Variations in culture pH affect the cloning efficiency and differentiation of progenitor cells in ex vivo haemopoiesis

Abstract

Summary.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.

Induction of anion exchanger-1 translation and its opposite roles in the carcinogenesis of gastric cancer cells and differentiation of K562 cells

“…Functional AE1 mediates the Cl À /HCO 3 À exchange across the plasma membrane, and so pH change could impact the differentiation of erythoid lineage cells. The importance of functional AE1 in the regulation of differentiated erythroid cells is supported by the funding that pH is a potent modulator and plays a role in the maintenance of the bone marrow microenvironment and development of erythroid cells (Endo et al, 1994;McAdams et al, 1997).…”

Storage of unprocessed G-CSF-mobilized whole blood in a modified Leibovitz's L15 medium preserves clonogenic capacity for at least 7 days

“…At 22°C, glucose, which is abundantly present in CPDA-1 (2.9 g/dl), is metabolized rapidly into lactate and CO 2 , resulting in a drop in pH. Deviation of culture pH has been shown to affect both the cloning efficiency and differentiation potential of progenitor cells, 36,37 and indeed in separate experiments with mononuclear cells, a pH of 6.8 or lower negatively influenced CFU-GM recovery. Therefore, we hypothesized that either a strong buffer or an alternative to glucose, resulting in less lactate production, should be used.…”

Three-StageEx VivoExpansion of High-Ploidy Megakaryocytic Cells: Toward Large-Scale Platelet Production

“…Our previous work demonstrated the importance of pH in Mk maturation and consistently showed that culturing primary Mks at higher pH in the range from 7.2 to 7.6 increased production of high-ploidy ( > 4N) Mks. 9,29 pH also modulates the differentiation of granulocytes, 41,59,60 erythrocytes, 60,61 and BM stromal cells. 62 Hematopoietic stem cells reside close to the bone surface and migrate toward the sinuses during maturation (Fig.…”