculum densities. WBMC cultures had elevated levels of endogenous EGF and PDGF production, which may have been responsible for the more extensive stromal development observed. Flow cytometric analysis showed that the final culture composition, with respect to T and B lymphocytes, monocytes, granulocytes, and erythrocytes, was not significantly affected by the inoculum composition and in all cases was comprised of multiple lineages. Therefore, each step in cell purification resulted in the loss of primitive and accessory cells, which in turn resulted in a net decrease in the number of expanded cells obtained per ml BM aspirate.
Koller
M R
MR
Aastrom Biosciences, Inc., Ann Arbor, MI 48106, USA.
Manchel
I
I
Newsom
B S
BS
Palsson
M A
MA
Palsson
B O
BO
D018952:Antigens, CD34 / Q000032:analysis*; D001853:Bone Marrow / Q000276:immunology; D001854:Bone Marrow Cells; D002452:Cell Count; D018929:Cell Culture Techniques / Q000295:instrumentation*; D019070:Cell Lineage; D002469:Cell Separation; D005434:Flow Cytometry; D006412:Hematopoietic Stem Cells / Q000166:cytology; D006801:Humans
D018952:Antigens, CD34