Kinetics and Mechanism of Intercellular Ice Propagation in a Micropatterned Tissue Construct

AbstractUnderstanding the effects of cell–cell interaction on intracellular ice formation (IIF) is required to design optimized protocols for cryopreservation of tissue. To determine the effects of cell–cell interactions during tissue freezing, without confounding effects from uncontrolled factors (such as time in culture, cell geometry, and cell–substrate interactions), HepG2 cells were cultured in pairs on glass coverslips micropatterned with polyethylene glycol disilane, such that each cell interacted with exactly one adjacent cell. Assuming the cell pair to be a finite state system, being either in an unfrozen state (no ice in either cell), a singlet state (IIF in one cell only), or a doublet state (IIF in both cells), the kinetics of state transitions were theoretically modeled and cryomicroscopically measured. The rate of intercellular ice propagation, estimated from the measured singlet state probability, increased in the first 24h of culture and remained steady thereafter. In cell pairs cultured for 24h and treated with the gap junction blocker 18β-glycyrrhetinic acid before freezing, the intercellular ice propagation rate was lower than in untreated controls (p<0.001), but significantly greater than zero (p