title: Variations in culture pH affect the cloning efficiency and differentiation of progenitor cells in ex vivo haemopoiesis.
abstract: Haemopoietic cultures may experience pH variations of as much as 0.5 units depending on culture duration and cell density. Since pH is a potent modulator of cellular proliferation and differentiation, we examined its effects on the performance of both semisolid and liquid haemopoietic 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 cultures). 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/Epo) 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 the expansion and cloning efficiency of progenitors in ex vivo liquid cultures. Cloning efficiencies of PB BFU-E in the erythroid combination were 9-fold higher at low pH (7.1) when compared to high pH (7.6). A small pH increase of 0.2 units over physiological values consistently produced significant reductions (42-85%) in cloning efficiencies for all cell types and cytokine combinations tested. Methylcellulose cultures 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 differentiation of erythroid progenitors (BFU-E) progressively increased as pH was increased from 6.95 (no colonies detected) to 7.4 (maximum colonies detected), to 7.6 (maximum haemoglobin content). Methylcellulose cultures using PB CD34+ cells exhibited similar patterns to the MNC cultures. We conclude that even small variations in pH substantially affected the performance of human haemopoietic cultures. The erythroid lineage was particularly sensitive, with its extent of differentiation increasing with increasing pH. PB progenitors are more sensitive to pH variations than CB progenitors.
authors.affiliation: Department of Chemical Engineering, Northwestern University, Evanston, Illinois 60208-3120, U.S.A.
authors.forename: T A
authors.initials: TA
authors.lastname: McAdams
authors.forename: W M
authors.initials: WM
authors.lastname: Miller
authors.forename: E T
authors.initials: ET
authors.lastname: Papoutsakis
mesh_terms: D018952:Antigens, CD34; D002454:Cell Differentiation; D002478:Cells, Cultured; D002999:Clone Cells; D003470:Culture Media; D016207:Cytokines / Q000494:pharmacology; D006412:Hematopoietic Stem Cells / Q000166:cytology*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008747:Methylcellulose / Q000737:chemistry
chemical_list: D018952:Antigens, CD34; D003470:Culture Media; D016207:Cytokines; D008747:Methylcellulose