A Scalable Suspension Platform for Generating High-Density Cultures of Universal Red Blood Cells from Human Induced Pluripotent Stem Cells

Abstract

Summary Universal red blood cells (RBCs) differentiated from O-negative human induced pluripotent stem cells (hiPSCs) could find applications in transfusion medicine. Given that each transfusion unit of blood requires 2 trillion RBCs, efficient bioprocesses need to be developed for large-scale in vitro generation of RBCs. We have developed a scalable suspension agitation culture platform for differentiating hiPSC-microcarrier aggregates into functional RBCs and have demonstrated scalability of the process starting with 6 well plates and finally demonstrating in 500 mL spinner flasks. Differentiation of the best-performing hiPSCs generated 0.85 billion erythroblasts in 50 mL cultures with cell densities approaching 1.7 × 10 7 cells/mL. Functional (oxygen binding, hemoglobin characterization, membrane integrity, and fluctuations) and transcriptomics evaluations showed minimal differences between hiPSC-derived and adult-derived RBCs. The scalable agitation suspension culture differentiation process we describe here could find applications in future large-scale production of RBCs in controlled bioreactors.

Industrially Compatible Transfusable iPSC-Derived RBCs: Progress, Challenges and Prospective Solutions

“…Recently, Sivalingam et al established a protocol for scalable differentiation for hiPSC-microcarrier aggregates in 500 mL spinner flasks, with about a 1000-fold expansion of the functional erythroblasts (see Table 1 ). Whilst hiPSC-derived erythroblasts exhibit low enucleation (6%), a co-culture with OP9 on the microcarriers resulted in up to 59.3% enucleation in vitro [ 72 ].…”

“…Additionally, the co-culture condition yielded a higher frequency of late-stage erythroblasts and even some levels of enucleation (see Table 1 ). Another mouse bone marrow stromal cell line, OP9, was able to reduce apoptosis of expanding iPSC-erythroblasts and improved enucleation by about three- to ten-fold [ 72 ]. More importantly, the authors also showed that OP9 can be grown on microcarriers (microbeads that greatly improve the growth area of adherent cells in a suspension culture) and thus demonstrating the scalability of their iPSC-erythrocyte platform.…”

“…Other groups have published methods with defined, GMP-compliant conditions for RBC expansion from PBMCs [ 51 ] and donor HSCs with high levels of enucleation and expansion [ 41 ]. Sivalingam et al achieved cell densities of 2 × 10 12 in suspension platforms by progressively scaling up the culture [ 72 ]. Here, we propose a practical solution to progressively expand hiPSCs and differentiate them into erythroid cells in increasing volumes of bioreactor cultures (see Figure 2 ).…”

“…One final, yet potentially significant issue is the tendency of hiPSC-derived RBCs to express predominantly fetal and embryonic globins (HbF, HbE) instead of the expected adult ones (HbA). Since fetal hemoglobin binds oxygen more tightly than its adult counterpart, this results in a significant left shift on an oxygen dissociation curve analysis compared to adult RBCs [ 72 ]. Research into the control of hemoglobin switching is ongoing and key findings have already begun to surface.…”

Expansion and differentiation ofex vivocultured erythroblasts in scalable stirred bioreactors

“…This includes roller bottles (Y. Zhang et al, 2017), rocking motion bioreactors (Boehm et al, 2010; Timmins et al, 2011), shake flasks (Aglialoro et al, 2021; Sivalingam et al, 2020), and spinner flasks (Griffiths et al, 2012; Kupzig et al, 2017; Sivalingam et al, 2020; Trakarnsanga et al, 2017). Stirred tank reactors (STRs) additionally allow for online monitoring and control of process parameters such as pH, dissolved oxygen and nutrient concentrations.…”