An optimum method for the introduction or removal of permeable cryoprotectants: Isolated cells

On the basis of a nonequilibrium model for the transport of water and permeable solute across cell membranes, an optimum method has been devised for the introduction and the removal of a permeable cryoprotectant from single, isolated cells so that potentially lethal drastic alterations in cellular volume are minimized. The method involves the simultaneous variation of both the permeable (an initial step change, followed by a linear variation with time which overshoots the terminal value, and a final step change to the terminal value) and impermeable (an initial step change in the opposite direction of the permeable solute concentration change, followed by a period where the concentration remains constant, and a final step change back to the initial value) extracellular solute concentrations in a prescribed manner such that both the cellular water content and the intracellular electrolyte concentration remain constant as the intracellular permeable solute (CPA) concentration is either raised or lowered. The results of our theoretical analysis indicate that the osmotic stresses and strains usually imposed upon cells during the introduction and the removal of permeable cryoprotectants can be minimized and that the resulting protocols are clinically the most efficient.     

Cryoprotectant delivery and removal from murine insulinomas at vitrification-relevant concentrations

Development of optimal cryopreservation protocols requires delivery and removal of cryoprotective agents (CPAs) in such a way that negative osmotic and cytotoxic effects on cells are minimized. This is especially true for vitrification, where high CPA concentrations are employed. In this study, we report on the determination of cell membrane permeability parameters for water (L(p)) and solute (P(s)), and on the design and experimental verification of CPA addition and removal protocols at vitrification-relevant concentrations for a murine insulinoma cell line, betaTC-tet cells. Using membrane permeability values and osmotic tolerance limits, mathematical modeling and computer simulations were used to design CPA addition and removal protocols at high concentrations. The cytotoxic effects of CPAs were also evaluated. Cells were able to tolerate the addition and removal of 2.5M dimethyl sulfoxide (DMSO) and 2.5M 1,2 propanediol (PD) in single steps, but required multi-step addition and removal with 3.0M DMSO, 3.0M PD, and a vitrification-relevant concentration of 3.0M DMSO+3.0M PD. Cytotoxicity studies revealed that betaTC-tet cells were able to tolerate the presence of single component 6.0M DMSO and 6.0M PD and to a lesser extent 3.0M DMSO+3.0M PD. These results determine the time and concentration domain of CPA exposure that cells can tolerate and are essential for designing cryopreservation protocols for free cells as well as cells in engineered tissues.     

Intracellular pH changes in isolated bovine articular chondrocytes during the loading and removal of cryoprotective agents

The addition and removal of a cryoprotective agent (CPA) are necessary steps in the cryopreservation of natural or engineered tissue products. However, the introduction and removal of CPAs induces dramatic chemical changes inside tissues and cells and these could cause irreversible damage. This study examined the effect of CPA loading and removal on the intracellular pH of isolated bovine articular chondrocytes using a fluorimetric technique. Chondrocytes that had been isolated from bovine articular cartilage were loaded with the pH-sensitive fluorophore 2('),7(')-bis(carboxyethyl)-5(6)-carboxyfluorescein. After removal of the extracellular fluorophore, the intensity of fluorescence was used to measure the intracellular pH according to a pre-determined calibration curve. Changes of intracellular pH in chondrocytes were measured following their exposure to dimethyl sulfoxide (Me(2)SO) and glycerol at concentrations of 0.6, 0.9, and 1.2M and later to the isotonic or hypertonic solutions that were used to remove the CPA. The effect of the presence of NaCl on the intracellular pH during CPA removal was also examined. The temperature was maintained at 37 degrees C. Trypan blue exclusion was used to quantify cell membrane integrity after the addition and removal of CPA. It was found that when the cells were exposed to CPA, the intracellular pH decreased quickly and recovered gradually later. During CPA removal, the intracellular pH rose following exposure to isotonic Hepes-buffered medium, but the opposite was observed if the Hepes buffer solution contained no NaCl; this was ascribed to the role of NaCl in cell membrane transport. It was noted that the change in intracellular pH correlated with the cell volume excursion, which could be estimated by the Kedem-Katchalsky model, and was linked to cell survival. The resulting alteration of pH inside the cells might contribute to cell damage and loss of function after cryopreservation.     

Synthetic polymers improve vitrification outcomes of macaque ovarian tissue as assessed by histological integrity and the in vitro development of secondary follicles

Long-term storage of natural tissues or tissue-engineered constructs is critical to allow off-the-shelf availability. Vitrification is a method of cryopreservation that eliminates ice formation, as ice may be detrimental to the function of natural or bioartificial tissues. In order to achieve the vitreous state, high concentrations of CPAs must be added and later removed. The high concentrations may be deleterious to cells as the CPAs are cytotoxic and single-step addition or removal will result in excessive osmotic excursions and cell death. A previously described mathematical model accounting for the mass transfer of CPAs through the sample matrix and cell membrane was expanded to incorporate heat transfer and CPA cytotoxicity. Simulations were performed for two systems, an encapsulated system of insulin-secreting cells and articular cartilage, each with different transport properties, geometry and size. Cytotoxicity and mass transfer are dependent on temperature, with a higher temperature allowing more rapid mass transfer but also causing increased cytotoxicity. The effects of temperature are exacerbated for articular cartilage, which has larger dimensions and slower mass transport through the matrix. Simulations indicate that addition and removal at 4°C is preferable to 25°C, as cell death is higher at 25°C due to increased cytotoxicity in spite of the faster mass transport. Additionally, the model indicates that less cytotoxic CPAs, especially at high temperature, would significantly improve the cryopreservation outcome. Overall, the mathematical model allows the design of addition and removal protocols that insure CPA equilibration throughout the sample while still minimizing CPA exposure and maximizing cell survival.     

Hemolysis in several animal species after rapid freezing of blood

The effect of various freezing rates on the extent of hemolysis in human, bovine and ovine erythrocytes, which are known to have different cell volumes, water contents and permeabilities, was investigated. Blood in stainless steel capillary tubes was frozen at various rates by abrupt immersion of the capillaries into cooling baths at temperatures ranging from ?20° to ?130°C. Minimum lysis values were obtained at freezing temperatures of ?40°, ?50° and ?70°C with, respectively, human, bovine and ovine blood. The smallest, highly permeable sheep erythrocytes were the least damaged at the highest freezing rates; the largest human cells with the highest water content, suffered the greatest damage; intermediate values were obtained with ox blood. At the lower freezing rates, the largest, human cells were the least damaged; the highest hemolysis values were obtained with the smallest, highly permeable sheep erythrocytes; ox blood again gave intermediate values. These results are in agreement with current views that, (1) very rapid freezing results in the formation of damaging intracellular ice; (2) injury associated with slow freezing is related to the extent of dehydration or to the increase in electrolyte concentration which accompanies ice formation; (3) minimum hemolysis is obtained under those freezing conditions in which osmotic dehydration has been sufficient to prevent the formation of intracellular ice, but has left enough water in the cells to prevent the damaging effects of dehydration and high electrolyte concentrations.     

Cryobiology of rat embryos II: A theoretical model for the development of interrupted slow freezing procedures

Current mammalian embryo cryopreservation protocols typically employ an interrupted slow freezing (ISF) procedure. In general, ISF consists of initial slow cooling, which raises the extracellular solute concentration, and results in cell dehydration. Permeating cryoprotective agents (CPAs), such as dimethyl sulfoxide (DMSO), are typically included in the medium to protect the cells against high solute concentrations. As this ISF procedure continues, slow cooling is terminated at an intermediate temperature (T(p)), followed by plunging into liquid nitrogen (LN(2)). If the slow cooling step allowed a critical concentration ([CPA](c)) of CPA to be reached within the cell, the CPA will interact with the remaining intracellular water during rapid cooling, resulting in the majority of the intracellular solution becoming vitrified and preventing damaging intracellular ice formation (IIF). This study presents a theoretical model to develop efficient ISF procedures, on the basis of previously developed data for the rat zygote. The model was used to select values of initial CPA concentrations and slow cooling rates (from initial estimated ranges of 0 to 4 molal DMSO and 0 to 2.5 degrees C/min cooling rates) that would allow the intracellular solute concentration to exceed the critical concentration. The optimal combination was then determined from this range based on minimizing the duration of slow cooling.     

Osmotically induced removal of water from fungal cells as determined by a spin probe technique

Effects of physical environment on plasma membrane semipermeability and osmotic induction of changes in aqueous cytoplasmic volume were studied in vegetative and spore cells of a plant pathogenic fungus, Fusarium sulphureum. A direct method, employing a spin probe molecule that partitioned between intracellular aqueous and hydrophobic phases, allowed measurement of reversible water movement out of macroconidial cells and chlamydospores exposed to solutions of high osmolarity. Equilibrium distribution of the spin probe between intracellular aqueous and lipid phases was more rapid than movement of water in and out of the cells. The extent of water removal was exponentially dependent on osmotic strength. Some cells became irreversibly permeable to divalent cations on treatment with sodium chloride above 1.5 osmolar but addition of sucrose to the suspension medium at equivalent osmolar concentrations caused water removal without adversely affecting the viability. Sucrose also protected the plasma membrane against damage during freeze-drying. Induction of plasma membrane damage by osmotic shock or freeze-drying permitted rapid permeation of nickel ions. Neither slow equilibration of intracellular components with divalent paramagnetic cations nor partial permeability of damaged plasma membranes to these ions was observed.     

The point of maximum cell water volume excursion in case of presence of an impermeable solute

A relativistic permeability model of cell osmotic response (Cryobiology 40:64-83; 41:366-367) is applied to a two-solute system with one impermeable solute. The use of the normalized water volume (w), and the amount of intracellular permeable solute (x), which is the product of the water volume and intracellular osmolality (y), as the main variables allowed us to obtain a homogeneous differential equation dx(Delta)/dw(Delta)=f(x(Delta)/w(Delta)), where w(Delta)=w-w(f), x(Delta)=x-x(f), and f refers to the final (equilibrium) values. The solution of this equation is an explicit function, w(Delta)=g(x(Delta)), which is given in the text. This approach allows us to obtain an analytical (exact) expression of the water volume at the moment of the maximum excursion (water extremum w(m)). Results are compared with numeration of basic osmotic equations and with approximation given in (Cryobiology 40:64-83). Assumption that, dw/dt approximately 0 gives good approximations of the kinetics of water and permeable CPA after the point of maximum volume excursion (the slow phase of osmotic response). Practical aspects of the relativistic permeability approach are also discussed.     

Differing actions of penetrating and nonpenetrating cryoprotective agents.

A two-step freezing technique has been used to examine the role of cryoprotective agents during cooling. Chinese hamster fibroblasts were cooled to various subzero holding temperatures and subsequently thawed or cooled to ?196 °C before thawing. Cells were suspended in various concentrations of dimethylsulfoxide (DMSO) or hydroxyethyl starch (HES) before freezing. The results indicated differing protective actions of DMSO and HES. These differences were verified using glycerol as either a penetrating or a nonpenetrating agent.The results are consistent with the concepts that cryoprotection is based on the avoidance or minimization of intracellular freezing and the minimization of damage to the cell from the environment of concentrated solutes during cooling, and that the colligative action of both penetrating and nonpenetrating agents allows the cells to survive the conditions for a reduction of cell water content during cooling thereby reducing the amount of intracellular freezing. The results indicate that penetrating and nonpenetrating agents accomplish this in different ways. Penetrating agents create the environment for a reduction of cell water content at temperatures sufficiently low to reduce the damaging effect of the concentrated solutes on the cells. Nonpenetrating agents osmotically “squeeze” water from the cells primarily during the initial phases of freezing at temperatures between ?10 and ?20 °C when these additives become concentrated in the extracellular regions.     

Cryopreservation of Preimplantation Embryos of Cattle, Sheep, and Goats

Preimplantation embryos from cattle, sheep, and goats may be cryopreserved for short- or long-term storage. Preimplantation embryos consist predominantly of water, and the avoidance of intracellular ice crystal formation during the cryopreservation process is of paramount importance to maintain embryo viability. Embryos are placed into a hypertonic solution (1.4 – 1.5 M) of a cryoprotective agent (CPA) such as ethylene glycol (EG) or glycerol (GLYC) to create an osmotic gradient that facilitates cellular dehydration. After embryos reach osmotic equilibrium in the CPA solution, they are individually loaded in the hypertonic CPA solution into 0.25 ml plastic straws for freezing. Embryos are placed into a controlled rate freezer at a temperature of -6°C. Ice crystal formation is induced in the CPA solution surrounding the embryo, and crystallization causes an increase in the concentration of CPA outside of the embryo, causing further cellular dehydration. Embryos are cooled at a rate of 0.5°C/min, enabling further dehydration, to a temperature of -34°C before being plunged into liquid nitrogen (-196°C). Cryopreserved embryos must be thawed prior to transfer to a recipient (surrogate) female. Straws containing the embryos are removed from the liquid nitrogen dewar, held in room temperature air for 3 to 5 sec, and placed into a 37°C water bath for 25 to 30 sec. Embryos cryopreserved in GLYC are placed into a 1 M solution of sucrose for 10 min for removal of the CPA before transfer to a recipient (surrogate) female. Embryos cryopreserved in EG, however, may be directly transferred to the uterus of a recipient.     

Mechanism of cryoprotection by extracellular polymeric solutes.

To elucidate the means by which polymer solutions protect cells from freezing injury, we cooled human monocytes to -80 degrees C or below in the presence of various polymers. Differential scanning calorimetric studies showed that those polymers which protect cells best have a limiting glass transition temperature (T'g) of approximately -20 degrees C; those with a T'g significantly higher or lower did not protect. Freeze-etch electron micrographs indicated that intracellular ice crystals had formed during this freezing procedure, but remained smaller than approximately 300 nm in the same proportion of cells as survived rapid thawing. We propose that cryoprotection of slowly frozen monocytes by polymers is a consequence of a T'g of -20 degrees C in the extracellular solution. In our hypothesis, the initial concentration and viscosity of protective polymer solutions reduce the extent and rate of cell water loss to extracellular ice and limit the injurious osmotic stress, which cells face during freezing at moderate rates to -20 degrees C. Below -20 degrees C, glass formation prevents further osmotic stress by isolating cells from extracellular ice crystals, virtually eliminating cell water loss at lower temperatures. On the other hand, the protective polymer solutions will allow some diffusion of water away from cells at temperatures above T'g. If conditions are correct, cells will concentrate the cytoplasm sufficiently during the initial cooling to T'g to avoid lethal intracellular freezing between T'g and the intracellular Tg, which has been depressed to low temperatures by that concentration. Thus, when polymers are used as cryoprotective agents, cell survival is contingent upon maintenance of osmotic stress within narrow limits.     

Apoptosis, the heat shock response, hyperthermia, birth defects, disease and cancer. Where are the common links?

Many cells die during normal prenatal development. Throughout postnatal life, production of new cells is balanced by death of older cells to maintain the normal mass of organs and tissues. In these situations, cell death is usually in the form of apoptosis, characterized morphologically by shrinkage of cellular contents within their membranes, condensation and margination of chromatin against the nuclear membrane and phagocytic removal by macrophages or adjacent cells of the organ. It is initiated and controlled by a complex set of gene-directed activities. The process is tidy and avoids the inflammatory effects of degenerating cellular contents on other tissues. The capacity to undergo this form of cell death is lost in neoplastic cell lines. In embryos the normal process of apoptosis has been termed programmed cell death, and in prenatal and mature animals a number of toxic agents can also cause morphologically typical forms of apoptosis.     

Modeling of the co-transport of cryoprotective agents in a porous medium as a model tissue

Cryopreservation is likely the choice for long-term preservation of natural and engineered tissues, and high concentration multiple cryoprotective agents (CPAs) are usually used in such a process. To achieve high cell viability after cryopreservation, cells at all locations within the tissue must be protected properly by the CPAs during freezing. It is hence essential to know the distribution and concentration of CPAs within the tissue during multiple-CPA addition, to maximize cell survival and minimize tissue damage. In this work, a model to describe the CPA transport during multiple-CPA addition in a one-dimensional porous medium, as a simplified model of living tissue, was developed on the basis of the Maxwell-Stefan (M-S) equations. The UNIFAC and UNIQUAC models were used to evaluate the activity coefficients, and the Siddiqi-Lucas correlation was used for estimation of Maxwell-Stefan diffusivities. Simulations were carried out to examine the effect of temperature, tissue property, CPA type and the interactions between solutes on the CPA transport within construct during the CPA addition. It was found that these parameters, especially the interactions between the different CPA molecules, which was neglected before, significantly affect the transport of each individual CPA component. It is hence concluded that the traditional single-component analysis on the CPA diffusion is not adequate to quantify the multiple-CPA distribution in the tissue, particularly when the CPA concentrations are relatively high.     

Osmotic consequences of cryoprotectant permeability and its relation to the survival of frozen-thawed embryos

The freezing of a living cell involves a complex physicochemical process of heat and water transport between the cell and its surrounding medium. Embryos survive cryopreservation only in the presence of a cryoprotectant in concentrations between 1 and 2M. During the addition and dilution of a permeating cryoprotectant, the cell undergoes osmotic changes in cell size. As a consequence, if the addition or particularly the dilution are carried out inappropriately, the viability of cells can be affected. Equations which model the influx and efflux of cryoprotectants in cells can be used to calculate the optimum and most practical addition and removal method. However, the equations require the permeability coefficient of the cryoprotectant, a quantity that has only experimentally determined for a few of the developmental stages of two species.     

Osmotic mass transfer in the yeast Saccharomyces cerevisiae.

This paper reviews the passive mechanisms involved in the response of a yeast to changes in medium concentration and osmotic pressure. The results presented here were collected in our laboratory during the last decade and are experimentally based on the measurement of cell volume variations in response to changes in the medium composition. In the presence of isoosmotic concentration gradients of solutes between intracellular and extracellular media, mass transfers were found to be governed by the diffusion rate of the solutes through the cell membrane and were achieved within a few seconds. In the presence of osmotic gradients, mass transfers mainly consisting in a water flow were found to be rate limited by the mixing systems used to generate a change in the medium osmotic pressure. The use of ultra-rapid mixing systems allowed us to show that yeast cells respond to osmotic upshifts within a few milliseconds and to determine a very high hydraulic permeability for yeast membrane (Lp>6.10(-11) m x sec)-1) x Pa(-1)). This value suggested that yeast membrane may contain facilitators for water transfers between intra and extracellular media, i.e. aquaporins. Cell volume variation in response to osmotic gradients was only observed for osmotic gradients that exceeded the cell turgor pressure and the maximum cell volume decrease, observed during an hyperosmotic stress, corresponded to 60% of the initial yeast volume. These results showed that yeast membrane is highly permeable to water and that an important fraction of the intracellular content was rapidly transferred between intracellular and extracellular media in order to restore water balance after hyperosmotic stresses. Mechanisms implied in cell death resulting from these stresses are then discussed.     

A network thermodynamic model of kidney perfusion with a cryoprotective agent

A network thermodynamic model has been devised to describe the coupled movement of water and a permeable additive within a kidney during perfusion under the combined action of diffusive, hydrodynamic, and mechanical processes. The model has been validated by simulating perfusions with Me2SO, glycerol, and sucrose and comparing predicted weight and vascular resistance with experimental results obtained by Pegg (1993). The flows of CPA, water, colloid, and cellular impermeants are governed by a combination of the individual osmotic potential and pressure differences between compartments of the kidney, the viscoelastic behavior of the tissue, and the momentum transferred between the flows. The model developed in this study presents an analytical tool for understanding the dynamics of the perfused kidney system and for modifying perfusion protocols to minimize the changes in cell volume, internal pressure build-up, and increases in vascular resistance that currently present barriers to the successful perfusion of organs.     

A Highly Thermosensitive and Permeable Mutant of the Marine Yeast Cryptococcus aureus G7a Potentially Useful for Single-Cell Protein Production and its Nutritive Components

The highly thermosensitive and permeable mutants are the mutants from which intracellular contents including proteins can be released when they are incubated both in the low osmolarity water and at the nonpermissive temperature (usually 37 degrees C). After mutagenesis by using nitrosoguanidine, a highly thermosensitive and permeable mutant named Z114 was obtained from the marine yeast Cryptococcus aureus G7a. Of the total protein, 65.3% was released from the mutant cells suspended in distilled water after they were treated at 37 degrees C overnight. However, only 12.3% of the total protein was released from the mutant cells suspended in 1.0 M sorbitol solution after they were treated at 37 degrees C overnight. We found that intracellular density of the mutant treated at 37 degrees C was greatly decreased, and cell volume of the mutant treated at 37 degrees C was increased due to the increased protein release. However, no significant changes in the intracellular density and cell volume of the mutant were observed when its cells suspended in 1.0 M sorbitol solution were treated at 37 degrees C. It was found that no big changes in cell growth, protein content, vitamin C content, nucleic acid content, fatty acids, and amino acid compositions of both the mutant and its wild type were detected. Therefore, the highly thermosensitive and permeable mutant still can be a good candidate as single-cell protein. This means that the method used in this study is a simple and efficient way to release protein from the highly thermosensitive and permeable yeast mutant cells with high protein content.     

Initiation and regulation of water deficit-induced abscisic acid accumulation in maize leaves and roots: cellular volume and water relations

Water deficit-induced ABA accumulation in relation to cellular water relations was investigated in maize root and leaf tissues. While polyethylene glycol (PEG) treatment led to a significant increase of ABA content in both root and leaf tissues, ethylene glycol (EG), a permeable monomer of PEG, had no effect on ABA accumulation at similar or much lower osmotic potentials. A rapid and massive accumulation of ABA in leaf tissues occurred at a specific threshold of PEG 6000 concentration, about 20% (w/v), and closely coincided with the start of the tissue weight loss and the obvious decrease of cellular osmotic potential. Pretreatment with EG lowered the cell sap osmotic potential and also lowered the capability of both root and leaf tissues to accumulate ABA in response to further air-drying or PEG treatment. When samples were dehydrated and incubated under pressure, a method to maintain high water potential and pressure potential during dehydration, ABA accumulation was similar to those dehydrated and incubated under atmospheric pressure. Such results suggest that both the absolute water potential and pressure potential per se had no direct effects on the dehydration-induced ABA accumulation. The results have provided evidence that the initiation of ABA accumulation is related to the weight loss of tissues or changes in cellular volume rather than the cell water relation parameters, and the capability of ABA accumulation can be regulated by cellular osmotic potential.     

Evaluation of five additives to mitigate toxicity of cryoprotective agents on porcine chondrocytes

Cryoprotective agents (CPAs) are used in cryopreservation protocols to achieve vitrification. However, the high CPA concentrations required to vitrify a tissue such as articular cartilage are a major drawback due to their cellular toxicity. Oxidation is one factor related to CPA toxicity to cells and tissues. Addition of antioxidants has proven to be beneficial to cell survival and cellular functions after cryopreservation. Investigation of additives for mitigating cellular CPA toxicity will aid in developing successful cryopreservation protocols. The current work shows that antioxidant additives can reduce the toxic effect of CPAs on porcine chondrocytes. Our findings showed that chondroitin sulphate, glucosamine, 2,3,5,6-tetramethylpyrazine and ascorbic acid improved chondrocyte cell survival after exposure to high concentrations of CPAs according to a live-dead cell viability assay. In addition, similar results were seen when additives were added during CPA removal and articular cartilage sample incubation post CPA exposure. Furthermore, we found that incubation of articular cartilage in the presence of additives for 2 days improved chondrocyte recovery compared with those incubated for 4 days. The current results indicated that the inclusion of antioxidant additives during exposure to high concentrations of CPAs is beneficial to chondrocyte survival and recovery in porcine articular cartilage and provided knowledge to improve vitrification protocols for tissue banking of articular cartilage.