Kidney tissue loading reduces the critical cooling and warming rates of VS55 and VMP cryoprotective solutions

Critical cooling and warming rates (CCR and CWR) are two important calorimetric properties of cryoprotective agents (CPA) solutions, and achieving these rates is generally regarded as the critical criterion for successful vitrification and rewarming. In 1996, Peyridieu et al. discovered that the measured critical rates are reduced inside kidney tissue equilibrated with 30 % (w/w) 2,3-butanediol compared to its free CPA solution. In general, they found a ∼5-fold reduction for CCR and a >100-fold reduction for CWR. However, to our knowledge, no follow-up studies have been conducted. We revisit this important concept, understanding that tissues never fully equilibrate with full-strength 100 % CPAs during perfusion. We therefore performed measurements in a range of dilutions of two commonly employed CPA cocktails, including 75-100 % VS55 (41.25-55.00 % w/v) and 90-100 % VMP (48.60-54.00 % w/v) equilibrated with kidney tissues vs. free solution. The measured reduction in the kidney was up to 5-fold for CCR and 9-fold for CWR. After discussing possible mechanisms for this effect, curves that fit the dilution to the observed reduction in critical rate were constructed to allow extrapolation for differentially loaded tissues, which can guide the follow-up studies to find the more concentrated CPA (>8.4 M VMP) in the M22 family to achieve human-sized kidney vitrification and rewarming.