Optimum conditions for cryoquenching of small tissue blocks in liquid coolants

Abstract

K E Y w ORDS. Cryoquenchant, propane, freeze substitution, Freon 22, ice crystal damage, cooling rates. SUMMARY Three .approaches were taken with the aim of defining the optimum conditions for rapid cryopreservation in liquid quenchants. In a theoretical approach, two mathematical models were used. The first is of value in defining the absolute maximum rates of cooling which could be achieved at various depths in the tissues. The second highlights the poor thermal properties of liquid coolants and therefore emphasizes the essential requirement for vigorous quenchant mixing and rapid specimen entry.Experimental work with thermocouples showed that fastest cooling rates occur at the leading edge of the object entering coolant. Of five liquid quenchants investigated, cooling rates were in the order, propane > Freon 22 > Freon 12 > liquid nitrogen slush > liquid nitrogen. Other considerations, however, may affect the choice of quenchant. For a given quenchant, cooling rate is maximal near the equilibrium freezing point. The consequences of quenching in the presence of thermal gradients either within the coolant or in the gas layer above it are shown. Cooling rate was found to be approximately proportional to entry velocity at least up to N 2 m s-1 in our system. Stereological analysis of rapidly quenched, freeze-substituted tissue samples, of geometry which imposed an approximately unidirectional heat flow, revealed four zones : (i) a narrow surface layer ( N 10 pm) of low image contrast and apparent absence of ice crystals; (ii) a zone of enhanced contrast with ice crystals whose size increased rapidly with depth from the surface (the 'slope'); (iii) a sharply defined zone (the 'ridge') of maximum ice crystal size beyond which there is (iv) an extensive 'plateau' with smaller ice crystals and no marked increase in size with depth. The 'ridge' of maximal ice-crystal damage was consistently found but varied considerably in depth from the surface ( N 25-120 pm) between samples. The existence of the deeper plateau region of relatively uniform ice-crystal-size may be of significance in X-ray microanalytical studies of physiological processes at some depth from the sample surface.In terms of our present understanding of the quenching process, the conditions for optimal cryofixation of small tissue samples are listed.

Temporal coincidence between synaptic vesicle fusion and quantal secretion of acetylcholine.

“…nents) and its low time resolution for physiological events. Among the various cryotechniques (for review see reference 40), pressing living tissues against a copper block cooled by liquid helium to a temperature of ~ 15*K appears to be one of the quickest way of freezing thick biological materials for excellent morphological-preservation (1,12,17,19,43). Indeed, to capture rapid morphological changes that occur concomitantly with transient physiological processes, such as synaptic transmission, one might require cooling rates higher than those needed to prevent formation of visible ice crystals.…”

Gap junction connexon configuration in rapidly frozen myocardium and isolated intercalated disks.

“…The tendon (which consists largely of collagen with some elastic elements, but no cardiac muscle cells) was cut above the forceps, allowing the muscle to be raised for clamping between thin, prealigned copper support plates (BU012-056-T, Balzers High Vacuum Ltd ., Berkhamsted, England) temporarily glued onto fine curved forceps . The muscle itself was then cut free at its base, and the sandwich was immediately plunged rapidly by hand into liquid propane under conditions designed to achieve optimal cooling rates (10,35,37) . After transfer into liquid nitrogen, the sandwich was freed from the forceps and stored .…”

Validation of Freeze-Drying to Visualize Percutaneous ³H-Estradiol Transport: The Influence of Skin Hydration on the Efficacy of the Method

“…Both skin from dry dose and patch experiments were suitable for freezedrying. The ultrastructural quality was limited to the outer 10-20 Ìm of the skin sample, which was expected, as it is the region that vitrifies during cryofixation [13]. Fortunately, this distance roughly matches the thickness of the layer of prime interest, the stratum corneum.…”

“…Skin Pharmacol Appl Skin Physiol 1998;11:11-22 13 wipes. Skin which had been in contact with the PBS formulation was rinsed briefly in PBS.…”