Successful survival in culture of spinal cord (SC) dorsal root ganglia (DRG) neurons and glia cells after automatic vitrification

Abstract

To assess the survival and re-growth of neuronal cells after cryopreservation with a new automatic vitrification system. Experimental research with SC and DRG isolated from rat fetuses. Stationary organotypic DRG and SC cultures were prepared from rat fetuses (gestational day 15, Lewis inbred, Harlan, Israel). mmediately after dissection, the DRG and SC were cut into small slices (400 micron) and seeded in 12 well culture plates containing 1 mL of NVR-Gel. Cultures were enriched once in gel with GDNF conjugated iron oxide nanoparticles (10 ng/mL) and subsequently with nutrient medium at each consecutive feeding. Monitoring of the DRG-SC growth pattern was done by daily phase contrast microscopic observations 24 hours after setting the cultures onward and by IF staining. The various neuronal samples (SC, DRG, glia cells) were cryopreserved with a new device for automatic vitrification (Sarah®, Fertilesafe, IL), consisting of a special capsule containing EM gold grids attached to 0.25ml straws (IMV,France). The straws loaded with neuronal tissue were placed into the mixing chamber of the device attached to a syringe pump dispensing timed and gradual increasing concentrations of equilibrium solution for 12-25 min. and vitrification solutions for 3-5 min., before plunginginto liquid nitrogen slush (VitMaster, IMT IL). After 1 week the samples were rewarmed according to the Origio’s kit instructions. To assess survival, the cells recovered were fixed (4% paraformaldehyde), permeabilized with 0.1% Triton X-100 in PBS and then immunoblocked with a 1% BSA in PBS for 1 h at RT prior to double staining with mouse anti S-100 antibodies (1:80, Acris Antibodies, glial cell marker) and rabbit anti neurofilament antibodies (NF, Novus Biologicals, 1:500, neuronal cell marker). The primary antibodies were diluted in 0.1% BSA and 0.05% Tween-20 in PBS and incubated with the specimens overnight at 4 °C. After rinsing, the DRG specimens were incubated for 1 h at RT with fluorescent secondary antibodies. Organotypic cultures of SC, DRG neurons and glia cells demonstrated a remarkable survival as well as nerve fiber regeneration after cryopreservation/rewarming. The SC neurons maintained their multipolar shape with re-growth of dendrites and axons. The round shaped DRG neurons exhibited euchromatic nuclei with prominent nucleoli and an active regeneration of nerve processes. This is the first report on successful vitrification of SC cells, DRG neurons and glia cells using an automatic freezing device. The remarkable resumption of growth for neuronal cells will greatly expand regenerative research opportunities.