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An effective detachment system for human induced pluripotent stem cells cultured on multilayered cultivation substrates using resonance vibrations.

ABSTRACT: Clinical application of human induced pluripotent stem cells (hiPSCs) has been hampered by the lack of a practical, scalable culture system. Stacked culture plates (SCPs) have recently attracted attention. However, final cell yields depend on the efficiency of cell detachment, and inefficient cell recovery from SCPs presents a major challenge to their use. We have developed an effective detachment method using resonance vibrations (RVs) of substrates with sweeping driving frequency. By exciting RVs that have 1-3 antinodes with ultra-low-density enzyme spread on each substrate of SCPs, 87.8% of hiPSCs were successfully detached from a 5-layer SCP compared to 30.8% detached by the conventional enzymatic method. hiPSC viability was similar after either method. Moreover, hiPSCs detached by the RV method maintained their undifferentiated state. Additionally, hiPSCs after long-term culture (10 passages) kept excellent detachment efficiency, had the normal karyotypes, and maintained the undifferentiated state and pluripotency. These results indicated that the RV method has definite advantages over the conventional enzymatic method in the scalable culture of hiPSCs using SCPs.

SUBMITTER: Terao Y

PROVIDER: S-EPMC6821886 | biostudies-literature | 2019 Oct

REPOSITORIES: biostudies-literature

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An effective detachment system for human induced pluripotent stem cells cultured on multilayered cultivation substrates using resonance vibrations.

Terao Yusuke Y Kurashina Yuta Y Tohyama Shugo S Fukuma Yuki Y Fukuda Keiichi K Fujita Jun J Takemura Kenjiro K

Scientific reports 20191030 1

Clinical application of human induced pluripotent stem cells (hiPSCs) has been hampered by the lack of a practical, scalable culture system. Stacked culture plates (SCPs) have recently attracted attention. However, final cell yields depend on the efficiency of cell detachment, and inefficient cell recovery from SCPs presents a major challenge to their use. We have developed an effective detachment method using resonance vibrations (RVs) of substrates with sweeping driving frequency. By exciting ...[more]

PMID: 31666563

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Project description:A prerequisite for the realization of human pluripotent stem cell (hPSC) therapies is the development of bioprocesses for generating clinically relevant quantities of undifferentiated hPSCs and their derivatives under xeno-free conditions. Microcarrier stirred-suspension bioreactors are an appealing modality for the scalable expansion and directed differentiation of hPSCs. Comparative analyses of commercially available microcarriers clearly show the need for developing synthetic substrates supporting the adhesion and growth of hPSCs in three-dimensional cultures under agitation-induced shear. Moreover, the low seeding efficiencies during microcarrier loading with hPSC clusters poses a significant process bottleneck. To that end, a novel protocol was developed increasing hPSC seeding efficiency from 30% to over 80% and substantially shortening the duration of microcarrier loading. Importantly, this method was combined with the engineering of polystyrene microcarriers by surface conjugation of a vitronectin-derived peptide, which was previously shown to support the growth of human embryonic stem cells. Cells proliferated on peptide-conjugated beads in static culture but widespread detachment was observed after exposure to stirring. This prompted additional treatment of the microcarriers with a synthetic polymer commonly used to enhance cell adhesion. hPSCs were successfully cultivated on these microcarriers in stirred suspension vessels for multiple consecutive passages with attachment efficiencies close to 40%. Cultured cells exhibited on average a 24-fold increase in concentration per 6-day passage, over 85% viability, and maintained a normal karyotype and the expression of pluripotency markers such as Nanog, Oct4, and SSEA4. When subjected to spontaneous differentiation in embryoid body cultures or directed differentiation to the three embryonic germ layers, the cells adopted respective fates displaying relevant markers. Lastly, engineered microcarriers were successfully utilized for the expansion and differentiation of hPSCs to mesoderm progeny in stirred suspension vessels. Hence, we demonstrate a strategy for the facile engineering of xeno-free microcarriers for stirred-suspension cultivation of hPSCs. Our findings support the use of microcarrier bioreactors for the scalable, xeno-free propagation and differentiation of human stem cells intended for therapies.

Dataset Information

An effective detachment system for human induced pluripotent stem cells cultured on multilayered cultivation substrates using resonance vibrations.

Publications

An effective detachment system for human induced pluripotent stem cells cultured on multilayered cultivation substrates using resonance vibrations.

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