A cryoprotectant solution took about nine months to permeate an entire fixed human brain

A cryoprotectant solution took about nine months to permeate an entire fixed human brain

On August 24, PLOS One published a protocol for storing whole human brains at −20 °C after their cellular structure had been stabilized by chemical fixation. The authors measured the solution's movement from the surface into deeper regions in three such brains. They assessed the procedure's effects on tissue using small samples from two canine brains.

Fixation chemically stabilizes tissue structure after death. Before cooling, the brain is gradually infused with a cryoprotectant solution. Ethylene glycol reduces the likelihood of ice formation, while sucrose increases the solution's viscosity. The final mixture contained 50% ethylene glycol and 30% sucrose.

In a whole organ, the solution reaches deep regions much more slowly than it reaches the surface. The authors performed several computed tomography scans of three fixed human brains and compared measurements from the cortex and deeper regions. They considered the solution evenly distributed once these measurements stopped changing. This had occurred throughout the entire brain volume by day 276 of the protocol. White matter, where nerve fibers are covered by a fatty sheath called myelin, became saturated more slowly than gray matter. The authors then stored one of the brains at −20 °C for two months and sectioned it without finding visible evidence of ice.

Microscopy was used to determine whether the tissue's fine structure remained intact after infusion, storage, and subsequent removal of the solution. In the first experiment, small samples from canine brains underwent three days of infusion. The white matter developed sharply defined voids and compressed fibers, a pattern consistent with artifacts caused by ice. In the second experiment, the researchers allowed more time for saturation, removed the solution in smaller concentration steps, and introduced glutaraldehyde earlier. Glutaraldehyde helps cells retain their shape as the solution concentration changes.

Light microscopy found no such voids in samples processed with the revised protocol. Under electron microscopy, preservation scores did not differ statistically between six cryopreserved samples and six controls. Computed tomography showed whether the solution had reached the deep regions of an entire brain. Microscopy showed whether the tissue's fine structure remained intact afterward.

In this protocol, the waiting period before cooling can be linked to the time required for the solution to become distributed throughout the brain volume.