TY - JOUR
T1 - Expansion and differentiation of ex vivo cultured erythroblasts in scalable stirred bioreactors
AU - Gallego-Murillo, Joan Sebastián
AU - Iacono, Giulia
AU - van der Wielen, Luuk A. M.
AU - van den Akker, Emile
AU - von Lindern, Marieke
AU - Wahl, Sebastian Aljoscha
N1 - Funding Information: We thank Queralt Farras Costa and Victur Puig I Laborda (Department of Biotechnology, Faculty of Applied Sciences, Delft University of Technology; Delft; The Netherlands) for their assistance on the determination of oxygen consumption rates. We also thank Yi Song, Dirk Geerts and Rob Kerste (Department of Biotechnology, Faculty of Applied Sciences, Delft University of Technology; Delft; The Netherlands) for their support on setting up the 0.5 L bioreactors. We thank Nurcan Yagci (Sanquin Research) who performed PBMC isolation and precultures. We thank Marten Hansen (Laboratory for Cell Therapies, Sanquin) for the support on setting up the 3 L bioreactors. We thank Tom van Arragon and Cristina Bernal Martínez (Applikon Biotechnology; Delft; The Netherlands) for technical help and advice on bioreactor cultures in the Applikon systems. This study was supported by the ZonMW TAS program (project 116003004), by the Landsteiner Foundation for Bloodtransfusion Research (LSBR project 1239), by Sanquin Blood Supply grants PPOC17‐28 and PPOC19‐14, and by European Union, Marie Skłodowska‐Curie Innovative Training Networks (860436; EVIDENCE). Funding Information: We thank Queralt Farras Costa and Victur Puig I Laborda (Department of Biotechnology, Faculty of Applied Sciences, Delft University of Technology; Delft; The Netherlands) for their assistance on the determination of oxygen consumption rates. We also thank Yi Song, Dirk Geerts and Rob Kerste (Department of Biotechnology, Faculty of Applied Sciences, Delft University of Technology; Delft; The Netherlands) for their support on setting up the 0.5 L bioreactors. We thank Nurcan Yagci (Sanquin Research) who performed PBMC isolation and precultures. We thank Marten Hansen (Laboratory for Cell Therapies, Sanquin) for the support on setting up the 3 L bioreactors. We thank Tom van Arragon and Cristina Bernal Martínez (Applikon Biotechnology; Delft; The Netherlands) for technical help and advice on bioreactor cultures in the Applikon systems. This study was supported by the ZonMW TAS program (project 116003004), by the Landsteiner Foundation for Bloodtransfusion Research (LSBR project 1239), by Sanquin Blood Supply grants PPOC17-28 and PPOC19-14, and by European Union, Marie Skłodowska-Curie Innovative Training Networks (860436; EVIDENCE). Publisher Copyright: © 2022 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals LLC.
PY - 2022/11
Y1 - 2022/11
N2 - Transfusion of donor-derived red blood cells (RBCs) is the most common form of cell therapy. Production of transfusion-ready cultured RBCs (cRBCs) is a promising replacement for the current, fully donor-dependent therapy. A single transfusion unit, however, contains 2 × 1012 RBC, which requires large scale production. Here, we report on the scale-up of cRBC production from static cultures of erythroblasts to 3 L stirred tank bioreactors, and identify the effect of operating conditions on the efficiency of the process. Oxygen requirement of proliferating erythroblasts (0.55–2.01 pg/cell/h) required sparging of air to maintain the dissolved oxygen concentration at the tested setpoint (2.88 mg O2/L). Erythroblasts could be cultured at dissolved oxygen concentrations as low as 0.7 O2 mg/ml without negative impact on proliferation, viability or differentiation dynamics. Stirring speeds of up to 600 rpm supported erythroblast proliferation, while 1800 rpm led to a transient halt in growth and accelerated differentiation followed by a recovery after 5 days of culture. Erythroblasts differentiated in bioreactors, with final enucleation levels and hemoglobin content similar to parallel cultures under static conditions.
AB - Transfusion of donor-derived red blood cells (RBCs) is the most common form of cell therapy. Production of transfusion-ready cultured RBCs (cRBCs) is a promising replacement for the current, fully donor-dependent therapy. A single transfusion unit, however, contains 2 × 1012 RBC, which requires large scale production. Here, we report on the scale-up of cRBC production from static cultures of erythroblasts to 3 L stirred tank bioreactors, and identify the effect of operating conditions on the efficiency of the process. Oxygen requirement of proliferating erythroblasts (0.55–2.01 pg/cell/h) required sparging of air to maintain the dissolved oxygen concentration at the tested setpoint (2.88 mg O2/L). Erythroblasts could be cultured at dissolved oxygen concentrations as low as 0.7 O2 mg/ml without negative impact on proliferation, viability or differentiation dynamics. Stirring speeds of up to 600 rpm supported erythroblast proliferation, while 1800 rpm led to a transient halt in growth and accelerated differentiation followed by a recovery after 5 days of culture. Erythroblasts differentiated in bioreactors, with final enucleation levels and hemoglobin content similar to parallel cultures under static conditions.
KW - cell culture
KW - cultured blood
KW - erythropoiesis
KW - red blood cell
KW - scale-up
KW - stirred tank bioreactor
UR - http://www.scopus.com/inward/record.url?scp=85135388952&partnerID=8YFLogxK
U2 - https://doi.org/10.1002/bit.28193
DO - https://doi.org/10.1002/bit.28193
M3 - Article
C2 - 35879812
SN - 0006-3592
VL - 119
SP - 3096
EP - 3116
JO - Biotechnology and bioengineering
JF - Biotechnology and bioengineering
IS - 11
ER -