Human Erythroblasts Generated in Vitro Remain Functional with a Normal Karyotype 8 Years after Cryopreservation: Implications for Ex Vivo Generated Erythroid Transfusion Products.
Authors: Sanchez Massimo , Leblanc Amanda , Mancini Annalisa , Masiello Francesca , Tirelli Valentina , Migliaccio Giovanni , Migliaccio Anna Rita , Najfeld Vesna , Whitsett Carolyn 
Published: April 2020

Abstract
The safety and adequacy of the blood supply is threatened by natural disasters, social and political events, epidemics, and emerging infections. During shortages, frozen blood is used to supplement the blood supply. Current regulations allow red blood cells to be stored frozen up to ten years; however, the shelf-life of such products is limited once blood is thawed. Cultured human erythroid cells derived in vitro from either fresh or cryopreserved CD34+ cells or peripheral blood mononuclear cells potentially represent an alternative source of erythrocytes for transfusion. However, it is unknown if normal erythroid cells undergoing ex-vivo expansion with growth factors will remain functional or develop genetic rearrangements in culture making them unsuitable for transfusion. We have compared the proliferative and differentiation potential of human erythroblasts obtained in culture from the peripheral blood mononuclear cells (PBMC) of adult donors. This analysis included freshly expanded erythroblasts as well as erythroblasts cryopreserved and stored for short (1 month) and long (8 years) periods.
PBMC from four volunteer blood donors were prepared using gradient-density centrifugation and cryopreserved in DMSO in June 2000. One months later, 2x107 PBMC from one of the donors were thawed and cultured under conditions that allow massive ex vivo generation of erythroblasts (HEMA culture, Migliaccio et al Blood Cells Mol Dis2002;28:169-80). These cultures were stimulated with recombinant hSCF (50ng/mL), hGM-CSF (1ng/ml), hIL3 (1U/mL), hEPO (1U/mL) and contained dexamethasone and estradiol (each 10−6 M). Twenty million PBMC from the three additional donors were thawed and cultured under HEMA conditions in 2008. In all the three cases, the day 9 cultures contained an average of 10x107 cells, 95% of which were erythroid by CD36 and CD235a staining. These day 9 cells were either cultured for 4 additional days or cryopreserved (&gt;10 individual vials per donor containing 5x106 each). Cells were subcultured and maintained either under HEMA conditions (to assess their proliferation ability) or stimulated with EPO alone (5U/ mL) (to assess maturation). In May 2008, aliquots of the erythroblasts obtained from all donors were thawed and cultured again and amplification and differentiation potential of the freshly expanded and thawed cells were compared. Cells thawed after few months or 8 years of cryopreservation gave similar results and the data were pooled. The viability of the erythroblasts after thawing was 60–70%. After 4 days under HEMA conditions, both freshly expanded and cryopreserved erythroblasts doubled in numbers and retained an immature erythroid phenotype (CD36highCD235alow). On the other hand, in cultures containing EPO alone, the erythroblasts remained constant in number but progressed to a mature CD36posCD235ahigh phenotype. The results are summarized in the following table:
Proliferation and Maturation Profile of Fresh and Cryopreserved Human Erythroblasts
Fold Increase Phenotype CD36highCD235alow CD36highCD235ahigh Fresh cells HEMA culture 2 53% 40% EPO alone 1 15% 80% Thawed Cells HEMA culture 2 46% 36% EPO alone 1 5% 90%
The eight-years cryopreserved erythroblasts expanded in culture were also cytogenetically evaluated. Karyotype and multicolor FISH analyses demonstrated a normal 46,XY karyotype with no obvious genomic rearrangements. To determine whether cells carried any known in utero leukemic genomic rearrangements, interphase FISH studies were performed for TEL/ETV6-AML1, MLL, 5q31 (EGR1) and 7q31 loci. In 800 evaluated interphase nuclei, all loci were present in disomy. This data indicates that human erythroblasts obtained in culture can be efficiently cryopreserved, remain functional in culture and do not acquire chromosomal abnormalities detectable by multicolor FISH analysis. These observations suggest that cultured erythroblasts should be further evaluated to determine if they represent a more suitable long term storage product than cryopreserved mature red blood cells.