A new, automated and miniaturized cryo-technique for the cryopreservation of rodent islet cells leads to markedly improved in vitro and in vivo graft function

In the past, we have reported on significant improvements of the conventional cryo-technique resulting in increased islet viability after cryopreservation. However, even with these optimized protocols, the islet cell quality was still markedly impaired after thawing, making its use for clinical transplantation unlikely. Recently we tried to develop a more effective cryo-protocol in cooperation with the Fraunhofer-Institut fur Biomedizinische Technik, St. Ingbert, Germany: the conventional cryovials were replaced by miniaturized micro-cryosubstrates with a better surface to volume-ratio. Further, a specific automated plotter-system was used allowing ultra-fine dispersion of the cryoprotectant (DMSO) during equilibrium. The concentration of the cytotoxic DMSO was reduced to 7%. We evaluated the effects of this innovative automated miniaturized cryotechnique (AMC) on rodent islet cells and compared it to our conventional cryotechnique (CC). With the CC post-thawing islet cell vitality was reduced by 50–60% whereas the AMT exhibited only a loss of 15–25% as evidenced by live-dead staining (n=10). Islet cell function was evaluated. Freshly isolated islets served as controls and showed a 7.6-fold glucose-induced stimulation of insulin secretion. With the CC a markedly reduced stimulation of 1.8 fold was observed (n=10) as compared to 5.1 fold stimulation in AMC (n=10). For the in vivo evaluation immune-deficient nude mice with streptozotocin-induced diabetes were used. Here, a volume of 2000 islet equivalents (IEQ) of freshly isolated rat islets (controls) were necessary to archive normoglycemia via xenotransplantation i.p. With the AMC only 3000 IEQ of croypreserved cells were needed to archive long-term (>10 weeks) normoglycemia (n=3 in each group). We conclude that the new AMC leads to a significantly reduced islet cell damage during the croypreservation process as compared to the conventional technique. These improvements make the clinical use of cryopreserved islets for islet cell banking and transplantation for the first time clinically effective and feasible.