Intracellular ice formation: cryomicroscopical observation and calorimetric measurement

Abstract

, differential scanning calorimetry, human lymphocytes, darkening and twitching types of intracellular ice formation, freezing injury, threshold cooling rate, crystallization temperature, influence of the cryo-additive dimethyl sulphoxide, nucleation mechanisms.The formation of ice crystals within biological cells is generally deleterious and results in a severe loss of cellular viability and function. With the aim of circumventing this lethal event, the mechanisms of nucleation and their dependence on governing parameters such as temperature, cooling rate and solute and/or additive concentration, and the correlation with the osmotically induced water transport across the cell membrane were investigated. Quantitative low-temperature light microscopy was used for this purpose as it offers the major advantage of studying the dynamics of the involved processes. T o substantiate further the visual observations of the morphological changes associated with intracellular ice formation, supplementary studies by differential scanning calorimetry (DSC) were performed under comparable conditions to measure the quantity of water actually transformed into the crystalline state due to the evolution of latent heat.Human lymphocytes were used as a biological model cell. In particular it could be shown that the twitching type of intracellular ice formation which is evident but difficult to observe under the cryomicroscope can be attributed to a liquid-solid phase change within the cells as determined by DSC. Good agreement was obtained between the results measured by both techniques with respect to the following dependencies of governing parameters: the fraction of cells exhibiting intracellular ice determined as a function of the cooling rate shows a sharp demarcation zone with an increase from 0 to loo",, at about the same threshold cooling rate. On the other hand, the temperatures at which intracellular ice forms were found to be only weakly dependent on the cooling rate. With respect to the effect of cryo-additive concentration at a fixed value of the cooling rate, the crystallization temperatures were seen to decrease with concentration. T h e D S C results may hence be regarded as a validation of the microscopic observations.

Polyvinylpyrrolidone (PVP) Mitigates the Damaging Effects of Intracellular Ice Formation in Adult Stem Cells

“…3c, 3d, 3g, and 3h). As expected, these results are in agreement with earlier studies that suggest that IIF is damaging to cells 1,6,7,15,16,21,24,[56][57][58] and in disagreement with other studies that suggest ice formation can be innocuous to cells. 2,3,26,37,41,42,45,60 Cooling Protocol 2 (in 19 PBS and 10% PVP)…”

“…This result is not surprising as it is well known that the size of ice crystals formed during nucleation is inversely correlated with the amount of super-cooling. 6,7,10,12,[14][15][16]24,32,36,43,44,48,[56][57][58] Most importantly, further cooling of these cells from À5°C to À40°C at various cooling rates (1,5,10,20, and 40°C/min) did result in re-nucleation or ''darkening/flashing'' in most of the cells, i.e. most of the frozenthawed cells exhibited IIF.…”

“…During cooling the formation of extra-and intra-cellular ice was visually observed by the associated changes in crystalline structure and the darkening/flashing of cells, respectively. 6,7,15,16,24,[56][57][58] Viability Experiments…”

“…6,7,10,12,15,16,24,32,36,43,44,48,[56][57][58] Briefly, P1 ASCs were detached using trypsin (ATCC, Manassa, VA) and transferred to a 1.5 mL centrifuge tube, spun down for 5 min at 1200 rpm, and re-suspended in 50 lL of either 19 phosphate buffered saline (PBS) or 10% PVP (w/v) in 19 PBS solution. Following this, 2.5 lL of the sample was then transferred to the high thermal conductivity quartz crucible on the BCS-196 cryostage (Linkam Scientific TM , Surrey, UK).…”

Freezing Characteristics of Genetically Modified Lymphocytes for the Treatment of MPS II

“…The formation of intracellular ice during the freezing of lymphocytes has been studied previously (18,32). The maximum cumulative fraction of cells with IIF as a function of cooling rate and the average temperature for IIF was measured.…”