Bioreactor Expansion of Human Bone Marrow: Comparison of Unprocessed, Density-Separated, and CD34-Enriched Cells

Abstract

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.

Ex Vivo Expansion of Cord Blood

“…[24][25][26][110][111][112][113][114] These systems were designed to allow larger volumes as well as to provide improved nutrient delivery and gas exchange. The secreted products of mature granulocytes and macrophages are toxic to progenitors, 115 and mature macrophages can directly damage cultured stroma and hematopoietic pro-genitors.…”

Clinical-scale human umbilical cord blood cell expansion in a novel automated perfusion culture system

“…16,17,43 Because the accessory cell composition of CB cultures is different from BM cultures (ie lacking an adherent stroma), the presence of mRNA encoding a number of growth factors was assessed by RT-PCR (Figure 7). Surprisingly, transcripts for most growth factors tested (G-CSF, GM-CSF, IL-1␤, IL-6, LIF, KL, FL, Tpo, Growth factor mRNA in bone marrow and cord blood expansion cultures.…”

“…Most of the recent literature on CB cell expansion has focused on the culture of CD34-enriched cells or subsets thereof. However, perfusion bioreactor systems have been proven capable of expanding non-enriched cells from BM [16][17][18] and mPB, 19 due to their ability to support high density accessory cell populations. 17,20 In fact, in the CB transplant setting, the potential advantages of CD34-enrichment prior to expansion, such as tumor purging and T cell depletion, are less relevant.…”

“…The ability to expand cells without CD34-enrichment would therefore provide an advantage, because considerable processing effort, cost and cell loss could be avoided. 16,21 Although perfusion culture parameters for BM cell expansion have been extensively optimized 17,20,22,23 and clinically tested, [24][25][26] potential differences between CB and BM cells warrant the study of several culture parameters for CB cell expansion within the perfused culture environment. In addition, the likely clinical use of cryopreserved and banked CB samples dictates the study of thawed cells in such culture systems.…”

Ex vivo expansion of hematopoietic cells from umbilical cord blood for clinical transplantation

“…There was a 73% loss of LTC-IC in the CD34 ϩ sample at the same time that total cell number expanded 269-fold. These authors concluded that CD34 ϩ cells can be expanded regardless of the extent of enrichment [32]. In fact, Xiao et al demonstrated that expansion from as little as one CD34 ϩ cell derived from UCB has been shown to occur with maintenance of self-renewal capacity for at least 2 weeks.…”