A hanging drop culture method to study terminal erythroid differentiation.
OBJECTIVE
To design a culture method allowing the quantitative and qualitative analysis of terminal erythroid differentiation.


METHODS
Primary erythroid progenitors derived either from mouse tissues or from human umbilical cord blood were differentiated using hanging drop cultures and compared to methylcellulose cultures. Cultured cells were analyzed by FACS to assess differentiation.


RESULTS
We describe a practical culture method by adapting the previously described hanging drop culture system to conditions allowing terminal differentiation of primary erythroid progenitors. Using minimal volumes of media and small numbers of cells, we obtained quantitative terminal erythroid differentiation within two days of culture in the case of murine cells and 4 days in the case of human cells.


CONCLUSIONS
The established methods for ex vivo culture of primary erythroid progenitors, such as methylcellulose-based burst-forming unit-erythroid (BFU-E) and colony-forming unit-erythroid (CFU-E) assays, allow the detection of committed erythroid progenitors but are of limited value to study terminal erythroid differentiation. We show that the application of hanging drop cultures is a practical alternative that, in combination with clonogenic assays, enables a comprehensive assessment of the behavior of primary erythroid cells ex vivo in the context of genetic and drug-induced perturbations.
Gutiérrez
Laura
L
Department of Cell Biology and Genetics, Erasmus University Medical Center, Rotterdam, The Netherlands.
Lindeboom
Fokke
F
Ferreira
Rita
R
Drissen
Roy
R
Grosveld
Frank
F
Whyatt
David
D
Philipsen
Sjaak
S
D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods; D002454:Cell Differentiation / Q000502:physiology*; D002478:Cells, Cultured; D015672:Erythroid Precursor Cells / Q000166:cytology / Q000502:physiology*; D004920:Erythropoiesis / Q000502:physiology*; D005434:Flow Cytometry / Q000379:methods; D051379:Mice; D008822:Mice, Transgenic