A Study of Intracellular Ice Formation in Jurkat Cells

2008 ◽  
Author(s):  
Tathagata Acharya ◽  
Ram V. Devireddy
Keyword(s):  
Cooling Rate ◽  
Jurkat Cells ◽  
Freezing Rate ◽  
Ice Formation ◽  
Substantial Evidence ◽  
Cooling Rates ◽  
Cryoprotective Agents ◽  
Intracellular Ice Formation ◽  
Intracellular Ice ◽  
Phosphate Buffered Saline

The objective of this study was to characterize the IIF behavior of Jurkat cells in isotonic conditions in the absence of any cryoprotective agents. The Jurkat cells were collected from culture and then washed and re-suspended in Dulbecco’s Phosphate Buffered Saline (PBS). The freezing experiments were carried out at defined freezing protocols and at various freezing rates of 5, 20, 30 and 50 °C/min. The results suggest there was no substantial evidence of intracellular ice formation at lower cooling rates of 5, 20 and 30° C/min. The first conspicuous indication of intracellular ice formation (IIF) was observed at a freezing rate of 50 °C/min. At this cooling rate, unlike the usual sudden blackening of cells, the cells suddenly grew and exploded suggesting the formation of intracellular ice, which was reminiscent of a prior observed phenomenon for IIF.

A Simple Cryomicroscopic Method to Measure the Size of Intracellular Ice Crystals

2009 ◽  
Author(s):  
Xu Han ◽  
Hongbin Ma ◽  
John K. Critser
Keyword(s):  
Melting Point ◽  
Ethylene Glycol ◽  
Cooling Rate ◽  
Ice Crystals ◽  
Ice Formation ◽  
Melting Point Depression ◽  
Critical Importance ◽  
Factors Influencing ◽  
Intracellular Ice Formation ◽  
Intracellular Ice

Investigating the factors influencing the characteristics of intracellular ice formation (IIF) is of critical importance for cryopreservation and cryosurgery techniques. However, for the detection of the size of intracellular ice crystals, ∼10nm-0.1μm, there exist serious technical and theoretical difficulties. In this study, a cryomicroscopic method was established to measure the size of intracellular ice crystals in mouse oocytes during their warming processes by investigating the melting point depression of the intracellular ice crystals from extracellular ones. Using the Gibbs-Thomson relation, the size of intracellular ice crystals was calculated and the results range from 4–28 nm, when the molality of the extracellular ethylene glycol and NaCl ranges from 0 to 4m and 0.15 to 0.6m, respectively, and the cooling rate is 100K/min.

scholarly journals Intracellular ice formation in mouse zygotes and early morulae vs. cooling rate and temperature-experimental vs. theory

Cryobiology ◽  
2016 ◽  
Vol 73 (2) ◽  
pp. 181-186 ◽  
Author(s):  
Bo Jin ◽  
Shinsuke Seki ◽  
Estefania Paredes ◽  
Juan Qiu ◽  
Yanbin Shi ◽  
...  
Keyword(s):  
Cooling Rate ◽  
Ice Formation ◽  
Intracellular Ice Formation ◽  
Intracellular Ice ◽  
Mouse Zygotes

Evaluation of Freezing Effects on Human Microvascular-Endothelial Cells (hMEC)

2001 ◽  
Author(s):  
Marwane S. Berrada ◽  
John C. Bischof
Keyword(s):  
Endothelial Cells ◽  
Predictive Ability ◽  
Cell Types ◽  
Cell System ◽  
Ice Formation ◽  
Microvascular Endothelial Cells ◽  
Cooling Rates ◽  
Intracellular Ice Formation ◽  
Intracellular Ice ◽  
Microvascular Endothelial

Abstract There is mounting evidence that the endothelium may play an important role in traditional cryosurgical treatments by acting to locally foster thrombi in the microvasculature of various tissues after freezing. Therefore, this study was designed to investigate, at the cellular level in human microvascular endothelial cells (hMEC), the various biophysical changes that occur during freezing and compare them with post-freeze viability. The hMECs were loaded on a cryomicroscope stage and freezing experiments at 5, 10, 15, 25, 100 and 130°C/min were performed to experimentally evaluate dehydration (water transport) as well as intracellular ice formation (IIF) within this cell system. The dehydration kinetics were found to be governed by a membrane permeability Lpg and activation energy ELp of 0.05 (μm/min.atm) and 14.8 (kcal/mole) respectively [R2 = 0.94]. These parameters were then tested for predictive ability against the experimentally measured behavior at 15°C/min with a good agreement [R2 = 0.98]. Intracellular Ice Formation (IIF) was found to occur at lower temperatures than many cell types (i.e. TIIF 50% ∼ −18°C) and at cooling rates greater than or equal to 25°C/min. At cooling rates above 50°C/min, two types of IIF, cell darkening and twitching, were both observed and quantified and were assumed to be governed by Surface Catalyzed Nucleation (SCN). IIF parameters Ωo, and κo were found to be 6.8 × 10−8 (m2.s)−1 and 8.3 × 10−9 (K5) [R2 = 0.94] respectively. Viability results suggest an inverted U-shape curve between 1 and 50°C/min (with a maximum at 10°C/min). But viability appears to increase again at cooling rates > 50°C/min (i.e. it does not continue to drop) which suggests that the traditional two factor hypothesis may not completely describe viability in this system. Additional cellular destruction was found by lowering the end-temperature to −30°C or below. At this temperature the majority of the cell population was destroyed regardless of the cooling rate.

A Determination of Biophysical Parameters Related to Freezing of an ELT-3 Cell Line

2000 ◽  
Author(s):  
Marwane S. Berrada ◽  
John C. Bischof
Keyword(s):  
Cell Line ◽  
Volume Fraction ◽  
Cell Types ◽  
Cellular Level ◽  
Ice Formation ◽  
Biophysical Parameters ◽  
Cooling Rates ◽  
Intracellular Ice Formation ◽  
Biophysical Study ◽  
Intracellular Ice

Abstract This study investigates two destructive biophysical mechanisms during freezing (extensive dehydration and intracellular ice formation) at the cellular level in the rodent ELT-3 uterine fibroid cell-line. The osmotically inactive volume fraction (Vb) of ELT-3 cells was approximated to 0.35 of the initial isotonic cell volume (Vo). The water transport characteristics of this cell-line are such that ELT-3 cells are highly permeable with a strong ability to lose water even at low subzero temperatures. The hydraulic reference permeability, Lpg and activation energy, Elp associated with Lp were found to be 0.13 (μm/min.atm) and 19.0 (kcal/mole) [R2 = 0.86] respectively. Intracellular Ice Formation (IIF) occurs at lower temperatures than many cell-types (i.e. TIIF 50% below −15°C) at cooling rates > 25 °C/min. Darkening IIF, which was assumed to occur by Surface Catalyzed Nucleation (SCN), is governed by kinetic Ωo and thermodynamic κo biophysical parameters, which were found to be 6.1×108(m2.s)−1 and 5.3×109(K5) [R2 = 0.94] respectively. At a cooling rate of 100°C/min, twitching IIF (non-darkening IIF) was observed. Viability data from a separate study (Bischof et. al., 2000) indicated that at cooling rates ≤ 1°C/min and ≥ 50°C/min with an end-temperature of −20°C, extensive damage to cells was observed. The current biophysical study shows that extensive dehydration occurs at 1°C/min while substantial IIF (77%) occurs at 50 °C/min. This data suggests that while biophysics can explain some of the destruction occurring at the investigated temperatures, other effects or mechanisms may be playing a role at lower end-temperatures.

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