Ex vivo expansion of hematopoietic cells is important for applications such as cancer treatment, gene therapy, and transfusion medicine. While cell culture systems are widely used to evaluate the biocompatibility of materials for implantation, the ability of materials to support proliferation of primary human cells in cultures for reinfusion into patients has not been addressed. We screened a variety of commercially available polymer (15 types), metal (four types), and glass substrates for their ability to support expansion of hematopoietic cells when cultured under conditions that would be encountered in a clinical setting. Cultures of peripheral blood (PB) CD34+ cells and mononuclear cells (MNC) were evaluated for expansion of total cells and colony-forming unit-granulocyte monocyte (CFU-GM; progenitors committed to the granulocyte and/or monocyte lineage). Human hematopoietic cultures in serum-free medium were found to be extremely sensitive to the substrate material. The only materials tested that supported expansion at or near the levels of polystyrene were tissue culture polystyrene, Teflon perfluoroalkoxy, Teflon fluorinated ethylene propylene, cellulose acetate, titanium, new polycarbonate, and new polymethylpentene. MNC were less sensitive to the substrate materials than the primitive CD34+ progenitors, although similar trends were seen for expansion of the two cell populations on the substrates tested. CFU-GM expansion was more sensitive to substrate materials than was total cell expansion. The detrimental effects of a number of the materials on hematopoietic cultures appear to be caused by protein adsorption and/or leaching of toxins. Factors such as cleaning, sterilization, and reuse significantly affected the performance of some materials as culture substrates. We also used PB CD34+ cell cultures to examine the biocompatibility of gas-permeable cell culture and blood storage bags and several types of tubing commonly used with biomedical equipment. While many of the culture bag materials gave satisfactory results, all of the tubing materials severely inhibited total cell and CFU-GM expansion. Taken together, our results show that many materials approved for blood contact or considered biocompatible are not suitable for use with hematopoietic cells cultured in serum-free medium. As hematopoietic cultures are scaled up for a variety of clinical applications, it will be essential to carefully examine the biocompatibility of all materials involved.

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.

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Summary. Physiological parameters such as pH and oxygen tension probably play significant roles in the regulation of haemopoiesis in the bone marrow microenvironment, but these roles have yet to be characterized in detail. We have found that changes in culture pH (0·2 units) can cause significant changes in the culture composition of mature cells and colony-forming cells (CFCs), especially in the presence of erythropoietin (Epo). Peripheral blood (PB) CD34þ cells cultured at different pH values (7·15-7·6) were characterized using total cell counts, colony assays, morphological analysis, haemoglobin staining, flow cytometry, immunocytochemical staining, and Western blots. Cultures performed at high (7·6) pH contained greater numbers of haemoglobin-positive and band-3-positive cells, and acquired these erythroid differentiation markers sooner than standard (7·35) and low (7·1) pH cultures. Flow cytometry using CD71 and CD45RA antigens also indicated that erythroid differentiation proceeds faster at high pH and is blocked at an intermediate stage by low pH. Morphological data confirmed that high pH cultures had been shifted towards late-stage erythroid compartments as compared to low and standard pH cultures. These findings have important implications both in elucidating the regulatory role of pH in the bone marrow microenvironment and for the design of in vitro systems to study the development of erythroid cells. Keywords: pH, ex vivo expansion, erythroid differentiation.It has been recognized since early this century that pH buffering is an important and complex control system in mammals. Cells subjected to different pH environments exhibit distinct responses in terms of differentiation and protein secretion patterns. In haemopoietic cells, changes in intracellular pH have been associated with activation of lymphocytes, neutrophils and platelets (Ozaki, 1992). Also, the proliferation of, and erythropoietin (Epo) secretion by, murine macrophages have been shown to be pHdependent, with optimal values at higher than physiological pH (7·6-8·0) (Rich, 1988). Although the connection between pH and haemopoietic cells is more difficult to make in vivo because of interactions with CO 2 , O 2 and bicarbonate, there are suggestions of a link between blood pH and the generation and function of erythroid cells. One such condition is end-stage renal failure, in which both acidosis (low blood pH) and anaemia (shortage of red blood cells) are primary symptoms (Segal et al, 1988). In addition, Yang (1992) and Yang et al (1995) have demonstrated that some respiratory failure patients who exhibit acidosis also have impaired function of the primary ion channel found in erythroid cells.In order to provide insight into these pathophysiological conditions and to further understand the effects of culture pH on the erythroid lineage, we have conducted experiments to examine in detail the effects of culture pH on the ex vivo differentiation of erythroid lineage cells. Since bone marrow pH in vivo is likely to be as low as 7·1, and dramati...

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