scholarly journals A Non-Parametric Method for Estimating Rates of Intracellular Ice Formation Due to Independent Competing Mechanisms

2021 ◽  
Author(s):  
Jens Karlsson
Keyword(s):  
Conventional Method ◽  
Marked Point ◽  
New Method ◽  
Cumulative Distribution ◽  
Ice Formation ◽  
Marked Point Process ◽  
Cumulative Number ◽  
Intracellular Ice Formation ◽  
Intracellular Ice ◽  
Non Parametric

Abstract The probability of intracellular ice formation (IIF) has conventionally been analyzed by counting the cumulative number of IIF events observed in a cell population, and normalizing to the total cell count to estimate the cumulative IIF probability. However, this method is invalid when attempting to distinguish among multiple, independent IIF mechanisms, because of confounding effects due to competition for a finite pool of unfrozen cells. Therefore, an alternative approach for analyzing IIF data is proposed, based on treating IIF as a marked point process, in which the points represent IIF events and the marks represent different mechanisms of IIF. Using the new method, it is possible to quantify the kinetics associated with any IIF mechanism for which corresponding events can be marked (experimentally distinguished from competing IIF mechanisms). The proposed approach is non-parametric, making possible characterization of IIF mechanisms that have not yet been fully elucidated. The new analytical approach was compared to the conventional method of IIF analysis using data from a simulated experiment, demonstrating that the new method yielded superior estimates of the cumulative distribution function of IIF times when two competing mechanisms of IIF were active. The proposed algorithm was also applied to cryomicroscopic IIF observations in adherent endothelial cells, yielding rate estimates for two concurrent IIF processes. Furthermore, a proof is presented to demonstrate that when the proposed data analysis algorithm is applied to IIF data from a single mechanism of IIF, the results are equivalent to those obtained by the conventional method of analysis.

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.

Survival of intracellular freezing by the Antarctic nematode Panagrolaimus davidi

1995 ◽  
Vol 198 (6) ◽  
pp. 1381-1387 ◽  
Author(s):  
D Wharton ◽  
D Ferns
Keyword(s):  
Mammalian Cells ◽  
Body Cavity ◽  
The Body ◽  
Ice Formation ◽  
Surrounding Water ◽  
Intracellular Ice Formation ◽  
Intracellular Compartments ◽  
Intracellular Ice ◽  
Body Compartments ◽  
The Antarctic

Animals are usually thought to survive ice formation in their bodies only if the ice is confined to the body cavity and to extracellular spaces. Intracellular ice formation is believed to be fatal. This conclusion is based on studies of the cryopreservation of mammalian cells. Intracellular freezing has been observed in some living insect cells but has not been observed in intact animals. Nematodes are transparent and so the location of ice in their bodies can be observed directly using a cryomicroscope stage. We have observed freezing and melting in all body compartments, including intracellular compartments, of the Antarctic nematode Panagrolaimus davidi. Inoculative freezing from the surrounding water occurs via the body openings, rather than across the cuticle; most frequently it occurs via the excretory pore. Individual nematodes that have frozen intracellularly will subsequently grow and reproduce in culture. Determining the mechanisms by which this nematode survives intracellular freezing could have important applications in the cryopreservation of a variety of biological materials.

The temperature and type of intracellular ice formation in preimplantation human embryos

Cryobiology ◽  
2018 ◽  
Vol 80 ◽  
pp. 188
Author(s):  
Feng Yuan ◽  
Qiu Juan ◽  
Chao Ma ◽  
Na Zou ◽  
Lei Wang ◽  
...  
Keyword(s):  
Ice Formation ◽  
Human Embryos ◽  
Intracellular Ice Formation ◽  
Intracellular Ice
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