Long-term storage does not alter functionality of in vitro generated human erythroblasts: Implications for ex vivo generated erythroid transfusion products

Background: Cultured human erythroid cells derived in vitro may represent alternative transfusion products. It is unknown, however, if these ex vivo expanded erythroid cells remain functional or develop genetic abnormalities after storage. Study design and methods: Using mononuclear cells from four adult blood donors, erythroblasts were generated ex vivo in expansion cultures supplemented with stem cell factor, interleukin-3, erythropoietin (EPO), dexamethasone, and estradiol. The viability and in vitro function of freshly expanded or short (1-2 months)- and long (8 years)-term-stored erythroblasts cryopreserved in dimethyl sulfoxide were compared. Erythroblast function was defined as ability to proliferate in expansion media and mature in response to EPO. Cell number was determined manually and expressed as fold increase. Viability was assessed by trypan blue and propidium iodide exclusion. Maturation was evaluated by morphologic analyses and CD36/CD235a expression profiling. Cytogenetic evaluation included karyotype and multicolor fluorescence in situ hybridization analyses. Results: Equivalent numbers (>80%) of erythroblasts were viable after short- and long-term storage. Freshly expanded and short- and long-term-stored erythroblasts equally doubled in number (fold increase, 2.4) retaining an immature phenotype (23% of the cells were CD36 ^highCD235a ^neg) when cultured for 4 days under expansion conditions. The numbers of freshly expanded and short-term-stored erythroblasts that matured when exposed for 4 days to EPO were also similar (approx. 22% of the cells became CD36 ^negCD235a ^high). In spite of the massive amplification, ex vivo generated erythroblasts demonstrated a normal (46,XY) karyotype with no obvious genomic rearrangements. Conclusion: Ex vivo expanded erythroblasts remain functional and genetically normal after long-term storage.