Cryopreservation of midbrain dopaminergic neural cells differentiated from human embryonic stem cells

Abstract

Recent advancements in protocols to differentiate human pluripotent stem cells into midbrain dopaminergic (mDA) neurons has improved the ability to model Parkinson's disease (PD) in a dish, and has provided a scalable source of donor cells for emerging PD cell replacement therapy (CRT). However, to facilitate reproducibility, collaboration, and clinical trials it would be highly beneficial to cryopreserve committed mDA neural precursors cells in a ready-to-use format. In terms of cell manufacturing for PD CRT trials, a cryopreserved transplantation-ready mDA cell product would provide a critical opportunity for quality control, efficacy testing, and safety assessments. To address this challenge, we have compared six (6) different clinical-grade cryopreservation media and different freezing conditions for mDA neural precursor cells differentiated from two human embryonic stem cell (ESC) lines, MasterShef7 and RC17. Significant differences in cell viability were observed at 24h post-thawing, but no differences were observed immediately upon thawing. This highlights the need to check cell viability over the first 24h after thawing, and that viability of freshly thawed cells is insufficient to gauge the success of a cryopreservation protocol. Considerable apoptosis occurs in the first 24h post-thawing, and significant differences between cryopreservation procedures were only revealed during this time period. The presence of ROCK inhibitors improved cell viability at 24h for all conditions tested. A faster cooling rate of 1-2°C/min was significantly better than 0.5°C/min for all conditions tested, while rapid thawing at 37°C was not always superior to slow thawing at 4°C. Indeed, the optimal cryopreservation and thawing conditions in this study, as determined by 24h post-thaw viability, were cells frozen in PSC Cryopreservation medium at a cooling rate of 1°C/min and slow thawing at 4°C. These conditions permitted recovery of 60%-70% live cells at 24h with respect to the starting number of cryopreserved cells. Importantly, cryopreservation of mDA neural precursor cells did not alter their potential to resume differentiation into mDA neurons. Highlights:• First systematic comparison of multiple clinical-grade cryopreservation media for human ESC-derived mDA neural precursor cells• Differences in cell viability were observed at 24h after thawing, but not immediately upon thawing• Cooling rates of 1°C/min or 2°C/min were significantly better than 0.5°C/min for all cryopreservation conditions tested • A slow thawing condition at 4°C was significantly better than quick thawing at 37°C for cells frozen in PSC Cryopreservation medium • Cryopreservation of mDA cells does not significantly alter their potential to differentiate into mDA neurons

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

“…Another study reported the cryopreservation of primary mouse motor neurons in DMSO and post-thaw cell recovery rate of 68.8% ( Parker et al, 2018 ). There were two accounts of cryopreserved dopaminergic progenitors and neurons derived from hiPSCs and human embryonic stem cells (hESCs), with the highest reported post-thaw recovery approaching 50% upon 24-h treatment in apoptosis inhibitors ( Wakeman et al, 2017 ; Drummond et al, 2020 ). In comparison, the present study demonstrated DMSO-free cryopreservation of neuronal cells, with post-thaw survival of D5 crest measured at 91.6% immediately post-thaw, 88.2% 24 h post-thaw, and of D7 SN measured at 93.6% immediately post-thaw, 84.1% 24 h post-thaw, all of which were significantly higher than 50% ( p < 0.05) even without the use of apoptosis inhibitor.…”

Cryopreservation of Human Midbrain Dopaminergic Neural Progenitor Cells Poised for Neuronal Differentiation

“…We thank Jennifer Colquhoun for artwork in Figure 2, and Prof. David Melton for critical comments in the study. This manuscript has been released as a pre-print at bioRxiv (Drummond et al, 2020).…”