TY - JOUR
T1 - Large-scale in vitro production of red blood cells from human peripheral blood mononuclear cells
AU - Heshusius, Steven
AU - Heideveld, Esther
AU - Burger, Patrick
AU - Thiel-Valkhof, Marijke
AU - Sellink, Erica
AU - Varga, Eszter
AU - Ovchynnikova, Elina
AU - Visser, Anna
AU - Martens, Joost H. A.
AU - von Lindern, Marieke
AU - van den Akker, Emile
N1 - Funding Information: The authors are grateful to Wilson Wolf Manufacturing (Saint Paul, MN) for providing the G-Rex bioreactors. The authors also thank Rob van Zwieten, Martijn Veldthuis, and Jeffrey Berghuis (Department of Blood Cell Research, Sanquin) for the technical assistance and data acquisition regarding the HPLC data and deformability assays, and the Central Facility of Sanquin for its assistance regarding flow cytometry analysis. This work was supported by grants from The Netherlands Organization for Health Research and Development (grants ZonMw-TOP, 40-00812-98-12128 [S.H.] and ZONMW-TAS, 40-41400-98-1327 [P.B., M.T.-V., and E.S.]), Landsteiner Foundation for Blood Transfusion Research (grant LSBR:1141) (E.A. and E.H.), and Sanquin (grant PPOR:15-30) (A.V.). Funding Information: This work was supported by grants from The Netherlands Organization for Health Research and Development (grants ZonMw-TOP, 40-00812-98-12128 [S.H.] and ZONMW-TAS, 40-41400-98-1327 [P.B., M.T.-V., and E.S.]), Landsteiner Foundation for Blood Transfusion Research (grant LSBR:1141) (E.A. and E.H.), and Sanquin (grant PPOR:15-30) (A.V.). Publisher Copyright: © 2019 by The American Society of Hematology
PY - 2019
Y1 - 2019
N2 - Transfusion of donor-derived red blood cells (RBC) is the most common form of cellular therapy. Donor availability and the potential risk of alloimmunization and other transfusion-related complications may, however, limit the availability of transfusion units, especially for chronically transfused patients. In vitro cultured, customizable RBC would negate these concerns and further increase precision medicine. Large-scale, cost-effective production depends on optimization of culture conditions. We developed a defined medium and adapted our protocols to good manufacturing practice (GMP) culture requirements, which reproducibly provided pure erythroid cultures from peripheral blood mononuclear cells without prior CD341 isolation, and a 3 3 107-fold increase in erythroblasts in 25 days (or from 100 million peripheral blood mononuclear cells, 2 to 4 mL packed red cells can be produced). Expanded erythroblast cultures could be differentiated to CD71dimCD235a1CD441CD1172DRAQ52 RBC in 12 days. More than 90% of the cells enucleated and expressed adult hemoglobin as well as the correct blood group antigens. Deformability and oxygen-binding capacity of cultured RBC was comparable to in vivo reticulocytes. Daily RNA sampling during differentiation followed by RNA-sequencing provided a high-resolution map/resource of changes occurring during terminal erythropoiesis. The culture process was compatible with upscaling using a G-Rex bioreactor with a capacity of 1 L per reactor, allowing transition toward clinical studies and small-scale applications.
AB - Transfusion of donor-derived red blood cells (RBC) is the most common form of cellular therapy. Donor availability and the potential risk of alloimmunization and other transfusion-related complications may, however, limit the availability of transfusion units, especially for chronically transfused patients. In vitro cultured, customizable RBC would negate these concerns and further increase precision medicine. Large-scale, cost-effective production depends on optimization of culture conditions. We developed a defined medium and adapted our protocols to good manufacturing practice (GMP) culture requirements, which reproducibly provided pure erythroid cultures from peripheral blood mononuclear cells without prior CD341 isolation, and a 3 3 107-fold increase in erythroblasts in 25 days (or from 100 million peripheral blood mononuclear cells, 2 to 4 mL packed red cells can be produced). Expanded erythroblast cultures could be differentiated to CD71dimCD235a1CD441CD1172DRAQ52 RBC in 12 days. More than 90% of the cells enucleated and expressed adult hemoglobin as well as the correct blood group antigens. Deformability and oxygen-binding capacity of cultured RBC was comparable to in vivo reticulocytes. Daily RNA sampling during differentiation followed by RNA-sequencing provided a high-resolution map/resource of changes occurring during terminal erythropoiesis. The culture process was compatible with upscaling using a G-Rex bioreactor with a capacity of 1 L per reactor, allowing transition toward clinical studies and small-scale applications.
UR - https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=85074924768&origin=inward
UR - https://www.ncbi.nlm.nih.gov/pubmed/31698463
U2 - https://doi.org/10.1182/bloodadvances.2019000689
DO - https://doi.org/10.1182/bloodadvances.2019000689
M3 - Article
C2 - 31698463
SN - 2473-9529
VL - 3
SP - 3337
EP - 3350
JO - Blood advances
JF - Blood advances
IS - 21
ER -