Background: An in vitro erythropoiesis assay is a powerful tool for investigating red blood cell (RBC) development and diseases of the erythroid lineage. Most assays, however, failed in either proliferation or terminal differentiation. Here two liquid cultures (LCs) for in vitro generation of RBCs from peripheral blood CD34+ cells were compared.
Study design and methods: Granulocyte-colony-stimulating factor-mobilized CD34+ cells were cultured for 16 days in a two-phase LC (2P-LC; Days 1-8, stem cell factor [SCF], erythropoietin [EPO], insulinlike growth factor [IGF]-1, and steroids; Days 9-16, EPO and insulin) and for 21 days in a three-phase LC (3P-LC; Days 1-7, SCF, thrombopoetin, and Flt3-ligand; Days 8-14, SCF, EPO, and IGF-1; Days 15-21, EPO and IGF-1). Maturation was analyzed by flow cytometry (CD36, CD71, glycophorin A [GPA]) and microscopy.
Results: In the 2P-LC, cell numbers increased from 0.5 x 10(6) to 25.7 x 10(6) +/- 15.1 x 10(6) cells per mL. More than 95 percent were GPA+ and showed morphologic characteristics of normoblasts (52 +/- 15%) and enucleated reticulocytes (43 +/- 18%). In the 3P-LC, a higher overall proliferation to 55.7 x 10(6) +/- 37.7 x 10(6) cells per mL was achieved (p < 0.05). This was also accompanied by a high degree of normoblasts (36 +/- 16%) and reticulocytes (48 +/- 24%). The amount of GPA+ cells was slightly lower (88.4 +/- 16.4%), associated with a significantly higher contamination by nonerythroid cells (15.8 +/- 19.3% vs. 3.9 +/- 2.9%, p < 0.05).
Conclusion: Both LCs were able to generate fully matured RBCs and represent powerful tools for fundamental research in erythroid development and diseases targeting the erythroid lineage. A slightly higher proliferation was achieved in the 3P-LC. This was associated with a limited homogeneity due to more nonerythroid cells, however. Therefore the 2P-LC is favored, also saving additional culture days and growth factors.