Table 2.

Procedural description, examples, and trade-offs among classes of structural brain preservation methods.

Method	Procedure	Upsides	Downsides
Unprotected cryopreservation	•Sub-zero cooling of brain tissue in the absence of cryoprotectants •Variations: cooling rate, storage temperature •Ex: (51)	•Widely available initial preservation procedure •Biomolecule distributions will be altered, but minimal direct chemical changes	•Inevitable ice damage causes morphologic artifacts •Any unplanned rewarming would damage cell morphology •Long-term storage requires significant cost
Cryopreservation with CPAs	•Cryoprotectants perfused during cooling, potentially allowing vitrification •Variations: CPAs used, cooling rate •Ex: (52)	•After CPAs are removed, tissue microstructure looks intact •CPAs tend to minimally alter biomolecules •Plausibly on the shortest path to suspended animation	•Cryoprotectant toxicity •Relies on high-quality perfusion to avoid ice damage, which can be challenging postmortem •Long-term storage requires significant cost
Fixation and cryopreservation	•Chemical fixation and subsequent CPA-based cryopreservation •Variations: CPAs perfused or immersed •Ex: (53)	•Connectome preservation by vitrification after fixation shown in mammals (Brain Preservation Foundation, 2018) •Fallback of chemical preservation if low temperature storage fails	•Can be reliant on perfusion quality •Long-term storage requires significant cost •Reversing crosslinks is well beyond our current technology (also applies to all methods below)
Fluid preservation	•Fixation, then long-term storage in a liquid preservative solution •Variations: fixation method, chemicals used, storage temperature •Ex: (54)	•Simple and inexpensive •Widely used with a large infrastructure in place •Appears to retain morphology and classes of biomolecules for decades	•Chemical reactions over time will alter biomolecules •Some degree of loss of biomolecules that are not directly crosslinked •Possible storage artifacts
Polymer embedding	•Fixation, then processing and embedding •Variations: paraffin, epoxy, polyester, etc. for embedding •Ex: (55)	•Can have excellent ultrastructural preservation •Minimal chemical reactions occur during storage	•Biomolecular extraction during embedding procedures •Challenging to perform on human brain scale without sectioning first •Can be expensive

CPA, cryoprotective agent; Ex, example.