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 × 10vs 1.17 ± 0.07 × 10 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 × 10vs 1.23 ± 0.07 × 10 CM/mL). The combination of AS addition and change in agitation regime resulted in a production yield of 1.50 ± 0.10 × 10 CM/mL which is comparable to that achieved in the rocker platform as described before (1.61 ± 0.36 × 10 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/10 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 × 10 to 3.50 ± 0.65 × 10 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 × 10 to 1.29 ± 0.42 × 10 CM/mL with 72.5 ± 8.35% to 83.12 ± 8.73% cardiac troponin-T positive cells were obtained from these cultures.

Selection of human induced pluripotent stem cells lines optimization of cardiomyocytes differentiation in an integrated suspension microcarrier bioreactor

“…Method for microcarrier coating have been reported earlier by our group [57,58]. Briefly, 20 mg/mL of Cytodex 1 (GE Healthcare, USA) was coated with (1:30 v/v) Geltrex® in DMEM/F12 medium (Thermo Fisher Scientific) overnight at 4°C.…”

“…Besides, although EBs with controlled size can be form by many methods [45,46,62], they might be limited to research scale [37]. In the past few years, our group has developed protocols for scale-up and production of de novo hPSC-/hiPSC-derived CMs based on the use of MC cultures in platforms such as rockers and spinners [7,8,34,35,38,[57][58][59]. However, in order to generate high-quantity and high-quality CMs, applying intermittent agitation during the first 3 days of cardiac differentiation phase is important [57,58], which makes it problematic in large-scale reactors.…”

“…In the past few years, our group has developed protocols for scale-up and production of de novo hPSC-/hiPSC-derived CMs based on the use of MC cultures in platforms such as rockers and spinners [7,8,34,35,38,[57][58][59]. However, in order to generate high-quantity and high-quality CMs, applying intermittent agitation during the first 3 days of cardiac differentiation phase is important [57,58], which makes it problematic in large-scale reactors. In addition, the continuous monitoring and control of the environment within a spinner culture are still challenging.…”

“…This problem becomes increasing important when cells reach high densities and pH can be highly acidic and oxygen availability to the cells can be hampered. In this study, we adapted our MC-based cardiac differentiation system from spinners [33,58] to establish a robust, scalable and controllable process for manufacturing CMs in a single enclosed industrial-type stirred tank reactor. Stirred tank bioreactors are commonly used for the production of cellular products, such as antibodies, enzymes, vaccines and virus in the biotechnology industry.…”

“…In this study, we attempt to integrate the phases of undifferentiated hiPSC expansion, CHIR99021-induced cardiac differentiation and lactate purification into a scalable MC suspension culture system using a stirred tank bioreactor under continuous agitated condition, providing a streamlined and controlled process for scaleup of pure CM production. Lower CM yields previously seen in a spinner flask under continuous agitation [57,58] were corrected with the process of cell line selection and by controlling the culture parameters in the bioreactor. A process for CM production that is amenable to good manufacturing practice (GMP) and scale-up was developed.…”