Selection of human induced pluripotent stem cells lines optimization of cardiomyocytes differentiation in an integrated suspension microcarrier bioreactor
Autor: | Elina Grishina, Poh-Loong Soong, Kun-Han Lin, Steve Oh, Alan Tin-Lun Lam, Valerie Ho, Tsung-Liang Woo, Gerine Tong, Filip Laco, Shaul Reuveny |
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Rok vydání: | 2020 |
Předmět: |
Induced Pluripotent Stem Cells
Cell Culture Techniques Medicine (miscellaneous) Continuous stirred-tank reactor Biochemistry Genetics and Molecular Biology (miscellaneous) Microcarriers lcsh:Biochemistry Bioreactors Bioreactor Humans Myocytes Cardiac lcsh:QD415-436 Stirred tank bioreactor Bioprocess Induced pluripotent stem cell Cells Cultured Cardiomyocytes lcsh:R5-920 Troponin T Bioprocessing Research technology industry and agriculture Microcarrier Cell Differentiation Human induced pluripotent stem cells Cell Biology Cell biology Cell culture Molecular Medicine OptioQUANT™ platform Stem cell lcsh:Medicine (General) |
Zdroj: | Stem Cell Research & Therapy, Vol 11, Iss 1, Pp 1-16 (2020) Stem Cell Research & Therapy |
ISSN: | 1757-6512 |
Popis: | Background The production of large quantities of cardiomyocyte is essential for the needs of cellular therapies. This study describes the selection of a human-induced pluripotent cell (hiPSC) line suitable for production of cardiomyocytes in a fully integrated bioprocess of stem cell expansion and differentiation in microcarrier stirred tank reactor. Methods Five hiPSC lines were evaluated first for their cardiac differentiation efficiency in monolayer cultures followed by their expansion and differentiation compatibility in microcarrier (MC) cultures under continuous stirring conditions. Results Three cell lines were highly cardiogenic but only one (FR202) of them was successfully expanded on continuous stirring MC cultures. FR202 was thus selected for cardiac differentiation in a 22-day integrated bioprocess under continuous stirring in a stirred tank bioreactor. In summary, we integrated a MC-based hiPSC expansion (phase 1), CHIR99021-induced cardiomyocyte differentiation step (phase 2), purification using the lactate-based treatment (phase 3) and cell recovery step (phase 4) into one process in one bioreactor, under restricted oxygen control (6 cells/mL) was achieved in the expansion phase. By controlling the stirring speed and DO levels in the bioreactor cultures, 7.36 ± 1.2 × 106 cells/mL cardiomyocytes with > 80% Troponin T were generated in the CHIR99021-induced differentiation phase. By adding lactate in glucose-free purification media, the purity of cardiomyocytes was enhanced (> 90% Troponin T), with minor cell loss as indicated by the increase in sub-G1 phase and the decrease of aggregate sizes. Lastly, we found that the recovery period is important for generating purer and functional cardiomyocytes (> 96% Troponin T). Three independent runs in a 300-ml working volume confirmed the robustness of this process. 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-compliant production process, to fulfill the quantity requirements of the cellular therapeutic industry. |
Databáze: | OpenAIRE |
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