Abstract Ex-vivo expansion of primary human hematopoietic progenitor and stem cells is an important technique for clinical applications in cancer and gene therapies. The importance of stromal cells for the cultivation of hematopoietic cells has been shown in numerous studies. Therefore we developed a cultivation system for immobilized co-culture of hematopoietic and stromal cells 1) and for screening purposes a miniaturized parallel system, the so-called miniaturized loop-bioreactor 2) . These cultivation systems are based on the mimikry of the natural compartment of the hematopoieses, the bone marrow. The in vivo -like co-cultivation with stromal cells obtains important biological effects like cell contact and the production of cytokines and extracellular matrix. The immobilization of the cells on macroporous collagen microcarriers simulates the three-dimensional structure of bone marrow. And the optimal feeding of the cells is ensured by the continuous perfusion in the reactor. The miniaturized loop-bioreactor consists of a special stainless steel insert and a magnetic stirbar that are placed in a conventional 12-well culture plate. The microcarriers are placed as a fixed bed in this insert and are flown through with medium by the agitation of the magnetic stirbar. It is possible to investigate in this system twelve parallel experiments. By co-culturing CD34 + cells purified from umbilical cord blood with the irradiated murine stromal cell line S1/S1 in the miniaturized loop-bioreactor we have optimized different parameters for the co-cultivation. The experiments were performed over 7 days under standardized conditions with serum containing medium (IMDM) supplemented with IL-3, TPO, FLT-3-ligand, SCF and anti TGF-beta. In first experiments different cell densities on the carrier were analyzed. The optimal cells density ranged from 2.5* 10 4 to 1* 10 5 CD34 + cells per reactor system. The cell ratio between CD34 + and stromal cells plays an important role in the support of hematopoiesis. The optimal ratio of 1:10 between these cell types was determined by expansion of the CAFC. Another relevant cultivation parameter is the feeding rate. Frequent feeding improved significantly the expansion of MNC, whereas the highest rate of 1/2 reactor volume per day, showed the best results. Based on this parameter optimization we have cultivated CD34 + cells in co-culture with S1/S1 (ratio 1:10) and without stroma, both with a feeding rate of 1/2 medium exchange per day for two weeks in the miniaturized loop-reactor. The positive effect of the stromal cells is demonstrated by a strong increase in the expansion of MNC (454 fold vs 180 fold stroma free) and CFC (26 fold day 10). The stroma containing system generated also the greater CAFC output compared to the stroma free system (4.5 fold vs 2 fold). These results indicate that the novel miniaturized loop-reactor is a very suitable system for the cultivation of primary human hematopoietic cells and for the screening of various culture parameters. Using this system the positive effect of a stromal based and frequently fed cultivation strategy was proven. 1) P. Meissner, B. Schroder, C. Herfuth, M. Biselli. Development of a fixed bed bioreactor for the expansion of human hematopoietic progenitor cells. Cytotechnology, Vol. 30, pp. 227-234, 1999. 2) Schmidt, S., Jelinek, N., Biselli, M.; patient application DE: 198 22 050 A1.