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Freeze-drying of red blood cells: how useful are freeze/thaw experiments for optimization of the cooling rate?

A red blood cell suspension, prepared according to a high-yield HES cryopreservation protocol, was frozen at selected cooling rates of 50, 220, 1250, 4200, and 13,500 K/min. After either thawing or vacuum-drying, the cell recovery was determined using a modified saline stability test. As expected, the recovery of thawed samples followed the theory of Mazur's two-factor hypothesis. The best result was found at a cooling rate of 220 K/min. In contrast, the recovery of freeze-dried and rehydrated samples was very poor at that rate, but maximal at 4200 K/min where thawing caused almost complete hemolysis. This discrepancy is attributed to different damaging mechanisms involved with the respective sample processing subsequent to freezing. While thawing leads to increased devitrification and recrystallization at supraoptimal cooling rates for cryopreservation, the resultant almost vitreous sample structure seems to be advantageous for vacuum-drying. It can be concluded that freeze/thaw experiments are not sufficient for optimization of the cooling rate for freeze-drying.