Layman Summary

Cooling complex tissues to ultra-low temperatures produces severe damage, as measured by cell viability tests and microscopy. To prevent this kind of damage, so called “cryoprotectants” are used to minimize ice formation. Vitrification agents use very high concentrations of cryoprotectant to completely inhibit ice formation. VM-1 is a vitrification agent that is optimized for brain cryopreservation. Isolated brain slices can be exposed to VM-1 and recover viability after cooling to cryogenic temperatures. VM-1 also permits ice-free cryopreservation of the whole brain if the agent is introduced through the circulatory system of a mammal. The objective of this research project is to optimize introduction and removal of VM-1 and preserve the fine structure of the brain. First, we compared images of normal brains against brains cooled to 5 degrees Celsius. Then we compared different chemical preservation protocols to prepare cryoprotected brains for electron microscopy. After optimizing a chemical preservation protocol, we compared several protocols to introduce and remove VM-1 from the brain. The best outcome was observed when the brain was chemically prepared for microscopy before removal of the cryoprotectant. This protocol still showed evidence of severe brain shrinking. Further testing and optimization of VM-1 cryoprotection protocols is desired to eliminate this phenomenon.

Scientific Summary

VM-1 is a vitrification agent optimized for the cryopreservation of central nervous tissue and consists of 32.5% (w/v) ethylene glycol and 32.5% (w/v) DMSO in a carrier solution of glucose, potassium chloride, and THAM buffer. Hippocampal brain slices cryopreserved with VM-1 can recover more than 80% cell viability as measured by the potassium / sodium ratio assay. In-situ perfusion of a 35.0% (w/v) ethylene glycol and 35.0% (w/v) DMSO allows for complete ice-free formation of the brain. In this project we aim to optimize an in-situ perfusion and chemical fixation protocol for electron microscopy. Prior efforts to produce VM-1 brain electron micrographs produced sub-optimal images characterized by evidence of membrane damage and cells without ground substance. We compared control images against images of brains cooled to 5 degrees Celsius, VM-1 protocols with and without blood brain barrier modification, and different chemical fixation protocols. The best results were obtained when VM-1 perfusion was followed by chemical fixation of formaldehyde and glutaraldehyde prior to unloading VM-1 from the brain. These VM-1 cryoprotected brains did show histological evidence of severe cryoprotectant-induced dehydration. Additional studies to optimize VM-1 cryoprotection after blood brain barrier modification and cryopreservation will be required to further validate VM-1 for routine use in neural cryobiology. Here you can read the full report.