Cryobanking of Embryoid Bodies to Facilitate Basic Research and Cell-Based Therapies

Abstract

Pluripotent stem cells offer unique opportunities for curing debilitating diseases. However, further comprehensive research is needed to better understand cell signaling during the differentiation of pluripotent cells into different cell lineages and accordingly to develop clinically applicable protocols. One of the limiting steps for differentiation studies is proper culture and expansion of pluripotent stem cells, which is labor intensive, expensive, and requires a great deal of expertise. This limiting step can be overcome by successful banking and distribution of embryoid bodies (EBs), which are aggregates of pluripotent stem cells and typically the starting point of differentiation protocols. The objective of this study was to investigate the feasibility of EB banking by studying survival and functionality of cryopreserved EBs. To this end, EBs were formed by culturing mouse 129 embryonic stem (ES) cells in the absence of leukemia inhibitory factor (LIF) in hanging drops and then subjected to different cryopreservation protocols. In a series of experiments, we first tested the postthaw survival of EBs as a function of dimethylsulfoxide (DMSO) and extracellular trehalose concentrations and cooling rates. Next, we studied the functionality of cryopreserved EBs by assessing their postthaw attachment, growth, and differentiation into various cell types. Higher (≥5%) DMSO concentrations alone or in combination with trehalose (0.1 M and 0.2 M) yielded good postthaw survival rates of >80%, whereas cooling of EBs at 1°C/min in the presence of 5% DMSO +0.1 M trehalose gave the best attachment and growth rates, with differentiation into cell lineages of three germ layers. Taken together, our results suggest that EBs are tolerant to cryopreservation-associated stresses and retain their differentiation potential after freezing and thawing. Furthermore, our experiments with dissociated EB cells and nondissociated EBs suggest that the extracellular matrix may play a beneficial role in the cryotolerance of EBs. Overall, our data support the feasibility of EB banking, which would facilitate advancement of cell-based therapies.

The Microenvironment of Embryoid Bodies Modulated the Commitment to Neural Lineage Postcryopreservation

“…47,48 The sugar components of the ECMs (e.g., glycosaminoglycans) also can exert cryo-protective effects (e.g., mimicking the protection effect of trehalose). 17 In this study, intact EBs retained expressions of laminin and fibronectin, which may contribute to better cell survival. 49 Therefore, the microenvironment of intact EBs may protect the cells from oxidative stress.…”

“…13,17 Various cryopreservation methods including slow-cooling and vitrification have been tested for aggregate-based stem cells. 19,20,30,31 Slow-cooling method is more amendable for large-scale cell banking due to its simplicity.…”

“…10,15,16 Cryo-banking of EBs for NPC derivation provides a necessary step for sample storage, process monitoring, and preservation of the intermediate cell populations. 17 During EB cryopreservation, the 3D cell organization is a critical parameter to maintain the recovered cell properties. 17 For adult neurospheres, disruption of 3D cell organization has been shown to reduce the efficiency of terminal neuronal differentiation.…”

“…17 During EB cryopreservation, the 3D cell organization is a critical parameter to maintain the recovered cell properties. 17 For adult neurospheres, disruption of 3D cell organization has been shown to reduce the efficiency of terminal neuronal differentiation. 18,19 For PSC-derived NPCs, cryopreservation of the dissociated single cells caused significant apoptosis and required treatment with Rho-associated protein kinase (ROCK) inhibitors or caspase inhibitors to maintain cell viability.…”

“…To date, there are only a few studies for cryopreservation of spontaneously differentiated EBs. 17,21 Especially, the impacts of EB organization and cryopreservation process on neural lineage commitment of EBs postthaw have not been fully characterized.…”