Large-scale expansion of human stem and progenitor cells from bone marrow mononuclear cells in continuous perfusion cultures

Abstract

There is a growing consensus that clinical practice in the areas of bone marrow (BM) transplantation and gene therapy will rely on the ex vivo expansion of hematopoietic cells. We report here on the development of continuously perfused culture systems (bioreactor systems) that expand human stem and progenitor cells from BM mononuclear cell (MNC) populations obtained without cell enrichment. In three separate experiments, 10 bioreactors were each inoculated with 3 x 10(7) BM MNC from patients undergoing marrow harvest for autologous transplantation. At various times thereafter (between days 6 and 16), duplicate bioreactors were harvested and cells were analyzed. The bioreactors contained three cell populations that were analyzed separately: nonadherent cells; cells that were loosely adherent to the endogenously formed stromal layer; and an adherent cell layer that required trypsinization for removal. Total cell numbers increased continuously to give an overall 10-fold (range, 8- to 11-fold) expansion by day 14. The adherent stromal layer significantly expanded to more than 2 x 10(7) cells, but remained less than 6% of the total cell population. Colony-forming unit-granulocyte-macrophage (CFU-GM) numbers expanded 21-fold (range, 12- to 34-fold) by day 14 and, because this expansion was greater than that for total cells, CFU-GM were enriched by as much as fourfold by day 14. Burst-forming unit-erythroid (BFU-E) numbers peaked earlier than did CFU-GM numbers, with a 12-fold (range, 6- to 18-fold) expansion obtained on day 8. In contrast to CFU- GM, which were predominantly nonadherent, BFU-E were more evenly distributed between the three cell populations. Stem cell activity was measured by the long-term culture-initiating cell (LTC-IC) limiting dilution assay. The number of LTC-IC per reactor consistently increased with time in all cultures, resulting in a 7.5-fold (range, 3.4- to 9.8- fold) expansion. In summary, more than 3 billion cells, containing 12 million CFU-GM, were reproducibly generated from the equivalent of a 10 to 15 ml BM aspirate. These data indicate that small numbers of BM MNC can be readily expanded ex vivo in continuous perfusion cultures, and that such ex vivo expansion may have direct applications in clinical and experimental BM transplantation.

Serum supplement, inoculum cell density, and accessory cell effects are dependent on the cytokine combination selected to expand human HPCs ex vivo

“…Methods of expanding HPCs ex vivo include the co‐cultivation of these cells by using stroma–cell contact, 10 stroma–cell noncontact, 11 and stroma‐free (i.e., suspension) cultures 12 . Stroma‐based culture systems offer the advantage of providing an environment that tends to mimic the interactions occurring during in vivo hematopoiesis.…”

Stem cell bioengineering strategies to widen the therapeutic applications of haematopoietic stem/progenitor cells from umbilical cord blood

“…Interestingly, Koller and colleagues have described an enrichment of the immature populations after culturing BM MNCs (Koller et al ., ), suggesting that complete CD34 + selection might not be strictly necessary to obtain net cell expansion, which also results in significant cell losses (Berenson et al ., ; Koller et al ., ). More recently, and in agreement with these results, highly dynamic culture events were reported regarding CD34 modulation in UCB HSCs‐MSC co‐cultures, prior to cell division, affecting cell cycle and proliferation status in culture and ultimately the final haematopoietic cell yield, and pointing to the need to establish a balance between the cell recovery upon purification and the stem/ progenitor cell proliferative potential of cultured cells (Andrade et al ., ).…”

“…This type of bioreactor system (Figure f), non‐mechanically stirred, was first used for the expansion of BM and UCB MNCs by Koller and colleagues (Koller et al ., , ; Palsson et al ., ), reporting a 20–25‐fold expansion of BM MNCs over a 2 week period (Palsson et al ., ) and a three‐fold increase in LTC‐ICs for UCB in a 7 day expansion (Koller et al ., ). Perfusion chambers, due to their low‐flow nature, better mimic the BM microenvironment by allowing a low‐shear environment that promotes the concurrent development of stroma, stem cells, progenitors, precursors and mature cells from a MNC fraction of BM or UCB (Koller et al ., ). Radial flow‐type chambers provide the most uniform environment, due to the absence of walls parallel to the flow path, which create slow flowing regions.…”

Cellular engineering for the production of blood components

“…Methods have been available for some time to increase the number of myeloid cells. Combinations of growth factors and cytokines can increase myeloid precursors ex vivo by 20‐ to 60‐fold after about 14 days 19–22 . This new myeloid cell blood component could be a supplement in marrow transplantation by providing myeloid cells during the initial period of neutropenia after transplantation.…”

“…Culture systems to produce large numbers of lymphocytes or immune cells ex vivo are discussed below. Attempts to increase the number of pluripotent HPCs ex vivo in this manner for transplantation without inducing cell maturation have not yet been successful.Although laboratory culture systems have been able to expand the number of progenitors ex vivo up to a 60‐fold increase in the number of CD34+ cells or CFU–GM colonies, this has involved different degrees of cell maturation 19–24 . This has not lead to the hoped‐for result of providing complete hematopoietic reconstitution from small numbers of originally collected cells.…”