(596ae) 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.