Dissolvable Microcarriers Allow Scalable Expansion And Harvesting Of Human Induced Pluripotent Stem Cells Under Xeno‐Free Conditions

Abstract

The development of bioprocesses capable of producing large numbers of human induced pluripotent stem cells (hiPSC) in a robust and safe manner is critical for the application of these cells in biotechnological and medical applications. Scalable expansion of hiPSC is often performed using polystyrene microcarriers, which have to be removed from the cell suspension using a separation step that causes loss of viable cells. In this study, application of novel xeno‐free dissolvable microcarriers (DM) for an efficient and integrated expansion and harvesting of hiPSC is demonstrated. After an initial screening under static conditions, hiPSC culture using DM is performed in dynamic culture, using spinner‐flasks. A maximum 4.0 ± 0.8‐fold expansion is achieved after 5 days of culture. These results are validated with a second cell line and the culture is successfully adapted to fully xeno‐free conditions. Afterwards, cell recovery is made within the spinner flask, being obtained a 92 ± 4% harvesting yield, which is significantly higher than the one obtained for the conventional filtration‐based method (45 ± 3%). Importantly, the expanded and harvested hiPSC maintain their pluripotency and multilineage differentiation potential. The results here described represent a significant improvement of the downstream processing after microcarrier‐based hiPSC expansion, leading to a more cost‐effective and efficient bioprocess.

Microcarriers for Upscaling Cultured Meat Production

“…Up to date, only one MC now commercialized by Corning, has been developed with the purpose of being totally and rapidly degraded for cell harvesting (161). It is made of crosslinked polygalacturonic acid (PGA) and can be easily dissolved within 10-20 min using an EDTA solution, which destabilizes the PGA crosslinking in combination with pectinase that digests the polymer.…”

“…Many other polymers including dextran, cellulose, collagen, pectin or gelatin could be theoretically enzymatically digested in a similar way. For instance, the dextran-based MC Cytodex 1 has not been specifically developed to be degradable, however Lindskog et al have reported complete dissolution of Cytodex 1 MCs using dextranase while maintaining high cell viability (161). Similarly, the degradation of alginate, which is generally slow in vivo, can be accelerated by the use of nonenzymatic chemicals, such as citrate or phosphate.…”

“…Thus, the cell suspension can be washed and used directly for downstream processing. However, it has to be noted that when using degradable MCs, cells are usually released as a sheet/cellclump, therefore proteolytic enzymes may be additionally needed to promote dissociation into single-cell suspension (161). Depending on the downstream processing of the SCs, however, a single-cell suspension may not be necessarily required and aggregates may be permitted.…”

Online measurement of oxygen enables continuous noninvasive evaluation of human‐induced pluripotent stem cell ( hiPSC ) culture in a perfused 3D hollow‐fiber bioreactor

“…The hiPSC expansion was influenced by the initially inoculated cell number; a low initial cell density (i.e., 3‐ml bioreactors with 10 × 10 6 inoculated cells or 17‐ml bioreactor inoculated with 50 × 10 6 cells) led to a longer expansion duration and lower overall glucose metabolism but higher expansion rates, whereas a high cell density (i.e., 3‐ml bioreactors inoculated with 50 × 10 6 cells) led to shorter expansion durations and higher glucose metabolism, but to lower expansion rates. In bioreactors inoculated with a low initial cell number, a more than 100‐fold expansion of hiPSCs was achieved during bioreactor cultures, which has rarely been reported by others; mostly, 3.5‐ to 12‐fold expansion rates have been described (Haraguchi, Matsuura, Shimizu, Yamato, & Okano, ; Meng, Liu, Poon, & Rancourt, ; Olmer et al, ; Rodrigues et al, ; Wang et al, ). To date, such high expansion yields as presented in this study have only been realized by Abecasis et al (), obtaining 10 10 hiPSCs and an 1100‐fold expansion rate within an 11‐day culture period in stirred‐tank bioreactors, and Kwok et al (), achieving a 125‐fold expansion rate and a cell number of 2 × 10 9 hiPSCs after 14 days of stirred suspension culture.…”

Scaled‐Up Inertial Microfluidics: Retention System for Microcarrier‐Based Suspension Cultures

“…The optimum concentration of MCs has not been reported, as it is dependent on cell type, MC properties, bioreactor type, and culture volume. However, a diverse range of ≈3000 MCs mL −1 to ≈15 000 MCs mL −1 is reported in the literature …”