Filip Laco1†, Alan Tin-Lun Lam1†, Tsung-Liang Woo1, Gerine Tong1, Valerie Ho1, Poh-Loong Soong2, Elina Grishina2, Kun-Han Lin2, Shaul Reuveny1 and Steve Kah-Weng Oh1
1 Bioprocessing Technology Institute, Agency for Science, Technology and Research (A*STAR), Singapore, Singapore
2 Ternion Biosciences, National Heart Centre of Singapore, Singapore
† Filip Laco and Alan Tin-Lun Lam contributed equally to this work
Published in Stem Cell Research and Therapy 2020 11: 118 (Online Version)
One of the leading causes of death worldwide are cardiac-related illnesses, such as heart attack. Because of the limited regenerative potential of the cardiac cells, heart transplantation for reconstituting the function of damaged heart is the only effective solution currently available. However, this is severely hindered mainly due to the shortage of donor organs. To that end, stem cell-derived cardiomyocytes (CMs) may serve as a renewable cellular source for repairing the heart. However, doses of up to 1 billion functional CMs are required for a single cell therapy for heart attack. It is a challenge to produce large quantities of high purity CMs due to the adherent characteristic of stem cells and their differentiation efficiency.
The goal of our study is to develop a scalable bioprocess for production and purification of CMs from human induced pluripotent stem cell (hiPSC) in a stirred tank bioreactor. The combination of large quantities of cells on microcarriers (MCs) from a bioreactor and purification of homogeneous cardiac population facilitates the transplantation of CMs derived from hiPSC for heart disease in the future. Selection of a hiPSC line with high potential for expansion and cardiac differentiation on MCs is essential. Among five different hiPSC lines, one generated from our lab, was selected for cardiac differentiation in a 22-day integrated bioprocess, which showed a higher compatibility for expansion and cardiac differentiation in our MC-based stirred suspension bioreactor.
In summary, we integrated four critical unit operations into a single process: scalable expansion (phase 1), cardiomyocyte differentiation step (phase 2), purification using the lactate-based treatment (phase 3) and cell recovery step (phase 4), under restricted oxygen control and continuous stirring with periodic batch type media exchanges. Overall, high quantity (>40 CM/hiPSC) and high quality (>96% Troponin T) cardiomyocytes were generated, which is at least 8-fold more than other reports. We also introduced a novel high throughput platform for imaging and quantifying subtle changes of membrane potentials in hiPSC-derived CMs, based on voltage sensitive dyes and high-speed microscopy. It could automatically quantify the action potential waveforms of more than 100 CMs simultaneously, which is not achievable via manual or automated patch clamping in the same duration.
In conclusion, a streamlined and controllable platform for large quantity manufacturing of pure functional atrial, ventricular and nodal cardiomyocytes on MCs in conventional type stirred tank bioreactors was established, which can be further scaled up and translated to a good manufacturing practice (GMP)-compliant production process, to fulfil the quantity requirements of the cellular therapeutic industry.