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.

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.

scholarly journals Effect of the expression of aquaporins 1 and 3 in mouse oocytes and compacted eight-cell embryos on the nucleation temperature for intracellular ice formation

Reproduction ◽  
2011 ◽  
Vol 142 (4) ◽  
pp. 505-515 ◽  
Author(s):  
Shinsuke Seki ◽  
Keisuke Edashige ◽  
Sakiko Wada ◽  
Peter Mazur
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Cell Types ◽  
Ice Nucleation ◽  
Nucleation Temperature ◽  
Ice Formation ◽  
Mouse Oocytes ◽  
Intracellular Ice Formation ◽  
Intracellular Ice ◽  
Mature Mouse

The occurrence of intracellular ice formation (IIF) is the most important factor determining whether cells survive a cryopreservation procedure. What is not clear is the mechanism or route by which an external ice crystal can traverse the plasma membrane and cause the heterogeneous nucleation of the supercooled solution within the cell. We have hypothesized that one route is through preexisting pores in aquaporin (AQP) proteins that span the plasma membranes of many cell types. Since the plasma membrane of mature mouse oocytes expresses little AQP, we compared the ice nucleation temperature of native oocytes with that of oocytes induced to express AQP1 and AQP3. The oocytes were suspended in 1.0 M ethylene glycol in PBS for 15 min, cooled in a Linkam cryostage to −7.0 °C, induced to freeze externally, and finally cooled at 20 °C/min to −70 °C. IIF that occurred during the 20 °C/min cooling is manifested by abrupt black flashing. The mean IIF temperatures for native oocytes, for oocytes sham injected with water, for oocytes expressing AQP1, and for those expressing AQP3 were −34, −40, −35, and −25 °C respectively. The fact that the ice nucleation temperature of oocytes expressing AQP3 was 10–15 °C higher than the others is consistent with our hypothesis. AQP3 pores can supposedly be closed by low pH or by treatment with double-strandedAqp3RNA. However, when morulae were subjected to such treatments, the IIF temperature still remained high. A possible explanation is suggested.

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.

KrioBlastTM: A Hyper-Fast Cooling and Thawing Scalable Device for Vitrification of Stem and Other Cells in Large Volumes

2012 ◽  
Author(s):  
Vladimir F. Bolyukh ◽  
Igor I. Katkov ◽  
Vsevolod Katkov ◽  
Ilya Yakhnenko
Keyword(s):  
Stem Cells ◽  
Small Sample Size ◽  
Small Sample ◽  
Ice Formation ◽  
Intracellular Ice Formation ◽  
Order Of Magnitude ◽  
Leidenfrost Effect ◽  
Intracellular Ice ◽  
Fast Cooling ◽  
New System

Kinetic (very rapid) vitrification (KVF) is a very promising approach in cryopreservation (CP) of biological materials as it is simple, avoids lethal intracellular ice formation (IIF) and minimizes damaging dehydration effects of extracellular crystallization. Moreover, achieving the ultra-high rates, which would prevent IIF during cooling and devitrification during resuscitation, and achieve KVF for practically any type of cells with one protocol of cooling and re-warming would be the “Holy Grail” of cell cryobiology [3]. However such hyperrapid rates currently require very small sample size which, however, is insufficient for many applications such as stem cells, blood or sperm. As the result, even smallest droplets of 0.25 microliters cannot be vitrified sufficiently fast to avoid the use of potentially toxic external vitrification agents such as DMSO or EG due to the Leidenfrost effect (LFE). In this presentation, we describe an entirely new system for hyperfast cooling of one-two order of magnitude larger samples that we call “KrioBlastTM”, which completely eliminates LFE. We have successfully vitrified up to 4,000 microliters of 15% glycerol solutions, which theoretically corresponds to the critical cooling rate of hundreds of thousands °C/min. We believe that such a system can revolutionize the future cryobiological paradigm.

scholarly journals Innocuous Intracellular Ice Improves Survival of Frozen Cells

2002 ◽  
Vol 11 (6) ◽  
pp. 563-571 ◽  
Author(s):  
Jason P. Acker ◽  
Locksley E. Mcgann
Keyword(s):  
Epithelial Cells ◽  
Cell Survival ◽  
Mdck Cells ◽  
Model Systems ◽  
Ice Formation ◽  
Long Term Storage ◽  
Intracellular Ice Formation ◽  
Intracellular Ice ◽  
Term Storage

Extensive efforts to avoid intracellular ice formation (IIF) during freezing have been central to current methods used for the preservation and long-term storage of cells and tissues. In this study, we examined the effect of intracellular ice formation on the postthaw survival of V-79W fibroblast and MDCK epithelial cells using convection cryomicroscopy and controlled-rate freezing. V-79W and MDCK cells were cultured as single attached cells or as confluent cell monolayers. Postthaw cell survival was assessed using three different indices: the presence of an intact plasma membrane, the ability to reduce alamarBlue, and the capacity to form colonies in culture. Regulating the isothermal nucleation temperature was used to control the incidence of IIF in the model systems. We report that the presence of intracellular ice in confluent monolayers at high subzero temperatures does not adversely affect postthaw cell survival. Further, we show that in the absence of chemical cryoprotectants, the formation of intracellular ice alone improves the postthaw survival of cultured V-79W fibroblast and MDCK epithelial cells. Improved long-term storage of cells and tissues will result by incorporating innocuous intracellular ice formation into current strategies for cryopreservation.

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