Quantitative Analysis of Cultured Erythroid Progenitors in Liquid System

Han, Jin Yeong; Kim, Tae Gyeom; Kim, Kyeong Hee; Kim, A Seong; Bae, Hae Rahn; Suh, Duk Joon; Kang, Seol Mi; Park, Joo In; Kim, In Hoo

Abstract

BACKGROUND: Hemopoiesis and erythropoiesis have been studied mainly in immortalized cell lines and semisolid medium. But cell lines do not represent normal erythropoiesis, besides, in semisolid medium the cells are immobilized that it is difficult to do additional immunologic, biochemical, and molecular biologic experiments. In the present study we used a two-phase liquid culture system to isolate and quantify erythroid progenitors from peripheral blood and cord blood.
METHODS
Peripheral and cord blood were obtained from three healthy donors and three full-term deliveries, respectively. Mononuclear cells were separated by density gradient centrifugation and were cultured in the first phase at a density of 5x106/mL in alpha- minimal essential medium ( -MEM). After 5~7 days of incubation at 37degrees C in an atmosphere of 5% CO2 with extra humidity, the nonadherent cells were harvested and recultured in the original volume of -MEM containing 10ng/mL stem cell factor and 1U/mL erythropoietin (EPO). Cellular morphology was observed by preparing cytocentrifuge slides stained with Wright Giemsa. On days 8, 10, 12, and 16 of the second phase, hemoglobin (Hb)- containing cells were counted on hemocytometer after staining with acid benzidine and glycophorin A-positive erythroid cells were scored by a flow cytometer.
RESULTS
Pronormoblasts first started to appear on days 4~5 in the secondary culture. On day 10 basophilic normoblasts could be seen and on days 12~14 orthochromatic normoblasts were present. Both from peripheral and cord blood the maximum number of benzidine and glycophorin A-positive cells were achieved after 10 days and the total erythroid cell yield was approximately 1x106/mL.
CONCLUSION
Using two-phase liquid culture, erythroid cell yield reached 1x106/mL both from peripheral and cord blood. In addition, this culture system permits the study of the effect of various culture conditions and components without terminating the culture, therefore it might provide us a useful experimental tool for studying pathogenesis and therapeutic modalities in genetic erythroid disorders as well as erythroid cell development.