IDENTIFICATION AND MINIMIZATION OF A TOXICITY COST FUNCTIONAL FOR THE OPTIMAL ADDITION AND REMOVAL OF CRYOPROTECTANTS
James D. Benson, Anthony J. Kearsley, Adam Higgins
Cryopreservation nearly universally depends on the equilibration of cells and tissues with high concentrations of permeating chemicals known as cryoprotective agents, or CPAs. Despite their protective properties, CPAs can cause damage as a result of osmotically-driven cell volume changes, as well as chemical toxicity. Only recently have non-heuristically optimized protocols been developed to account for these two competing factors, and previous optimization strategies have relied on the simplifying assumption that minimizing protocol duration is equivalent to minimizing toxicity. However, besides duration of exposure, there are several other factors that are known to affect toxicity, including the CPA concentration. In this study, we have used previously published data to determine a concentration-dependent toxicity cost functional, a quantity that represents the cumulative damage caused by toxicity. We then used this cost functional to define and numerically solve the optimal control problem for CPA equilibration. The resulting toxicity-optimal procedures are predicted to yield significantly less toxicity than conventional stepwise procedures. In particular, our results show that toxicity is more pronounced during CPA addition than removal, and that toxicity is minimized during addition by inducing the cell to swell to its maximum tolerable volume and then loading it with CPA while in the swollen state. This counterintuitive result is considerably different from the conventional stepwise strategy, which involves exposure to successively higher CPA concentrations in order to avoid excessive shrinkage. The procedures identified in the present study have the potential to significantly reduce damage due to toxicity and warrant further investigation.