VARIATIONS IN CULTURE PH AFFECT THE CLONING EFFICIENCY AND DIFFERENTIATION OF PROGENITOR CELLS IN EX-VIVO HEMATOPOIESIS

Haemopoietic cultures may experience pH variations of as much as 0.5 u nits depending on culture duration and cell density. Since pH is a pot ent modulator of cellular proliferation and differentiation, we examin ed its effects on the performance of both semisolid and liquid haemopo ietic cultures. Culture pH was found to have substantial effects both on progenitor cloning efficiency (as measured in liquid cultures) and on progenitor cell differentiation (as measured in methylcellulose cul tures). Liquid cultures were conducted with both peripheral blood (PB) mononuclear cells (MNCs) and cord blood (CB) MNCs using growth factor combinations that promote either erythroid expansion (IL-3/IL-6/SCF/E po) or granulocyte/macrophage expansion (IL-3/IL-6/SCF/G-CSF/GM-CSF). Reduced pH was found to have either a positive or neutral effect on th e expansion and cloning efficiency of progenitors in ex vivo liquid cu ltures. Cloning efficiencies of PB BPU-E in the erythroid combination were 9-fold higher at low pH (7.1) when compared to high pH (7.6). A s mall pH increase of 0.2 units over physiological values consistently p roduced significant reductions (42-85%) in cloning efficiencies for al l cell types and cytokine combinations tested. Methylcellulose culture s conducted using CB MNC and PB MNC indicated that differentiation of CFU-GM into progeny was optimal between pH 7.2 and 7.4. The differenti ation of erythroid progenitors (BFU-E) progressively increased as pH w as increased from 6.95 (no colonies detected) to 7.4 (maximum colonies detected), to 7.6 (maximum haemoglobin content). Methylcellulose cult ures using PB CD34(+) cells exhibited similar patterns to the MNC cult ures. We conclude that even small variations in pH substantially affec ted the performance of human haemopoietic cultures. The erythroid line age was particularly sensitive, with its extent of differentiation inc reasing with increasing pH. PB progenitors are more sensitive to pH va riations than CB progenitors.