Sources of hematopoietic cells for bone marrow transplantation are limited by the supply of compatible donors, the possibility of viral infection, the painful bone marrow harvest procedure, autologous (patient) marrow that is depleted from prior therapy or has leukemic involvement, and the small number of progenitor cells in umbilical cord blood. An in vitro system to amplify hematopoietic progenitor cells could increase the number of patients eligible for autologous transplantation, allow use of cord blood hematopoietic cells to repopulate an adult, reduce the amount of bone marrow required for transplantation, and allow collection of peripheral blood stem and progenitor cells to replace the bone marrow harvest process. Present methods for long-term bone marrow (hematopoietic) culture (LTHC) on stromal layers in flasks lack a well-controlled growth environment. The proposed research will improve the prospects for use of LTHC in bone marrow transplantation by identifying conditions that allow for rapid expansion of cord blood hematopoietic progenitors in a perfusion bioreactor without the need for stromal cells. Eliminating the stromal layer will eliminate the need for a stomal cell donor, reduce culture time, and simplify culture conditions. Existing perfusion chambers suitable for culture of cells on a stromal layer will be modified for operation without stromal cells by adding a nylon mesh to better retain nonadherent cells. Analysis of the bone marrow environment suggests that further improvements in progenitor cell expansion may be obtained by manipulating the culture oxygen (O2) tension, pH, and temperature (T), and by developing more effective combinations of cytokines for LTHC. Progenitor cell expansion is enhanced at reduced O2 tensions. The effects of culture in medium saturated with 2.5-10% O2 will be examined via assays for colony-forming cells and the primitive long-term culture- initiating cells (LTC-IC). Mechanisms responsible for enhanced hematopoiesis at reduced O2 tension will be investigated by monitoring levels of oxidizing species and cytokines in the medium. The cytokine mixture IL-3/IL-6/stem cell factor, which has proved successful in expanding primitive cells, will be used as the base formulation to test the effectiveness of other cytokine combinations which will include both positive (IL-1,G-CSF) and negative (TNF-alpha, TGF-Beta) regulators of hematopoiesis. These cytokine combinations will be tested first in a static suspension culture assay. The best combinations will be then examined under perfusion conditions. While 33 degrees C is generally considered better than 37 degrees C for static LTHC, this may be due to less rapid nutrient depletion at 33 degrees C in these infrequently- fed cultures. We will compare perfusion cultures at 35 degrees C and 37 degrees C against the control 33 degrees C culture for effects on progenitor cell expansion. pH effects have not been examined in LTHC even though extracellular and/or intracellular pH are important regulators of proliferation, function and differentiation for a variety of cell types. Cultures at three pH values from 6.9 to 7.8 will be compared against the control culture at pH 7.35.
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