ASH 1998 b



Cytomatrix212 West Cummings Park Woburn, MA 01801 *Phone:* 781-939-0995		*Study of Cytokines Effects on Stem Cells Using Novel Bioreactor*

		N. Banu, J. Bagley, M. Rosenzweig, H. Kim, and M. Pykett Cytomatrix, Cambridge, MA 023139; New England Regional Primate Center, Harvard Medical School, Boston, MA, USA
		Studies aimed at the in vitro expansion of hematopoietic progenitor cells (HPCs) have faced tradeoffs between increase in cell number and loss of long term repopulating ability. We have developed a long term bone marrow bioreactor culture system that resembles the bone marrow microenvironment in which HPCs are cultured in an inert, three dimensional, porous biomatrix termed Cellfoam^TM. Previous studies have shown that short term culture of CD34⁺ cells in Cellfoam^TM improved the maintenance and multipotency of hematopoietic stem cells compared to cells cultured on bone marrow stroma (BMS) or fibronectin-coated plastic dishes. In this study, we examined the effects of low concentrations of cytokines (picogram/ml and nanogram/ml) including SCF, IL-3, Flk-2/Flt-3 ligand and Il-6, alone or in combination, on the maintenance, preservation and multipotency of CD34⁺ cells cultured for 3 to 6 weeks in Cellfoam^TM, compared to culture in BMS or plastic cultures. Analysis of CD45⁺ cell yield showed that CD45⁺, CD45⁺34⁺ and CD45^-34⁺38^- cell yields are uniformly higher in Cellfoam^TM cultures compared to BMS or plastic cultures. In single cytokine supplementation experiments, of 32 possible comparisons of cell number vs. single cytokine-tie datum points in Cellfoam^TM and BMS cultures, higher CD45⁺34⁺ and CD45⁺34⁺38^- cells were observed in Cellfoam^TM in 25 (78%). With combinations of cytokines, all 16 comparisons of cell number vs. cytokine-concentration-time-datum points were higher in Cellfoam^TM (100%). Similar findings were observed in comparison with plastic cultures. The function of cultured cells was evaluated in standard methylcellulose CFU-assays. The data demonstrated that im most of the cytokine supplemented cultures, Cellfoam^TM led to greater increases in colony activity than BMS or plastic cultures. Analysis of CAFC and LTC-IC activity also demonstrated that primitive progenitors were maintained to a greater extent in cytokine-supplemented Cellfoam^TM cultures than in parallel BMS or plastic cultures. In conclusion, the culture of HPCs in Celfoam^TM for extended period may benefit form the addition of low levels of IL-3 (picogram/ml) SCF (nanogram/ml) and Flk-2/Flt-3 ligand resulting in high yields of cell that are enriched for primitive progenitors.