Abstract
Fetal rat dorsal root ganglia (DRG) and spinal cords (SC) slices from rat fetuses were vitrified in a new semiautomatic vitrification system, cooled in sterile slush liquid air (SLA) and stored in a special sterile sealed container in liquid nitrogen (LN). Upon warming organotypic stationary cultures were performed in using NVR-Gel (composed mainly from hyaluronic acid and laminin) and enriched with neuronal factors conjugated to iron oxide nanoparticles. Evaluation of cultures was made by daily phase-contrast microscopy observations and by immune fluorescent staining. Results revealed that SC neurons maintained their multipolar shape and regrew dendrites and axons. The round shape DRG neurons exhibited euchromatic nuclei with prominent nucleoli and an active regeneration of nerve processes. Migration of both neurons and flat cells (fibroblasts and glia Schwann cells) started within 48 hours after seeding and intensified in the upcoming days. In conclusion, it can be said that the using a semi-automatic vitrification, sterile vitrification and sterile storage of neuronal tissues from the CNS and the PNS is a successful advanced technology for the preservation of neurons and glial cells, as shown in the regain of a full regular growth pattern in culture. This may an important step towards clinical use in the reconstruction of severe peripheral nerves and spinal cord injuries.
Highlights
Vitrification is an ice free cryopreservation method which includes a step wise exposure of the sample to high concentrations of cryoprotectants (i.e. DMSO and Ethylene glycol) followed by rapid cooling to the temperature of liquid nitrogen [1,2,3,4]
It can be said that using a semi-automatic vitrification, sterile vitrification and sterile storage of neuronal tissues from the Central Nervous (CNS) and the PNS is a successful advanced technology for the preservation of neurons and glial cells, as shown in the regain of a full regular growth pattern in culture
In order to simplify the vitrification process and to overcome these potential risks of contamination we developed three devices; (1) A semi-automatic system that allows a step wise exposure to vitrification solutions and cooling within the same device (Sarah®, FertileSafe, Nes-Ziona, Israel)
Summary
Vitrification is an ice free cryopreservation method which includes a step wise exposure of the sample to high concentrations of cryoprotectants (i.e. DMSO and Ethylene glycol) followed by rapid cooling to the temperature of liquid nitrogen [1,2,3,4]. Cryopreservation of stem cells and progeny tissue by direct exposure into liquid nitrogen (LN) and their storage in standard LN tanks encompass in it the risk of potential contamination and crosscontamination by viruses, bacteria, fungi and spores that survive in LN and pose a real threat [6,7,8]. In order to simplify the vitrification process and to overcome these potential risks of contamination we developed three devices; (1) A semi-automatic system that allows a step wise exposure to vitrification solutions and cooling within the same device (Sarah®, FertileSafe, Nes-Ziona, Israel). In order to simplify the vitrification process and to overcome these potential risks of contamination we developed three devices; (1) A semi-automatic system that allows a step wise exposure to vitrification solutions and cooling within the same device (Sarah®, FertileSafe, Nes-Ziona, Israel). (2) A device for producing clean liquid air having the same temperature and properties as LN (CLAir®, FertileSafe, NesZiona, Israel). (3) A sterile storage device which enables storing samples in a sterile manner within the standard LN tanks, preserving the LN temperature and insulating the samples from being in contact with the surroundings (Esther®, FertileSafe, Nes-Ziona, Israel)
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