Algae Permeability to Me2SO from −3 to 23°C

Biphasic transport of water and dimethyl sulfoxide (Me₂SO), a common cryoprotective agent (CPA), in algal cells was induced and measured on a cryoperfusion stage. A two-step experimental protocol provided data for the volumetric response of Chlorococcum (C.) texanum to impermeable and permeable solutes. First, the cells were exposed to a 500-mOsm sucrose solution, causing immediate shrinkage of the cell to a minimum equilibrium volume. Then an isoosmotic 200-mOsm/300-mOsm CPA/sucrose solution was introduced to the cells, resulting in increased cell volume to a new equilibrium state. Experiments were conducted at temperatures between −3 and 23°C. Cell volumes were measured off-line by computer analysis of video images. A network thermodynamic model was fit to the transient volume data to determine permeabilities of C. texanum to water and Me₂SO over the full temperature range, and results were calculated with two numeric methods. Biphasic transport was found to be slower at colder temperatures, with water entering the cell faster than Me₂SO. Experimental results were also compared with data from similar experiments using methanol (MeOH) as the CPA. MeOH influx was calculated to be a magnitude larger than that of water. Additionally, MeOH permeability was at least three orders of magnitude greater than Me₂SO permeability, and the difference in these solute permeabilities increased as temperature decreased.     

Suitability of cryoprotectants and impregnation protocols for embryos of Japanese whiting Sillago japonica

The purpose of this study was to examine the suitability of cryoprotectant agent (CPA) impregnation protocols for the embryos of Japanese whiting (Sillago japonica), a small-sized, easy-to-rear, and prolific marine fish which may constitute a suitable experimental material for the development of cryopreservation methods for fish embryos. Our immediate goals were to assess the toxicity and permeability of various CPAs to whiting embryos of different developmental stages. Exposure of gastrula, somites, tail elongation, and pre-hatching embryos to 10%, 15%, and 20% solutions of propylene glycol (PG), methanol (MeOH), dimethyl sulfoxide (Me2SO), dimethylformamide (DFA), ethylene glycol (EG), and glycerol (Gly) in artificial sea water (ASW; 33 psu) for 20 min revealed that CPA toxicity for whiting embryos increased in the order of PG相似文献   

Measurement and simulation of water and methanol transport in algal cells

BACKGROUND: Experimental data and a complementary biophysical model are presented to describe the dynamic response of a unicellular microalga to osmotic processes encountered during cryopreservation. METHOD OF APPROACH: Chlorococcum texanum (C. texanum) were mounted on a cryoperfusion microscope stage and exposed sequentially to various solutions of sucrose and methanol. Transient volumetric excursions were determined by capturing images of cells in real time and utilizing image analysis software to calculate cell volumes. A biophysical model was applied to the data via inverse analysis in order to determine the plasma membrane permeability to water and to methanol. The data were also used to determine the elastic modulus of the cell wall and its effect on cell volume. A three-parameter (hydraulic conductivity (Lp), solute permeability; (omega), and reflection coefficient, (sigma)) membrane transport model was fit to data obtained during methanol perfusion to obtain constitutive property values. These results were compared with the property values obtained for a two coefficient (Lp and omega) model. RESULTS: The three-parameter model gave a value for sigma not consistent with practical physical interpretation. Thus, the two-coefficient model is the preferred approach for describing simultaneous water and methanol transport. The rate of both water and methanol transport were strongly dependent on temperature over the measured temperature range (25 degrees C to -5 degrees C) and cells were appreciably more permeable to methanol than to water at all measured temperatures. CONCLUSION: These results may explain in part why methanol is an effective cryoprotective agent for microalgae.     

Determination of bull sperm membrane permeability to water and cryoprotectants using a concentration-dependent self-quenching fluorophore

The objective of this study was to determine the membrane permeability characteristics of bovine spermatozoa. These included the hydraulic conductivity (Lp), the permeability coefficients (Ps) of four common cryoprotective agents (CPAs) and the associated reflection coefficients (sigma). Stopped-flow fluorometry was applied in order to capture rapid cell volume changes under anisosmotic conditions in the absence or presence of permeant solutes (CPAs). This technique utilizes a concentration-dependent self-quenching entrapped fluorophore. The resulting cell volume changes were used in three-parameter fitting calculations to compute Lp in the absence of permeant solutes and Ps and Lp in the presence of permeating solutes (CPAs) at 22 degrees C. The hydraulic conductivity in the absence of permeating solutes was estimated to be 0.68+/-0.05 microm/min/atm (mean+/-SEM). Hydraulic conductivity (Lp) in the presence of CPAs was 0.91+/-0.27 (mean+/-SEM), 0.29+/-0.04, 0.42+/-0.05, and 0.39+/-0.03 microm/min/atm in the presence of dimethylsulfoxide (Me(2)SO), glycerol, propylene glycol (PG), and ethylene glycol (EG), respectively. The values for Ps were estimated to be 1.72+/-0.36, 1.75+/-0.03, 2.47+/-0.24, and 1.49+/-0.33 x 10(-3)cm/min for Me(2)SO, glycerol, PG, and EG, respectively. The data were used to simulate volume excursions during addition and removal of CPA, to predict the different effects of the four CPAs.     

Fundamental cryobiology of rat immature and mature oocytes: hydraulic conductivity in the presence of Me(2)SO, Me(2)SO permeability, and their activation energies

The hydraulic conductivity in the presence of dimethyl sulfoxide Me(2)SO (L(p)(Me(2)SO)), Me(2)SO (P(Me(2)SO)) permeability and reflection coefficient (sigma) of immature (germinal vesicle; GV) and mature (metaphase II; MII) rat oocytes were determined at various temperatures. A temperature controlled micropipette perfusion technique was used to conduct experiments at five different temperatures (30, 20, 10, 4, and -3 degrees C). Kedem and Katchalsky membrane transport theory was used to describe the cell volume kinetics. The cell volumetric changes of oocytes were calculated from the measurement of two oocyte diameters, assuming a spherical shape. The activation energies (E(a)) of L(p)(Me(2)SO) and P(Me(2)SO) were calculated using the Arrhenius equation. Activation energies of L(p)(Me(2)SO) for GV and MII oocytes were 34.30 Kcal/mol and 16.29 Kcal/mol, respectively; while the corresponding E(a)s of P(Me(2)SO) were 19.87 Kcal/mol and 21.85 Kcal/mol, respectively. These permeability parameters were then used to calculate cell water loss in rat oocytes during cooling at subzero temperatures. Based on these values, the predicted optimal cooling rate required to maintain extra- and intracellular water in near equilibrium for rat GV stage oocytes was found to be between 0.05 degrees C/min and 0. 025; while for rat MII oocytes, the corresponding cooling rate was 1 degrees C/min. These data suggest that standard cooling rates used for mouse oocytes (e.g., 0.5-1 degrees C/min) can also be employed to cryopreserve rat MII oocytes. However, the corresponding cooling rate required to avoid damage must be significantly slower for the GV stage rat oocyte. J. Exp. Zool. 286:523-533, 2000.     

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.     

Cell volume measured by total internal reflection microfluorimetry: application to water and solute transport in cells transfected with water channel homologs.

Total internal reflection (TIR) microfluorimetry was established as a method to measure continuously the volume of adherent cells and applied to measure membrane permeabilities in cells transfected with water channel homologs. Cytosol was labeled with the membrane-impermeant fluorophore calcein. Fluorescence was excited by the TIR evanescent field in a thin section of cytosol (approximately 150 nm) adjacent to the cell-substrate interface. Because cytosolic fluorophore number per cell remains constant, the TIR fluorescence signal should be inversely related to cell volume. For small volume changes in Sf-9 and LLC-PK1 cells, relative TIR fluorescence was nearly equal to inverse relative cell volume; deviations from the ideal were modeled theoretically. To measure plasma membrane osmotic water permeability, Pf, the time course of osmotically induced cell volume change was inferred from the TIR fluorescence signal. LLC-PK1 cells expressing the CHIP28 water channel had an HgCl2-sensitive, threefold increase in Pf compared to nontransfected cells (Pf = 0.0043 cm/s at 10 degrees C). Solute permeability was measured from the TIR fluorescence time course in response to solute gradients. Glycerol permeability in Sf-9 cells expressing the water channel homolog GLIP was (1.3 +/- 0.2) x 10(-5) cm/s (22 degrees C), greater than that of (0.36 +/- 0.04) x 10(-5) cm/s (n = 4, p < 0.05) for control cells, indicating functional expression of GLIP. Water and urea permeabilities were similar in GLIP-expressing and control cells. The TIR method should be applicable to the study of water and solute permeabilities and cell volume regulation in cells of arbitrary shape and size.     

Temperature-dependent osmotic behavior of germinal vesicle and metaphase II stage bovine oocytes in the presence of Me2SO in relationship to cryobiology

Plasma membrane permeability coefficients and their activation energies (Ea) for water (Lp) and dimethyl sulfoxide (PMe2SO) as well as the reflection coefficient (sigma) were determined for germinal vesicle (GV) and metaphase II (MII) bovine oocytes. A micropipette perfusion technique was used with a temperature controlled circulation chamber, which was adapted to a micromanipulator. Experiments were performed at five different temperatures (30, 20, 10, 4 and -3 degrees C). The Kedem and Katchalsky model was assumed and L(p), P(Me2SO) and sigma were estimated. Estimated permeability values from the experimental temperatures were then applied to Arrhenius plots In(Lp) or In(PMe2SO) vs 1/Temperature (K) to estimate the activation energies (Ea) for L(p)Me2SO and P(Me2SO). The estimated E(a) for L(p)Me2SO for GV and MII oocytes were 23.84 Kcal/mol and 8.46 Kcal/mol, respectively. The E(a) for P(Me2SO) were 21.0 Kcal/mol and 23.20 Kcal/mol, respectively. The correlation (r2) for these linear regression plots for GV oocytes were 0.83 and 0.95 for L(p)Me2SO and P(Me2SO), respectively. For MII oocytes, r2 values were 0.95 and 0.99 for L(p)Me2SO and P(Me2SO), respectively. There was a possible discontinuity detected in the Arrhenius plot for L(p)Me2SO for GV oocytes. A significant decrease of the reflection coefficient was observed at 10 degrees C compared to other experimental temperatures. These data provide a fundamental basis that should be taken into account for low temperature preservation of bovine oocytes in the presence of Me2SO.     

Toxicity and protective efficiency of cryoprotectants to flounder (Paralichthys olivaceus) embryos

With the purpose of finding an ideal cryoprotectant or combination of cryoprotectants in a suitable concentration for flounder (Paralichthys olivaceus) embryo cryopreservation, we tested the toxicities, at culture temperature (16 degrees C), of five most commonly used cryoprotectants-dimethyl sulfoxide (Me2SO), glycerol, methanol (MeOH), 1,2-propylene glycol (PG) and ethylene glycol (EG). In addition, cryoprotective efficiency to flounder embryos of individual and combined cryoprotectants were tested at -15 degrees C for 60 min. Five different concentrations of each of the five cryoprotectants and 20 different combinations of these cryoprotectants were tested for their protective efficiency. The results showed that the toxicity to flounder embryos of the five cryoprotectants are in the following sequence: PG < MeOH < Me2SO < glycerol < EG (P < 0.05); whereas the protective efficiency of each cryoprotectant, at -15 degrees C for a period of 60 min, are in the following sequence: PG > Me2SO approximately MeOH approximately glycerol > EG (greater symbols mean P < 0.05, and approximate symbols mean P > 0.05). Methanol combined with any one of the other cryoprotectants gave the best protection, while ethylene glycol combined with any one of the other cryoprotectants gave the poorest protection at -15 degrees C. Toxicity effect was concentration dependent with the lowest concentration being the least toxic for all five cryoprotectants at 16 degrees C. For PG, MeOH and glycerol, 20% solutions gave the best protection at -15 degrees C; whereas a 15% solution of Me2SO, and a 10% solution of EG, gave the best protection at -15 degrees C.     

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.     

Aquaporin proteins in murine trophectoderm mediate transepithelial water movements during cavitation

Mammalian blastocyst formation is dependent on establishment of trophectoderm (TE) ion and fluid transport mechanisms. We have examined the expression and function of aquaporin (AQP) water channels during murine preimplantation development. AQP 3, 8, and 9 proteins demonstrated cell margin-associated staining starting at the 8-cell (AQP 9) or compacted morula (AQP 3 and 8) stages. In blastocysts, AQP 3 and 8 were detected in the basolateral membrane domains of the trophectoderm, while AQP3 was also observed in cell margins of all inner cell mass (ICM) cells. In contrast, AQP 9 was predominantly observed within the apical membrane domains of the TE. Murine blastocysts exposed to hyperosmotic culture media (1800 mOsm; 10% glycerol) demonstrated a rapid volume decrease followed by recovery to approximately 80% of initial volume over 5 min. Treatment of blastocysts with p-chloromercuriphenylsulfonic acid (pCMPS, > or =100 microM) for 5 min significantly impaired (P < 0.05) volume recovery, indicating the involvement of AQPs in fluid transport across the TE. Blastocysts exposure to an 1800-mOsm sucrose/KSOMaa solution did not demonstrate volume recovery as observed following treatment with glycerol containing medium, indicating glycerol permeability via AQPs 3 and 9. These findings support the hypothesis that aquaporins mediate trans-trophectodermal water movements during cavitation.     

Dimethyl sulfoxide influx in turbot embryos exposed to a vitrification protocol

The particular characteristics of fish embryos require the development of specific methods for cryopreservation. One of the main obstacles is related to the presence of membranes and compartments with different water and cryoprotectant permeability. To assess dimethyl sulfoxide (Me2SO4) permeability, we exposed turbot embryos (Scophthalmus maximus) at F stage (tail bud) to the cryoprotectant solutions used in a vitrification protocol and then evaluated the Me2SO4 content inside the embryo using high-performance liquid chromatography (HPLC). The Me2SO4 influx was analyzed in normal embryos and in embryos treated with pronase (2mg/ml) in order to increase chorion permeability. The evaluation was made after each step of cryoprotectant incorporation and removal. Three embryo compartments were distinguished: the perivitelline space (PVS), the yolk sac (YS) and the cellular compartment (CC), and the relative volumes of each, estimated using stereoscopic microscopy imaging, were 11.37, 81.23 and 7.40%, respectively. The Me2SO4 concentration inside the embryos was calculated based on their entrance into one, two or three compartments. Results suggest high entrance of Me2SO4 into the PVS and a low concentration of this cryoprotectant inside the other compartments. Pronase did not significantly increase Me2SO4 influx, but facilitated its elimination during the washing steps.     

Factors influencing renal cryopreservation. II. Toxic effects of three cryoprotectants in combination with three vehicle solutions in nonfrozen rabbit cortical slices

The [K+]/[Na+] ratio of rabbit renal cortical slices was used to examine, at 25 degrees C, the effects on viability of three cryoprotectant agents (CPA) (dimethyl sulfoxide (Me2SO), ethylene glycol, and glycerol) in combination with three vehicle solutions (Krebs-Henseleit (K-H), solution A, and RPS-2). Viability assessment by [K+]/[Na+] for all test solutions was made after incubating the slices in modified Cross-Taggart solution (C-T). With K-H and solution A, all concentrations of ethylene glycol and glycerol resulted in lowered ratios, whereas with Me2SO, concentrations greater than 1.4 M are required to reduce [K+]/[Na+]. With RPS-2 no decrease in the ratios was found until concentrations greater than 2.8 M were reached for all three CPAs. Binding of Me2SO to albumin, studied using [14C]Me2SO, was inhibited by RPS-2 when compared to K-H. Introduction and removal of Me2SO at 10 degrees C allowed an improvement in viability, at higher Me2SO concentrations, as compared to 25 degrees C.     

Freezing response of white bass (Morone chrysops) sperm cells

The water transport response during freezing of sperm cells of Morone chrysops (white bass, WB) was obtained using a shape-independent differential scanning calorimeter (DSC) technique. Sperm cell suspensions were frozen at a cooling rate of 20 degrees C/min in two different media: (1) without cryoprotective agents (CPAs), or (2) with 5% (v/v) dimethyl sulfoxide (Me2SO). For calculations, the sperm cell was modeled as a cylinder of length 24.8 microm and diameter of 0.305 microm, while the osmotically inactive cell volume (Vb) was assumed to be 0.6 Vo, where Vo was the isotonic or the initial cell volume. By fitting a model of water transport to the experimentally determined water transport data, the best fit membrane permeability parameters (reference membrane permeability to water, Lpg or Lpg[cpa] and the activation energy, ELp or ELp[cpa]) were determined, and ranged from Lpg = 0.51-1.7 x 10(-15) m3/Ns (0.003-0.01 microm/min-atm), and ELp = 83.6-131.3 kJ/mol (20.0-31.4 kcal/mol). The parameters obtained in this study suggest that the optimal rate of cooling for M. chrysops sperm cells is approximately 22 degrees C/min, a value that compares closely with experimentally determined optimal rates of cooling (approximately 16 degrees C/min).     

Cryoprotectants lead to phenotypic adaptation to freeze-thaw stress in Lactobacillus delbrueckii ssp. bulgaricus CIP 101027T

This study was conducted to investigate the ability of cryoprotective chemicals to induce phenotypic cryoadaptation in Lactobacillus delbrueckii ssp. bulgaricus CIP 101027T. Tolerance to negative temperature stress (freezing at -20 degrees C and thawing at 37 degrees C) was induced by pretreatment with Me(2)SO, glycerol, lactose, sucrose, and trehalose. Interestingly, Me(2)SO has a significantly greater cryoprotective effect than glycerol. Furthermore, lactose, sucrose, and trehalose, often referred to as osmotica, were shown to have greater cryoadaptive than cryoprotective properties. These results suggest that bacteria such as L. delbrueckii ssp. bulgaricus could be phenotypically adapted to freezing and thawing by an osmotic stress applied prior to freeze-thaw stress.     

Ca2+ transport and permeability in inside-out red cell membrane vesicles after freezing

To evaluate the effects of freezing and thawing on Ca2+ transport and permeability, inside-out red cell membrane vesicles (IORCMV) are examined. Exposure to the cryoprotectant Me2SO as well as different cooling regimes on unprotected and cryoprotected vesicles do not affect the membrane Ca2+ transport. However, freezing and thawing increase the membrane permeability to sucrose.     

Chilling sensitivity of carp (Cyprinus carpio) embryos at different developmental stages in the presence or absence of cryoprotectants: work in progress

The unsolved problem of cryopreservation of the yolk-rich teleost embryos may be related, in part, to their sensitivity to chilling and cryoprotective agents. The aim of this study was to gain data on the sensitivity of carp embryos to low temperatures at different developmental stages and on the possible protective and toxic effects of cryoprotectants. A total of 86,400 morulae, half-epiboly and heartbeat-stage embryos was selected and then placed in water or in 1 M methanol, dimethyl sulfoxide (Me2SO), glycerol or 0.1 M sucrose solution at 0, 4 or 24 degrees C for 5 min or 1 h. Following these treatments, the embryos were held in a 24 degrees C water bath until the evaluation of hatching rates. In every developmental stage a significant decrease of hatching rates following exposure to 4 or 0 degree C was detected. Sensitivity to chilling changed significantly with development (heartbeat < morula < half-epiboly). Half-epiboly stage embryos were less sensitive to a short period of exposure to cryoprotectants than morula and heartbeat stages. A 1-h exposure to cryoprotectants revealed a stage dependent sensitivity. Toxicity increased in the order of methanol < Me2SO < glycerol in morula and half-epiboly stages, and methanol < glycerol < Me2SO in the heartbeat stage. The results show morulae are partially protected against chilling in Me2SO and sucrose, half-epiboly in Me2SO, sucrose and methanol, and heartbeat-stage in methanol and glycerol. The results further suggest that carp embryos are sensitive to chilling and that toxicity and protective effects against chilling of cryoprotectants are stage-dependent. The finding on the low chilling sensitivity of heartbeat-stage embryos and the protective effect of certain cryoprotectants may be useful in designing cryopreservation protocols.     

Vitrification media: toxicity,permeability, and dielectric properties

The aim of this study was to select a cryoprotectant for use in attempts to preserve tissues and organs by vitrification. The first step was to select a cell line with which to compare the toxicity of a range of commonly used cryoprotectants. An immortal vascular endothelial cell (ECV304) was exposed to vitrifying concentrations of four cryoprotectants: dimethyl sulfoxide (Me(2)SO; 45% w/w); 2,3 butanediol (BD; 32%); 1,2-propanediol (PD; 45%); and ethanediol (ED; 45%). Three times of exposure (1, 3, and 9 min) and two temperatures (22 and 2-4 degrees C) were studied. After removal of the cryoprotectant, the ability of the cells to adhere and divide in culture over a 2-day period was measured and expressed as a Cell Survival Index (CSI). There was no measurable loss of cells after exposure to the four cryoprotectants but 3-min exposure to BD, PD, or Me(2)SO at room temperature completely destroyed the ability of the cells to adhere and divide in culture. In contrast, exposure to all four cryoprotectants at 2-4 degrees C for up to 9 min permitted the retention of significant cell function, the CSIs, as a proportion of control, being 76.3+/-7.0% for BD, 63.6+/-7.1% for PD, 37.0+/-4.1 for Me(2)SO, and 33.2+/-3.0 for ED. The permeability properties of the cells for these four cryoprotectants was also measured at each temperature. Permeability to water was high, L(p) approximately equal 10(-7) cm/s/atm at 2-4 degrees C with all the cryoprotectants, but there were substantial differences in solute permeability: BD and PD were the most permeable at 2-4 degrees C (P(s)=4.1 and 3.0 x 10(-6) cm/s, respectively). Equilibration of intracellular cryoprotectant concentration was rapid, due in part to high water permeability; the cells were approximately 80% of their physiological volume after 10 min. Treatment at 2-4 degrees C with BD was the least damaging, but PD was not significantly worse. Exposure to vitrifying concentrations of ED and Me(2)SO, even at 2-4 degrees C, was severely damaging. Segments of rabbit carotid artery were treated with vitrifying concentrations of each of the two most favorable cryoprotectants, BD and PD, for 9 min. It was shown that each cryoprotectant reduced smooth muscle maximum contractility to a similar extent and abolished the acetylcholine response. However, vital staining revealed that exposure to BD also caused substantial damage to the endothelial lining, whereas the endothelium was completely intact after PD exposure, raising the possibility that the effect of PD on NO release may be reversible. In later stages of this project it is planned to use dielectric heating to rewarm the tissues and thereby avoid devitrification. The effects of each cryoprotectant on this mode of heating was therefore studied. Gelatin spheres containing vitrifiable concentrations of each cryoprotectant were rewarmed from -60 degrees C in a radiofrequency applicator. Because the uniformity of heating is related to the dielectric properties of the material, these properties were also measured. PD was the most suitable. These physical measurements, combined with the measurements of toxicity and permeability, indicate that PD is the most favorable cryoprotectant of those tested for use in subsequent stages of this study.     

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.     

Modulation of the cryopreservation cap: elevated survival with reduced dimethyl sulfoxide concentration

The development of cryopreservation (CP) strategies has traditionally focused on the cellular chemo-osmometric characteristics attendant to the freeze-thaw process. This approach coupled with a limited understanding of cellular physiological and biochemical responses to the CP process often yields sub-optimal cell survival. Recently, we have reported on the benefits of the utilization of an intracellular-like preservation solution, HypoThermosol (HTS), as well as incorporating a molecular approach to improving CP outcome [In Vitro Cell. Dev. Biol. Anim. 36(4) (2000) 262]. We now report on the elucidation of a cryoprotective agent (CPA)-dependent survival limit (cap) associated with standard CP methodologies. We further demonstrate an elevation and shift in the CP cap through the utilization of HTS coupled with a reduction in CPA levels necessary to achieve "successful" cell preservation. METHODS: Human fibroblasts, keratinocytes, hepatic, and renal cells were cryopreserved in a standard fashion (approximately 1 degrees C min-1 cooling and storage in LN2) in culture media (serum-free) or HTS with varying levels of dimethyl sulfoxide (Me2SO). Samples were allowed to recover for 24-h prior to survival assessment. Survival was assessed using alamarBlue (metabolic activity indicator) and calcien-AM (membrane integrity stain) in comparison with non-frozen controls. RESULTS: (1) A limit in cell survival was identified following CP in media-based CP solutions yielding a cell-type specific CPA-dependent survival limit, (2) peak cell survival resulted in the identification of "optimal" Me2SO concentrations for CP of each cell type, (3) incorporation of HTS as the carrier medium at typical Me2SO concentrations substantially elevated survival, and (4) utilization of HTS allowed for the successful preservation of all systems examined at significantly reduced Me2SO levels. CONCLUSION: The data presented in this study illustrate that the utilization of HTS as the carrier medium during CP facilitated a significant improvement in efficacy at reduced Me2SO levels. Further, the utilization of HTS offers the potential for successful Me2SO-free CP. These findings may prove significant to the advancement in the development of cell-based clinical therapies by providing an improved biocompatible CP methodology.