Cryomicroscopic Investigations of Freezing Processes in Cell Suspensions

Acharya, Tathagata; Devireddy, Ram V.

doi:10.2174/1874070701004010026

Cited by 17 publications

(6 citation statements)

References 57 publications

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“…In sharp contrast at the microscopic level, both ice crystallization and gas emboli have been proposed as the root cause of “blackening” or “darkening” of the intracellular compartment of individual cells observed by cryomicroscopy during cooling. This so-called “flashing” has conventionally been adopted as the definitive sign of intracellular freezing [1,15,34] and is clearly associated with a change of refractive index in the cytosol due to a physical change of state. These well documented phenomena are associated with events at the microscopic (sub-cellular) level and do not necessarily translate to the physical events now observed at the macroscopic level using the Cryomacroscope-III device.…”

Section: Resultsmentioning

confidence: 99%

A new cryomacroscope device (Type III) for visualization of physical events in cryopreservation with applications to vitrification and synthetic ice modulators

et al. 2013

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The objective of the current study is to develop a new cryomacroscope prototype for the study of vitrification in large-size specimens. The unique contribution in the current study is in developing a cryomacroscope setup as an add-on device to a commercial controlled-rate cooler and in demonstration of physical events in cryoprotective cocktails containing synthetic ice modulators (SIM)—compounds which hinder ice crystal growth. Cryopreservation by vitrification is a highly complex application, where the likelihood of crystallization, fracture formation, degradation of the biomaterial quality, and other physical events are dependent not only upon the instantaneous cryogenic conditions, but more significantly upon the evolution of conditions along the cryogenic protocol. Nevertheless, cryopreservation success is most frequently assessed by evaluating the cryopreserved product at its end states—either at the cryogenic storage temperature or room temperature. The cryomacroscope is the only available device for visualization of large-size specimens along the thermal protocol, in an effort to correlate the quality of the cryopreserved product with physical events. Compared with earlier cryomacroscope prototypes, the new Cryomacroscope-III evaluated here benefits from a higher resolution color camera, improved illumination, digital recording capabilities, and high repeatability in tested thermal conditions via a commercial controlled-rate cooler. A specialized software package was developed in the current study, having two modes of operation: (a) experimentation mode to control the operation of the camera, record camera frames sequentially, log thermal data from sensors, and save case-specific information; and (b) post-processing mode to generate a compact file integrating images, elapsed time, and thermal data for each experiment. The benefits of the Cryomacroscope-III are demonstrated using various tested mixtures of SIMs with the cryoprotective cocktail DP6, which were found effective in preventing ice growth, even at significantly subcritical cooling rates with reference to the pure DP6.

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Section: Resultsmentioning

confidence: 99%

A new cryomacroscope device (Type III) for visualization of physical events in cryopreservation with applications to vitrification and synthetic ice modulators

et al. 2013

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“…During cryomicroscopy, a sudden change in opacity of the cytoplasm is often described as “blackening” or “flashing” (Acharya and Devireddy 2010 ; Day et al 2000 ; Scheiwe and Korber 1984 ; Smith and Smiles 1953 ). The cause for blackening is not completely understood.…”

Section: Discussionmentioning

confidence: 99%

Impact of drying and cooling rate on the survival of the desiccation-sensitive wheat pollen

2022

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Key message Fast-drying and cooling induce fast intracellular water loss and reduced ice-crystal formation, which may promote the formation of intracellular glasses that might improve the likelihood of wheat pollen survival. Abstract Long-term storage of pollen is important for the fertilization of spatially or temporally isolated female parents, especially in hybrid breeding. Wheat pollen is dehydration-sensitive and rapidly loses viability after shedding. To preserve wheat pollen, we hypothesized that fast-drying and cooling rates would increase the rate of intracellular water content (WC) removal, decrease intracellular ice-crystal formation, and increase viability after exposure to ultra-low temperatures. Therefore, we compared slow air-drying with fast-drying (dry air flow) and found significant correlations between pollen WC and viability (r = 0.92, P < 0.001); significant differences in WCs after specific drying times; and comparable viabilities after drying to specific WCs. Fast-drying to WCs at which ice melting events were not detected (ΔH = 0 J mg−1 DW, < 0.28 mg H2O mg−1 DW) reduced pollen viability to 1.2 ± 1.0%, but when drying to 0.39 mg H2O mg−1 DW, some viable pollen was detected (39.4 ± 17.9%). Fast cooling (150 °C min−1) of fast-dried pollen to 0.91 ± 0.11 mg H2O mg−1 DW induced less and a delay of ice-crystal formation during cryomicroscopic-video-recordings compared to slow cooling (1 °C min−1), but viability was low (4.5–6.1%) and comparable between cooling rates. Our data support that the combination of fast-drying and cooling rates may enable the survival of wheat pollen likely due to (1) a reduction of the time pollen would be exposed to drying-related deleterious biochemical changes and (2) an inhibition of intracellular ice-crystal formation, but additional research is needed to obtain higher pollen survival after cooling.

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“…This model is based on the catalytic mechanisms including both surface-catalyzed nucleation (SCN) and volumecatalyzed nucleation (VCN) [15,22]. However, this model assumes that apparent ice should form in all cells (i.e., PIF = 1) if they are frozen to low enough temperature, which is not necessary tenable according to several recent experimental observations [14,26,27]. Therefore, it is of significance to further improve the PIF model for better prediction and understanding of ice nucleation in living cells during freezing.…”

Section: Introductionmentioning

confidence: 99%

An Improved Model for Nucleation-Limited Ice Formation in Living Cells during Freezing

Liang

Zhao

et al. 2014

PLoS ONE

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Ice formation in living cells is a lethal event during freezing and its characterization is important to the development of optimal protocols for not only cryopreservation but also cryotherapy applications. Although the model for probability of ice formation (PIF) in cells developed by Toner et al. has been widely used to predict nucleation-limited intracellular ice formation (IIF), our data of freezing Hela cells suggest that this model could give misleading prediction of PIF when the maximum PIF in cells during freezing is less than 1 (PIF ranges from 0 to 1). We introduce a new model to overcome this problem by incorporating a critical cell volume to modify the Toner's original model. We further reveal that this critical cell volume is dependent on the mechanisms of ice nucleation in cells during freezing, i.e., surface-catalyzed nucleation (SCN) and volume-catalyzed nucleation (VCN). Taken together, the improved PIF model may be valuable for better understanding of the mechanisms of ice nucleation in cells during freezing and more accurate prediction of PIF for cryopreservation and cryotherapy applications.

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Cryomicroscopic Investigations of Freezing Processes in Cell Suspensions

Cited by 17 publications

References 57 publications

A new cryomacroscope device (Type III) for visualization of physical events in cryopreservation with applications to vitrification and synthetic ice modulators

A new cryomacroscope device (Type III) for visualization of physical events in cryopreservation with applications to vitrification and synthetic ice modulators

Impact of drying and cooling rate on the survival of the desiccation-sensitive wheat pollen

An Improved Model for Nucleation-Limited Ice Formation in Living Cells during Freezing

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