This lack of modeling to minimize toxicity is surprising in

This lack of modeling to minimize toxicity is surprising in some ways, because CPA toxicity has been highlighted as one of the chief impediments to successful cryopreservation of tissues and organs [28]. This, coupled with the fact that great gains have been made in the area of single cell suspension equilibration protocol optimization, including human and bovine oocytes [9], [24], [32], plated endothelial nm to lb [14], sperm [18], [19] and others, suggests that gains in CPA toxicity minimization may be made in tissues as well. This gain can be seen in Fig. 8, where the accumulated toxicities as a function of protocol are shown for each tissue type. Here we can see that the standard stepwise approach is neither faster nor less toxic than the toxicity optimal protocol, and that, if CPA toxicity is not a concern, time optimal protocols can be 4–6 times shorter than the non-optimized standard stepwise approach and 2–3 times shorter than their respective toxicity optimal protocols. Our approach relied on measurement of bulk solute diffusivity in the three tissue types of interest. Our diffusivity values (Table 3) are on the order of 10−6 cm2/s and are comparable to the PG diffusion coefficient in water at infinite dilution, which is about 10−5 cm2/s at room temperature, as well as the diffusivity at a PG mole fraction of 0.2, which is about 5 × 10−6 cm2/s [38]. We also note that the skin tissue has a diffusivity value that is half that of the other tissues. This might be related to the dense packing of the epidermis layer in skin when compared with myometrial and fibroid tissues. While our desorption curves were well-fit by our simple linear diffusion model, we note that, at the very least, diffusivity is typically considered a function of concentration [38] and that we do not model the interaction among the CPA and the base media nonpermeating solutes. It would be more appropriate to use a transport model that accounts for the salt, water, and CPA concentrations throughout. In fact, our approach neglecting the movement of salt and water is limiting in some ways because our previous work on CPA equilibration optimization [5], [8], [9], [14] relied on media containing only CPA, instead of including standard nonpermeating solutes. By omitting the nonpermeating solute in these studies, an additional 300 mOsm of permeating solute could be used at each step, increasing the speed at which equilibration occurred. This more complete model is a subject of our future work. In the present study we demonstrate that a clear choice exists between minimal time and minimal toxicity protocols. Minimal time protocols are simply constrained by the osmotic tolerance limit constraint: in their absence a minimal time optimal equilibration protocol would be to place the tissue in a media of concentration Cmax until the mass transfer model predicts that the center of the tissue has concentration CD. In fact, because of this, minimal time protocols can be predicted using the much simpler single cell dynamics. One could then use the minimal time optimal protocols that use continuous concentration controls instead of multistep protocols (see e.g. Benson et al. [9]). Considerable gains in time-saving could be made in this case.