29. Cell perimeter modulates the kinetics of intracellular ice formation and frequency of paracellular ice formation in micropatterned endothelial cells

Cryobiology ◽  
2006 ◽  
Vol 53 (3) ◽  
pp. 379-380 ◽  
Author(s):  
Shannon L. Stott ◽  
Jens O.M. Karlsson
Keyword(s):  
Endothelial Cells ◽  
Ice Formation ◽  
Intracellular Ice Formation ◽  
Intracellular Ice ◽  
Kinetics Of

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.

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.

Sign in / Sign up

Export Citation Format

Share Document