Differentiation of Human iPS Cells Into Sensory Neurons Exhibits Developmental Stage-Specific Cryopreservation Challenges.

Rui Li,Ashley Petersen,Allison Hubel,Vincent Truong,James R Dutton,Patrick Walsh

doi:10.3389/fcell.2021.796960

Abstract

Differentiation of human induced pluripotent stem cells (hiPSCs) generates cell phenotypes valuable for cell therapy and personalized medicine. Successful translation of these hiPSC-derived therapeutic products will rely upon effective cryopreservation at multiple stages of the manufacturing cycle. From the perspective of cryobiology, we attempted to understand how the challenge of cryopreservation evolves between cell phenotypes along an hiPSC-to-sensory neuron differentiation trajectory. Cells were cultivated at three different stages to represent intermediate, differentiated, and matured cell products. All cell stages remained ≥90% viable in a dimethyl sulfoxide (DMSO)-free formulation but suffered ≥50% loss in DMSO before freezing. Raman spectroscopy revealed higher sensitivity to undercooling in hiPSC-derived neuronal cells with lower membrane fluidity and higher sensitivity to suboptimal cooling rates in stem cell developmental stages with larger cell bodies. Highly viable and functional sensory neurons were obtained following DMSO-free cryopreservation. Our study also demonstrated that dissociating adherent cultures plays an important role in the ability of cells to survive and function after cryopreservation.

Highlights

Human induced pluripotent stem cells can be manufactured from a range of somatic cell types and further differentiated into cells valuable for drug discovery (Ko and Gelb, 2014), cell therapy (Yamanaka, 2020), and tissue engineering (Mazzola and Di Pasquale, 2020) applications
A non-dimethyl sulfoxide (DMSO) cryoprotective agent (CPA) solution composed of sucrose, glycerol, L-isoleucine, poloxamer 188 (P188), and HSA and previously developed for human induced pluripotent stem cells (hiPSCs) (Li et al, 2020) was compared with a DMSO-based solution commercially available and commonly used for hiPSC-derived cells (i.e., CS10) in terms of their ability to maintain the viability of different stages of neuronal cells before freezing
Compared with the non-DMSO CPA, the DMSO solution resulted in significant cell loss by both loss of membrane integrity and lysis for all cell stages within 30 min of exposure

Summary

Introduction

Human induced pluripotent stem cells (hiPSCs) can be manufactured from a range of somatic cell types and further differentiated into cells valuable for drug discovery (Ko and Gelb, 2014), cell therapy (Yamanaka, 2020), and tissue engineering (Mazzola and Di Pasquale, 2020) applications. There is significant added value in derisking the manufacturing supply chain by being able to efficiently cryopreserve cells at multiple stages along the differentiation trajectory, from the originally isolated somatic tissue through the pluripotent stem cell stage, intermediate progenitor, and differentiated phenotypes, to the terminal mature product. While results of cryopreservation have been reported in some studies of hiPSCderived cell types (Linville et al, 2020; van den Brink et al, 2020), we lack understanding of scientific principles on formulating cryopreservation media, freezing, thawing, cryoinjury mechanisms for many intermediate and mature cell product phenotypes, whether and how they change from one cell type to another, especially in relation to the expanding variety of cell types in this dynamic field of hiPSC-based manufacturing

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Conclusion