Effect of the Cell Alive System on nerve tissue cryopreservation

Abstract

Effective cellular cryopreservation while maintaining high cell viability is achieved by preventing intracellular and extracellular ice crystal formation using the Cells Alive System (CAS), a programmed freezer that applies a magnetic field. Here, the optimal temperature settings of the CAS were determined using rat sciatic nerves as a model tissue. Firstly, it was found that Schwann cell survival was increased by pre-cooling the samples in the ice crystal formation zone, increasing the freeze–thaw speed, and freezing–thawing in a magnetic field. Secondly, the setting (intensity and frequency) of the magnetic field at freezing–thawing was changed, and the optimum magnetic field strength was determined by evaluating cell viability. At the set temperature excluding previous studies, the minimum temperature was set to − 50 °C and kept frozen for 15 min, and then thawed immediately. The highest cell viability (27%) was achieved at 0.67 mT (intensity 3 [29.6 V] and frequency setting 10 [60 Hz]). The effects of the freeze–thaw program were assessed using transplanted sciatic nerve tissues removed after 2, 4, and 8 weeks. Anterior tibial muscle wet weight increased at 8 weeks in the control (without freezing) and after freezing–thawing in a magnetic field, compared to that without a magnetic field. Fluorescence staining of the sciatic nerve with anti-S100 antibodies revealed that Schwann cell counts increased at the transplanted site (at 8 weeks) of nerves that were freeze–thawed in a magnetic field. Overall, the CAS prevented ice crystal formation in rat sciatic nerves and could be used to maintain cell viability during cryopreservation.