Optimization of freezing, storage, and thawing conditions for the preparation of metabolically active synaptosomes from frozen rat and human brain

Abstract

Samples of rat and human cerebral cortex were frozen, stored, and thawed under a variety of conditions to define further the optimal procedure for storing human brain samples for subsequent metabolic and functional studies that use incubated synaptosomes. Tissue samples were best preserved by immersing them in isotonic sucrose prior to slow freezing, but there was no advantage in first chopping up the material. High concentrations of sucrose, rather than exerting a cryoprotective effect, were detrimental to subsequent synaptosomal performance (oxygen uptake, K⁺ accumulation, stimulus-induced release of amino acid neurotransmitters). However, good activity was observed in preparations from rat brain frozen in the absence of fluid. This result was confirmed by studies on the uptake of γ-aminobutyrate (GABA) into an osmotically sensitive compartment in synaptosomes prepared from frozen human autopsy material transshipped from the brain tissue collection (“brain bank”) at Harvard Medical School, MA, USA, to Sydney, Australia. Although activity was slowly lost over a 3-mo period in rat tissue samples stored at −20°C, there was little or no such loss at −70°C.