Simple method for cryopreservation of an anaerobic rumen fungus using ethylene glycol and rumen fluid

Abstract The cryopreservation of an anaerobic rumen fungus, Piromyces communis OTS1, was examined at −84 °C using dimethyl sulfoxide, propylene glycol or ethylene glycol as cryoprotectants. Ethylene glycol was the most effective agent, combining high survival and low toxicity, followed by dimethyl sulfoxide and propylene glycol. Cell-free rumen fluid in the cryopreservation medium decreased the toxicity of the cryoprotectant agents and also had a protective action per se. A survival of 80% after 1 year storage was obtained when samples with an initial zoospore density of 5 × 10⁴ zoospores/ml were equilibrated for 15 min in medium containing 0.64 M ethylene glycol and 5% cell-free rumen fluid, then frozen with dry ice and stored at −84 °C.     

Harvesting and cryopreservation of lymphatic endothelial cells for lymphatic tissue engineering

In order to provide a suitable source of cells for lymphatic tissue engineering, the present study was designed to investigate techniques for harvesting and cryopreservation of human dermal lymphatic endothelial cells (LECs) in vitro. The LECs were isolated from children’s foreskins and then cultured in endothelial growth medium-2 MV (EGM-2-MV) with 5% FBS. The second passage LECs were suspended in cryopreservation solution containing 40% FBS and 10% Me₂SO in EGM-2-MV, cooled to −80 °C at about 1 °C/min and stored in liquid nitrogen. Samples were thawed quickly in a 37 °C water bath, and the cryoprotectant was removed by serial elution. The membrane integrity of thawed LECs was determined by trypan blue staining exclusion, and their proliferation was evaluated using the MTT method. The expanded cells of two groups were identified using immunofluorescence staining and RT-PCR with lymphatic-specific markers such as Podoplanin and VEGFR-3. Uptake of fluorescent DiI-Ac-LDL and microtubular formation in three-dimensional cultures were used to detect the function of LECs. Flow cytometry was applied to identify cells and to measure the apoptosis rate as well. Cryopreservation resulted in a retrieval of 67 ± 4% and an intact cell rate of 80 ± 3%. The early apoptosis rate of thawed LECs (9.15 ± 0.34%) was higher than that of fresh control LECs (5.31 ± 0.23%). The growth curves of thawed LECs were similar to those of fresh LECs. The thawed LECs were propagated for at least 6-7 passages without alterations in phenotype and function. Highly purified LECs can be isolated by immunomagnetic beads from human dermis. The cryopreserved/thawed and recultivated LECs are proven to have high vitality and growth potential in vitro and may be considered suitable seed cells for lymphatic tissue engineering.     

The osmotic characteristics of human fetal liver-derived hematopoietic stem cell candidates

Hematopoietic stem cells derived from fetal liver have promising therapeutic potential for allotransplantation but require a specific protocol to minimize the damage produced by cryopreservation procedures. In this study, a fundamental approach was applied for designing a cell preservation protocol. To this end, the biophysical characteristics that describe the osmotic reaction of CD34(+)CD38(-) human fetal liver stem cell candidates were studied using fluorescent microscopy. The osmotically inactive volume of the stem cell candidates was determined as 48% of the isotonic volume. The permeability coefficients for water and Me(2)SO were determined at T = +22 degree C: L(p) = 0.27 +/- 0.03 microm x min(-1)atm(-1), P(Me(2)SO)) = 2.09 +/- 0.85 x 10 (-4) cm x min(-1), sigma (Me(2)SO)) = 0.63 +/- 0.03 and at T = +12 degree C: L(p) = 0.15 +/-0.02 microm x min(-1)atm(-1), P(Me(2)SO)) = 6.44 +/-1.42 x 10 (-5) cm x min(-1), sigma (Me(2)SO)) = 0.46 +/- 0.05. The results obtained suggest that post-hypertonic and hypotonic stress are the possible reasons for damage to a CD34(+)CD38(-) cell during the cryopreservation procedure.     

Cryopreservation of articular cartilage. Part 1: conventional cryopreservation methods

There is increasing interest in the possibility of treating diseased or damaged areas of synovial joint surfaces by grafts of healthy allogeneic cartilage. Such grafts could be obtained from cadaver tissue donors or in the future they might be manufactured by 'tissue engineering' methods. Cartilage is an avascular tissue and hence is immunologically privileged but to take advantage of this is the graft must contain living cells. Preservation methods that achieve this are required to build up operational stocks of grafts, to provide a buffer between procurement and use, and to enable living grafts of a practical size to be provided at the right time for patient and surgeon. Review of the literature shows that it has been relatively straightforward to cryopreserve living isolated chondrocytes, but at the present time there is no satisfactory method to preserve cartilage between the time of procurement or manufacture and surgical use. In this paper, we review the relevant literature and we confirm that isolated ovine chondrocytes in suspension can be effectively cryopreserved by standard methods yet the survival of chondrocytes in situ in cartilage tissue is inadequate and extremely variable.     

Manual cryopreservation of human alveolar periosteal tissue segments: Effects of pre-culture on recovery rate

Cultured human periosteal sheets constitute a promising grafting material for periodontal tissue regenerative therapy. However, preparation of these sheets usually requires six weeks or longer, and this lengthy commitment and delay limits both clinical applicability and availability. The aim of this study is to develop an efficient, practical, cost-effective cryopreservation method for periosteal tissue segments (PTSs). Human PTSs were aseptically excised from alveolar bone and pre-cultured in Medium 199 + 10% fetal bovine serum (FBS) for the indicated number of days before they were slowly frozen down to −75 °C in a commercial freezing vessel using medium containing 10% dimethyl sulfoxide (Me₂SO) and various concentrations of FBS. After fast-thawing at 37 °C, PTSs were again cultured, and their growth and responses to standard osteogenic induction were evaluated (vs. freshly excised PTSs). Proliferating cells were obtained at the highest levels from cryopreserved PTSs that were pre-cultured for 14 days before freezing. When a concentration of 50% or more FBS was included in the cryopreservation solution, cells migrated out more actively and grew faster. Importantly, osteoinduction up-regulated alkaline phosphatase (ALP) activity and osteoblastic marker mRNAs in cryopreserved PTS-derived sheets just as effectively as it did in native PTS-derived ones. These data suggest that pre-conditioned PTSs can be efficiently cryopreserved in a freezing solution containing high FBS by traditional manual cryopreservation methods without aid of a program freezer or more elaborate equipment.     

Survival of mint shoot tips after exposure to cryoprotectant solution components

Many plant species can be cryopreserved by treating shoot tips with complex cryoprotectant solutions before rapidly cooling them to liquid nitrogen temperatures. Plant vitrification solution 2 (PVS2), a commonly selected cryoprotectant, can be lethal with extended exposure times. To determine potentially toxic combinations, we have exposed mint shoot tips to one-, two-, three-, and four-component solutions of PVS2 chemicals (30% glycerol, 15% ethylene glycol, 15% dimethyl sulfoxide, and 0.4 M sucrose) at 0 and 22 degrees C. Overall, solution exposures at 22 degrees C were more damaging than exposures at 0 degree C. Solutions with glycerol, particularly in combination with ethylene glycol and dimethyl sulfoxide, were also damaging. Cryoprotectant solutions PGluD (10% PEG8000, 10% glucose, and 10% dimethyl sulfoxide) and PVS3 (50% glycerol, 50% sucrose) were less damaging than PVS2 at 22 degrees C. When plant cryoprotectants are characterized on a toxicological and biophysical basis, less damaging cryoprotectant solutions could be developed.     

Toxicity of cryoprotectants to honey bee semen and queens

Given the threats to the intraspecific biodiversity of Apis mellifera and the pressure on bee breeding to come up with disease-tolerant lines, techniques to cryopreserve drone semen are of great interest. Freeze-thawed drone semen of high viability and/or motility has repeatedly been obtained, but fertility of such semen, when it was measured, was always low. The cryoprotective agent (CPA) most frequently used with drone semen is dimethyl sulfoxide (DMSO), although this substance has been suspected of causing genetic damage in sperm. No form of sperm washing is currently performed. Using a membrane permeability assay, we measured the short-term toxicity of four possible replacements for DMSO, 1,3-propane diol, 2,3-butane diol, ethylene glycol, and dimethyl formamide. We also tested whether the practice of inseminating queens with CPA-containing semen affects sperm numbers in the storage organs of queens, or sperm fertility. Finally, we tested whether CPA-toxicity in vivo can be reduced by using mixtures of two CPAs, DMSO, and ethylene glycol. Our results show that, although short-term toxicity of all CPAs tested was low, the presence of single CPAs in insemination mixtures at concentrations required for slow freezing greatly reduced the number of sperm reaching the spermatheca. Contrary to earlier reports, this was also true for DMSO. Ethylene glycol was additionally shown to reduce the viability of spermatozoa reaching the storage organ. Mixtures of DMSO and EthGly performed better than either substance used singly at the same concentration. We conclude that the toxicity of CPAs, including DMSO, on honey bee semen and/or queens has been underestimated in the past. This could partly explain the discrepancy between in vitro and in vivo quality of cryopreserved drone semen, described by others. Combinations of several CPAs and techniques to partly remove CPAs after thawing could help to solve this problem.     

Comparison of actual vs. synthesized ternary phase diagrams for solutes of cryobiological interest

Phase diagrams are of great utility in cryobiology, especially, those consisting of a cryoprotective agent (CPA) dissolved in a physiological salt solution. These ternary phase diagrams consist of plots of the freezing points of increasing concentrations of solutions of cryoprotective agents (CPA) plus NaCl. Because they are time-consuming to generate, ternary diagrams are only available for a small number of CPAs. We wanted to determine whether accurate ternary phase diagrams could be synthesized by adding together the freezing point depressions of binary solutions of CPA/water and NaCl/water which match the corresponding solute molality concentrations in the ternary solution. We begin with a low concentration of a solution of CPA+salt of given R (CPA/salt) weight ratio. Ice formation in that solution is mimicked by withdrawing water from it which increases the concentrations of both the CPA and the NaCl. We compute the individual solute concentrations, determine their freezing points from published binary phase diagrams, and sum the freezing points. These yield the synthesized ternary phase diagram for a solution of given R. They were compared with published experimental ternary phase diagrams for glycerol, dimethyl sulfoxide (DMSO), sucrose, and ethylene glycol (EG) plus NaCl in water. For the first three, the synthesized and experimental phase diagrams agreed closely, with some divergence occurring as wt% concentrations exceeded 30% for DMSO and 55% for glycerol, and sucrose. However, in the case of EG there were substantial differences over nearly the entire range of concentrations which we attribute to systematic errors in the experimental EG data. New experimental EG work will be required to resolve this issue.     

Expansion and cryopreservation of porcine and human corneal endothelial cells

Impairment of the corneal endothelium causes blindness that afflicts millions worldwide and constitutes the most often cited indication for corneal transplants. The scarcity of donor corneas has prompted the alternative use of tissue-engineered grafts which requires the ex vivo expansion and cryopreservation of corneal endothelial cells. The aims of this study are to culture and identify the conditions that will yield viable and functional corneal endothelial cells after cryopreservation. Previously, using human umbilical vein endothelial cells (HUVECs), we employed a systematic approach to optimize the post-thaw recovery of cells with high membrane integrity and functionality. Here, we investigated whether improved protocols for HUVECs translate to the cryopreservation of corneal endothelial cells, despite the differences in function and embryonic origin of these cell types. First, we isolated endothelial cells from pig corneas and then applied an interrupted slow cooling protocol in the presence of dimethyl sulfoxide (Me₂SO), with or without hydroxyethyl starch (HES). Next, we isolated and expanded endothelial cells from human corneas and applied the best protocol verified using porcine cells. We found that slow cooling at 1 °C/min in the presence of 5% Me₂SO and 6% HES, followed by rapid thawing after liquid nitrogen storage, yields membrane-intact cells that could form monolayers expressing the tight junction marker ZO-1 and cytoskeleton F-actin, and could form tubes in reconstituted basement membrane matrix. Thus, we show that a cryopreservation protocol optimized for HUVECs can be applied successfully to corneal endothelial cells, and this could provide a means to address the need for off-the-shelf cryopreserved cells for corneal tissue engineering and regenerative medicine.     

Physiological evaluation of a rabbit kidney perfused with VS41A

The current report compares the renal physiological impact of a standard vitrification solution, VS41A, as measured by normothermic blood perfusion, to the physiological effects of VS4, a related but more dilute vitrification solution previously shown to be consistently compatible with life support function of transplanted rabbit kidneys. VS41A, which allows survival of only about half of the kidneys perfused with it, also appeared to be more damaging than VS4 based on in vitro functional indices and histology in one rabbit kidney so evaluated.     

The effect of osmotic stress on the cell volume, metaphase II spindle and developmental potential of in vitro matured porcine oocytes

Porcine animal models are used to advance our understanding of human physiology. Current research is also directed at methods to produce transgenic pigs. Cryobanking gametes and embryos can facilitate the preservation of valuable genotypes, yet cryopreserving oocytes from pigs has proven very challenging. The current study was designed to understand the effects of anisotonic solutions on in vitro matured porcine oocytes as a first step toward designing improved cryopreservation procedures. We hypothesized that the proportion of oocytes demonstrating a normal spindle apparatus and in vitro developmental potential would be proportional to the solution osmolality. Oocytes were incubated for 10 min at 38 degrees C in various hypo- or hypertonic solutions, and an isotonic control solution and then assessed for these two parameters. Our results support the hypothesis, with an increasing proportion of spindles showing a disrupted structure as the levels of anisotonic exposure diverge from isotonic. Only about half of the oocytes maintained developmental potential after exposure to anisotonic solutions compared to untreated controls. Oocyte volume displayed a linear response to anisotonic solutions as expected, with an estimated relative osmotically inactive cell volume of 0.178. The results from this study provide initial biophysical data to characterize porcine oocytes. The results from future experiments designed to determine the membrane permeability to various cryoprotectants will allow predictive modeling of optimal cryopreservation parameters and provide a basis for designing improved cryopreservation procedures.     

Optimization of cryoprotectant loading into murine and human oocytes

Loading of cryoprotectants into oocytes is an important step of the cryopreservation process, in which the cells are exposed to potentially damaging osmotic stresses and chemical toxicity. Thus, we investigated the use of physics-based mathematical optimization to guide design of cryoprotectant loading methods for mouse and human oocytes. We first examined loading of 1.5 M dimethyl sulfoxide (Me₂SO) into mouse oocytes at 23 °C. Conventional one-step loading resulted in rates of fertilization (34%) and embryonic development (60%) that were significantly lower than those of untreated controls (95% and 94%, respectively). In contrast, the mathematically optimized two-step method yielded much higher rates of fertilization (85%) and development (87%). To examine the causes for oocyte damage, we performed experiments to separate the effects of cell shrinkage and Me₂SO exposure time, revealing that neither shrinkage nor Me₂SO exposure single-handedly impairs the fertilization and development rates. Thus, damage during one-step Me₂SO addition appears to result from interactions between the effects of Me₂SO toxicity and osmotic stress. We also investigated Me₂SO loading into mouse oocytes at 30 °C. At this temperature, fertilization rates were again lower after one-step loading (8%) in comparison to mathematically optimized two-step loading (86%) and untreated controls (96%). Furthermore, our computer algorithm generated an effective strategy for reducing Me₂SO exposure time, using hypotonic diluents for cryoprotectant solutions. With this technique, 1.5 M Me₂SO was successfully loaded in only 2.5 min, with 92% fertilizability. Based on these promising results, we propose new methods to load cryoprotectants into human oocytes, designed using our mathematical optimization approach.     

Beyond membrane integrity: Assessing the functionality of human umbilical vein endothelial cells after cryopreservation

Assessment of cell membrane integrity is one of the most widely used methods to measure post-cryopreservation viability of cells such as human umbilical vein endothelial cells (HUVECs). However, an evaluation of cell function provides a better measure of cell quality following cryopreservation. The tube formation assay mimics angiogenesis in vitro and can be used to quantitate the ability of endothelial cells to form capillary-like tubular structures when cultured on reconstituted basement membrane (Matrigel). We compared the membrane integrity (measured by flow cytometry) and tube forming ability of HUVEC suspensions exposed to 10% dimethyl sulfoxide (Me₂SO), cooled at 1 °C/min to various sub-zero temperatures, plunged directly into liquid nitrogen, stored for an hour, and thawed rapidly. We found that as membrane integrity increased so did the various parameters associated with the extent of in vitro angiogenesis; however, in comparison to fresh cells with a similar percentage of membrane-intact cells, the extent of tube formation, expressed as total tube length, is significantly lower in previously frozen cells for the lower range of post-thaw membrane integrities. Our findings underscore the value of an assay that quantifies a specific function that a cell is known to perform in vivo to measure the success of cryopreservation protocols.     

An investigation of oxidative stress and antioxidant biomarkers during Greenshell mussel (Perna canaliculus) oocyte cryopreservation

Oxidative damage to proteins and lipids, the enzymatic and nonenzymatic antioxidants' response, and the fertilization and development capability of Perna canaliculus oocytes were investigated at critical treatment steps in a previously published controlled-rate cryopreservation protocol. The cryoprotectant (CPA) from this protocol comprises 10% ethylene glycol (v:v) and 0.2 M trehalose (wt/vol) final concentration. Critical treatment steps included (1) seawater control, (2) CPA addition, (3) CPA addition followed by cooling to −6 °C, (4) CPA addition and cooling to −10 °C, and (5) CPA addition and cooling to −35 °C and immersion in liquid nitrogen (LN). The percentage of fertilized oocytes was 53.8 ± 13.3% in the seawater control but was reduced to 26.0 ± 15.6% after −35 °C + LN treatment, whereas development to D-larvae was 21.0 ± 6.4% in the seawater control reduced to 4.8 ± 2.9% after cooling to −6 °C, and was zero at all the subsequent cooling steps. All oxidative damage biomarkers, protein carbonyls (PCs) and lipid hydroperoxides (LPs), and antioxidants, superoxide dismutase (SOD), catalase, glutathione peroxidase, percent reduced glutathione (%GSH), and total glutathione (defined as glutathione; reduced [GSH] plus glutathione disulphide; derived from two molecules of GSH [GSSG]) were measured over all treatments on unfertilized oocytes over a post-treatment recovery period of 0 to 240 minutes in seawater. An ANOVA showed that both treatment and post-treatment periods had significant effects on the concentrations of all biomarkers (P < 0.05). Protein carbonyls and LPs increased very little after CPA addition and cooling treatments, when compared with the seawater control, but large increases up to sixfold occurred between 0 and 240 minutes for the −35 °C + LN treatment. Concentrations of SOD, catalase, total glutathione, and %GSH at 0 minutes decreased by −31.2%, −26.9%, −21.9%, and −25.0%, respectively, between the seawater control and the −35 °C + LN treatment. In contrast, glutathione peroxidase concentrations at 0 minutes increased by 34.3% between the seawater control and the −35 °C + LN treatment. Although most biochemical biomarkers showed an increasing trend over time (0–240 minutes), total glutathione decreased in concentration over time in all treatments, with the greatest decrease after the −35 °C + LN treatment (−41.2%). Significant correlations between biomarkers and D-larvae yield were negative for LPs and positive for SOD, total glutathione, and %GSH (P < 0.05). This is the first report of an investigation using these oxidative stress biomarkers and antioxidant responses on mussel oocytes, and the results have proved useful in characterizing cellular injury during the cryopreservation process.     

Rationally optimized cryopreservation of multiple mouse embryonic stem cell lines: I—Comparative fundamental cryobiology of multiple mouse embryonic stem cell lines and the implications for embryonic stem cell cryopreservation protocols

The post-thaw recovery of mouse embryonic stem cells (mESCs) is often assumed to be adequate with current methods. However as this publication will show, this recovery of viable cells actually varies significantly by genetic background. Therefore there is a need to improve the efficiency and reduce the variability of current mESC cryopreservation methods. To address this need, we employed the principles of fundamental cryobiology to improve the cryopreservation protocol of four mESC lines from different genetic backgrounds (BALB/c, CBA, FVB, and 129R1 mESCs) through a comparative study characterizing the membrane permeability characteristics and membrane integrity osmotic tolerance limits of each cell line. In the companion paper, these values were used to predict optimal cryoprotectants, cooling rates, warming rates, and plunge temperatures, and then these predicted optimal protocols were validated against standard freezing protocols.     

Effects of synthetic antifreeze glycoprotein analogue on islet cell survival and function during cryopreservation

The antifreeze glycoprotein (AFGP), found in the blood of polar fish, is known to prevent ice crystal growth and to depress the freezing temperature, which may in turn protect tissues from freezing injury. The chemical synthesis of AFGP is an attractive alternative to its difficult isolation from natural sources, and this would permit quality control and mass production. In spite of recent success in islet transplantation for the treatment of type 1 diabetes mellitus, existing methods for the long-term preservation of islets are considered to be suboptimal and inadequate, which indicates the need for the development of improved methods. Rat islets were isolated from male Wistar rats, using intraductal collagenase distention, mechanical dissociation, and Ficoll-Conray gradient purification. Islets were cultured overnight and then cryopreserved in RPMI1640 in the presence of dimethyl sulfoxide (Me2SO) and 10% FCS with various concentrations of syAFGP, followed by slow cooling (0.3 degrees C/min) and rapid thawing (200 degrees C/min) as described by Rajotte. The freezing process was observed by cryomicroscopy. Islet recovery post-cryopreservation was 85.0 +/- 6.2% with syAFGP and 63.3 +/- 14.2% without syAFGP, both compared with the pre-cryopreservation counts (P < 0.05). The in vitro islet function measured by insulin release was equivalent to a static stimulation index of 3.86+/-0.43 for the islets that were frozen-and-thawed with syAFGP, compared to 2.98 +/- 0.22 without syAFGP (P < 0.05). At a concentration of around 500 microg/ml syAFGP, a strong attenuation of ice crystal growth and formation was observed by cryomicroscopy and these ice crystals did not cause cryoinjury. In conclusion, the attenuation of ice crystallization by syAFGP improves islet survival and function following cryopreservation and thawing.     

Water status and thermal analysis of alginate beads used in cryopreservation of plant germplasm

Encapsulation and dehydration techniques using alginate beads are used increasingly for the pre-treatment of various plant materials for cryopreservation to improve survival post-cryogenic storage. This study reports the effects of the water content of beads (formed with 3% (w/v) alginic acid in liquid S-RIB), polymerisation time (in 100 mM calcium chloride solution), osmotic dehydration (in 0.75 M sucrose solution), and evaporative air desiccation on the thermal properties of alginate beads used in cryopreservation protocols. Experimental beads were assayed using a differential scanning calorimeter (DSC) with a cooling programme to -150 degrees C, followed by re-warming. Resultant thermograms were evaluated with particular reference to the onset temperature and enthalpy of the melt endotherm from which the quantities of frozen and unfrozen water were calculated. Treatments were applied sequentially to samples of beads and their thermal features evaluated at each stage of the protocol. Using 'standard' beads (40-55 mg fresh weight), formed using plastic disposable pipettes, the degree of polymerisation (>10 min) proportionally reduced their dry weight and increased their water content. Thermal characteristics of the beads were unaffected by polymerisation times >10 min, but the maximum level (23%) of unfrozen (osmotically inactive) water was achieved after 15 min polymerisation. Osmotic dehydration using 0.75 M sucrose significantly lowered bead water content and mean dry weight approximately doubled with 20-24 h immersion time. Bead desiccation in still air reduced their water content by 83% of fresh weight, whilst dry weight remained constant. After 8 h desiccation in air between 27 and 37% of the water in the bead was osmotically inactive (unfrozen) in DSC scans. Desiccation >18 h reduced this fraction to zero. The melt onset temperature and the enthalpy of melting were directly related to bead water content. The unfrozen water fraction increased substantially with reduced water content of the beads (from 23 to 37% of total water content), concomitant with a reduction in the ratio of unfrozen to frozen water from 1:3 to 1:2. For successful vitrification and the production of a glass that did not destabilise on rewarming, a bead water content of ca. 26% of fresh weight (0.4 g waterg(-1) dry weight) was required, much of which was osmotically inactive water. These data are discussed in relation to optimal pre-treatments for alginate bead encapsulation techniques used in the cryopreservation of a range of plant germplasm. It is proposed that increased standardisation of alginate beads, in terms of volume, fresh weight, and water content, is required to reduce the variability in physical and thermal features, which in turn will improve survival of plant samples post-cryopreservation.     

3-D measurement of osmotic dehydration of isolated and adhered PC-3 cells

Cell dehydration during freezing results from an elevated concentration of electrolytes in the extracellular medium that is deeply involved in cellular injury. We undertook real-time threedimensional (3-D) observation of osmotic dehydration of cells, motivated by a comparison of cellular responses between isolated cells in suspension and cultured cells adhering to a surface since several studies have suggested a difference in freeze tolerance between cell suspensions and monolayers. A laser confocal scanner was used with a perfusion microscope to capture sectional images of chloromethylbenzamido (DiI)-stained PC-3 cells that were exposed to an increase in NaCl concentration from 0.15 to 0.5 M at 23 °C. Change in cell volume was determined from reconstructed 3-D images taken every 2.5 s. When cells were exposed to an elevated NaCl concentration, isolated cells contracted and markedly distorted from their original spherical shape. In contrast, adhered cells showed only a reduction in height and kept their basal area constant. Apparent membrane hydraulic conductivity did not vary considerably between isolated and adhered cells, suggesting a negligible effect of the cytoskeletal structure on the rate of water transport. The surface area that contributed to water transport in adhered PC-3 cells was nearly equal to or slightly smaller than that present in isolated cells. Therefore, the similarity in properties and dimensions between isolated and adhered cells indicate that there will be similar extents of dehydration, resulting in a similar degree of supercooling during freezing.