Meticulous optimization of cardiomyocyte yields in a 3-stage continuous integrated agitation bioprocess

Abstract

Human pluripotent stem cells (hPSCs) can be a renewable source for generating cardiomyocyte (CM) for treating myocardial infraction. In our previous publication, we described an integrated microcarrier-based wave reactor process for the expansion and differentiation of hPSCs to CMs on a rocker based platform. However, this platform is limited in terms of linear scalability and CMs purity. The present study describes ways to overcome these limitations by the use of a stirred scalable platform and incorporation of an additional lactate based purification step which increases CM purity.Efficient CM differentiation in stirred spinners was achieved by (1) Addition of ascorbic acid (AS) during the differentiation phase which resulted in an increase of 38.42% in CM yield (0.84 ± 0.03 × 106vs 1.17 ± 0.07 × 106 CM/mL for cultures without AS and with AS respectively) and (2) Change of agitation regime to a shorter static intervals one (from 66 min off/6 min on (66/6) to 8 min off/1 min on (8/1)) during the first 3 days of differentiation which resulted in 22% increase in CM yield (1.50 ± 0.10 × 106vs 1.23 ± 0.07 × 106 CM/mL). The combination of AS addition and change in agitation regime resulted in a production yield of 1.50 ± 0.10 × 106 CM/mL which is comparable to that achieved in the rocker platform as described before (1.61 ± 0.36 × 106 CM/mL).Increase in CM purity was achieved by changing of culture medium to RPMI1640 (without glucose) + 5 mM lactate +0.6 mM AS at day 10 of differentiation which resulted in 44.5% increase in CM purity at day 15. The increase in purity of CMs was due to the death of the non-CM cells (~76% of cell death). It is important to note that in the absence of glucose, lactate was consumed at a rate of 0.01 mmol/106 cells/h. Addition of glucose, even in small amounts, during the purification step prevents the process of CM purification, due to the growth of the non-CM cell population.In summary, hPSC (hESC-HES3 and hiPSC-IMR90) can be efficiently differentiated to CMs in a scalable spinner process which integrates 7 days of expansion (3.01 ± 0.51 × 106 to 3.50 ± 0.65 × 106 cells/mL) followed by 10 days of WNT modulated CM differentiation and 5 days of lactate based purification. CM yield of 1.38 ± 0.22 × 106 to 1.29 ± 0.42 × 106 CM/mL with 72.5 ± 8.35% to 83.12 ± 8.73% cardiac troponin-T positive cells were obtained from these cultures. Keywords: Human pluripotent stem cells, Microcarriers, Spinner platform, Cardiomyocytes differentiation, Lactate purification, Ascorbic acid