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Beschreibung:
The ex vivo expansion of hematopoietic cells is an important technique for many medical applications. The cultivation concept in this study was the ex vivo simulation of the human bone marrow by co-culture of hematopoietic and stromal cells in a fixed-bed bioreactor . Thereby the cells are immobilized on macroporous collagen microcarriers. A miniaturized loop-reactor was established for screening purposes. For the full supply with primary CD34 + - cells a cord blood bank was built up. In preliminary investigations two stromal cellines were characterized and the harvest of immobilized cells established by enzymatic lysis of the carriers. The relevant parameters for the co-culture were optimized in the miniaturized loop- reactor. In this system the mononuclear cells (MNC) were expanded up to 454 fold, the colony-forming-cells (CFC) up to 26 fold and the cobblestone-area-forming cells (CAFC) up to 18 fold. In this system the SCm-mouse repopulating cells (SRC) were maintained in contrast to a parallel cultivation in a suspension system. This demonstrates the positive effect of the stromal based, immobilized culture compared to the suspension culture of this cells. Based on the optimization in the screening system the hematopoietic cells were successfully expanded in the fixed-bed bioreactor. The co-culture with the stromal cellline SVSl and the cytokines IL-3, TPO, SCF and FL led to the best results. In this system the MNC were expanded maximal 103 fold, the CFU-GM 114 fold and the CAFC 15 fold. These data correspond the medical yardstick demonstrating the clinical relevance of the results presented in this study.