A reproducible and versatile system for the dynamic expansion of human pluripotent stem cells in suspension

Abstract

Reprogramming of patient cells to human induced pluripotent stem cells (hiPSC) has facilitated in vitro disease modeling studies aiming at deciphering the molecular and cellular mechanisms that contribute to disease pathogenesis and progression. To fully exploit the potential of hiPSC for biomedical applications, technologies that enable the standardized generation and expansion of hiPSC from large numbers of donors are required. Paralleled automated processes for the expansion of hiPSC could provide an opportunity to maximize the generation of hiPSC collections from patient cohorts while minimizing hands-on time and costs. In order to develop a simple method for the parallel expansion of human pluripotent stem cells (hPSC) we established a protocol for their cultivation as undifferentiated aggregates in a bench-top bioreactor system (BioLevitator™). We show that long-term expansion (10 passages) of hPSCs either in mTeSR or E8 medium preserved a normal karyotype, three-germ-layer differentiation potential and high expression of pluripotency-associated markers. The system enables the expansion from low inoculation densities (0.3 × 10(5) cells/mL) and provides a simplified, cost-efficient and time-saving method for the provision of hiPSC at midi-scale. Implementation of this protocol in cell production schemes has the potential to advance cell manufacturing in many areas of hiPSC-based medical research.

Scalable stirred suspension culture for the generation of billions of human induced pluripotent stem cells using single‐use bioreactors

“…To circumvent the loss of these small aggregates, it was hypothesized that leaving aggregates to grow for a longer period before the first medium exchange would result in larger aggregates, requiring less time for sedimentation and therefore avoiding both the problems of agglomeration and loss of small aggregates. Elanzew et al (2015) employed a medium supplementation strategy without medium removal and harvested hiPSCs after 4 days of culture, and such a strategy was adapted for the current protocol.…”

“…The stirred suspension culture technique is one promising method for the large-scale bioprocessing of pluripotent stem cells and maintaining their pluripotency (Amit et al, 2011;Badenes et al, 2015;Chen et al, 2015;Elanzew, Sommer, Pusch-Klein, Brüstle, & Haupt, 2015;Zweigerdt, Olmer, Singh, Haverich, & Martin, 2011). The aim of this study was to demonstrate the scalability of the technique in a singleuse stirred bioreactor (Kaiser, Eibl, & Eibl, 2011), and to develop a protocol for the expansion of billions of hiPSCs using stirred suspension culture vessels, while maintaining the undifferentiated, self-renewing state and pluripotency of the hiPSCs.…”

Application of Bioreactor in Stem Cell Culture

“…Suspension SCs like hematopoietic progenitor cells without requirement of surface attachment for growth were successfully cultured in stirring bioreactors. Adherent SCs, such as ESCs, iPSCs and MSCs, are attached to cell microcarriers for their suspension and are cultured in stirring bioreactors [19,20]. The growth efficiency and homogeneous of cultured SCs in stirring bioreactor were improved in somehow, due to homogeneous nature of culture system.…”

“…The magnetic core in the microcarrier makes the medium change and cell collection convenient and simplifies the manipulation [30]. This bioreactor with 3-D culture environment produces small shear force and thus is applicable for the culture of adherent SCs [20].…”

Editorial: Stem Cell Engineering

“…Elanzew et al [7] developed a process for long-term expansion (10 passages) of undifferentiated human pluripotent stem cells (hPSCs) in aggregates in suspension in chemically-defined conditions. Fernandes et al [8] describe a defined protocol employing dual SMAD inhibition on a vitronectin extracellular matrix for differentiating hPSCs to neural lineages.…”