Size-Dependent Cryopreservation of Pluripotent Stem Cell Aggregates

Size-dependent Cryopreservation of Pluripotent Stem Cell Aggregates

Sebastien Sart, Teng Ma, Yan Li*

In recent years, pluripotent stem cells (PSC) are expanded as aggregates in suspension culture, which enables the large scale production in stirred bioreactors.  During the production, PSCs are usually thawed from one vial of cells from Master Cell Bank (MCB) or Working Cell Bank (WCB).  Integrating the culture system with cryopreservation process improves the feasibility of large-scale PSC banking and expansion.  The optimized cryopreservation method also improves cell expansion and the differentiation.  In this study, cryopreservation of PSC aggregates as dissociated single cells and intact aggregates was compared.  It was found that intact aggregates quickly recovered after thawing in suspension.  The size of aggregates affected the recovery and small aggregates (80-90 µm in diameter) recovered better than medium and large aggregates (average diameter 180 µm and 300 µm).  Labeling with CellTracker Red showed the aggregate size-dependent diffusion.  The production of reactive oxygen species (ROS) for the aggregates post-thaw was also investigated.  It was observed that ROS production was initiated from aggregate center and small aggregates generated significant less ROS compared to medium and large aggregates.  Different cryoprotectants were compared and PSC aggregate can be frozen in defined xeno-free formulation.  The post-thawed aggregates maintained extracellular matrix (ECM) secretion, pluripotent marker expression, and the ability to differentiate into three-germ layers.  The developed method can be used for establishing MCB and WCB for large scale PSC-derived cell production.  This study also provides the insight for microtissue cryopreservation toward tissue engineering applications.