Billion-Scale Expansion of Functional hiPSC-Derived Cardiomyocytes in Bioreactors Through Oxygen Control and
Pedro Vicente1,2, Lara R Inocêncio1,2, Asier Ullate-Agote3
1iBET, Instituto de Biologia Experimental e Tecnológica, Apartado 12, Oeiras, 2780901, Portugal.
Insights
Generating large quantities of human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) is now faster and cheaper. A new stirred-tank bioreactor process maximizes hiPSC-CM expansion using Wnt pathway activation and mild hypoxia.
Area of Science:
- Biotechnology
- Stem Cell Biology
- Cardiovascular Research
Background:
- Large-scale production of human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) is crucial for therapeutic and testing applications but remains costly and time-intensive.
- Existing methods face challenges in scalability and efficiency, hindering widespread use.
Purpose of the Study:
- To develop a scalable and cost-effective bioprocess for the high-yield generation of hiPSC-CM.
- To optimize expansion conditions in stirred-tank bioreactors (STB) for maximizing hiPSC-CM production.
Main Methods:
- Development of a stirred-tank bioreactor (STB) bioprocess for hiPSC-CM expansion.
- Continuous activation of the Wnt pathway via CHIR99021 perfusion.
- Cultivation under mild hypoxia (10% O2) and controlled power input per volume.
Main Results:
- Achieved expansion of 4 billion pure hiPSC-CM in a 2L STB.
- Mild hypoxia (10% O2) increased expansion rates tenfold compared to normoxia by reducing reactive oxygen species and upregulating proliferation genes.
- Demonstrated constant power input per volume as a critical scale-up criterion.
Conclusions:
- The novel bioprocess enables time- and cost-effective generation of large quantities of hiPSC-CM.
- Optimized conditions promote hiPSC-CM expansion and subsequent maturation, evidenced by improved transcriptional signatures, sarcomere alignment, and calcium handling.
- This scalable method facilitates the production of hiPSC-CM for diverse applications.
Abstract:
Generation of upscaled quantities of human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM), for therapeutic or testing applications, is both expensive and time-consuming. Herein, a scalable bioprocess for hiPSC-CM expansion in stirred-tank bioreactors (STB) is developed. By combining the continuous activation of the Wnt pathway, through perfusion of CHIR99021, within a mild hypoxia environment, the expansion of hiPSC-CM as aggregates is maximized, reaching 4 billion of pure hiPSC-CM in 2L STB. In particular, the importance of i) controlling the dissolved oxygen at 10% O2 to reduce reactive oxygen species production and upregulate genes involved in cell proliferation, resulting in higher expansion rates (tenfold) compared to normoxic conditions, and ii) maintaining constant power input per volume as a scale-up criteria is demonstrated. After expansion, hiPSC-CM further mature in culture, revealing more mature transcriptional signatures, higher sarcomere alignment and improved calcium handling. This new bioprocess opens the door to time- and cost-effective generation of hiPSC-CM.
