Layman summary

Organ cryopreservation is a branch of cryobiology, its central purpose being to cryopreserve organs in liquid nitrogen in order to keep its biological properties for as long as possible. So far, reanimation of cryopreserved mammalian organs has not been achieved. In this context our main objective is to optimize cryopreservation technology in rat brains, used as a model of a mammalian organ. Specifically, the present project is intended as a contribution to assessing the effectiveness of the cryopreservation procedure known as vitrification which is based on the replacement of the animals’ blood by a cryopreservation solution that will not damage brain tissue after freezing and thawing as aqueous media like blood do. After thawing, the structural integrity of brain anatomy and brain cells was assessed by techniques currently in use in our laboratory. During the first year of the project our results revealed that serially embedding the brains in ethylene glycol (EG)-based cryopreservation solutions which are progressively richer in EG, up to 70%, fully preserve the anatomical features of the organ. At tissue level we observed some deterioration of certain types of neurons but not of others. Next year we will compare the effectiveness of our procedure on brains of different ages.

Scientific Summary

The aim of the project is to assess the degree of histologic and anatomical preservation of rat brains after pre-mortem perfusion with a vitrification protocol used in human patients, scaled down for young rats, followed by a 3-stage cooling protocol that ends in liquid nitrogen. This protocol will be followed, between 2 and 7 days later, by rewarming and standard fixation, sectioning, and Nissl and immunohistochemical staining of relevant brain regions. Young adult Sprague–Dawley female rats will be placed under deep anesthesia and intracardially perfused with three cryopreservation solutions progressively enriched in ethylene glycol (EG), which will range from 10 to 70% EG. The third solution (termed VM1) includes 15% dimethyl sulfoxide (DMSO) as a permeabilizing agent. Different experimental groups of 4-5 rats each will be used. A control group which will be perfused with fixative (paraformaldehyde 4% in buffered saline) and not frozen (standard procedure) and the vitrified groups, which will be perfused with the vitrification solutions and frozen as indicated. Comparisons will be made for the different parameters under study in the different experimental groups, which will be perfused immediately or 90 minutes after death. This should clarify the impact of the length of time between death and vitrification on the preservation of brain structural integrity.

Here you can read the first year report.