Reference Membrane Permeability Research Articles

Effect of cryoprotectant on optimal cooling rate during cryopreservation

The effect of initial Me2SO concentration (cgi) inside the cell lines on the optimal cooling rate is studied using a well established water transport model. A correlation formula is proposed for the determination of optimal cooling rate of freezing biological systems which depends on the cell activation energy, reference membrane permeability, initial Me2SO concentration, and the cell geometrical parameters. Here, the optimal cooling rate is defined as the highest cooling rate for which amount of trapped water inside the cell is equal to 5% of the initial cell water content at an end temperature of −40°C. It is found that the optimal cooling rate varies linearly with the reference membrane permeability and the ratio of surface area for water transport to the initial volume of intracellular water. The developed correlation is valid for cell activation energy between 20 and 80kcal/mole and initial Me2SO concentration between 0.1 and 1.3M. It has been observed that the optimal cooling rate does not follow a single trend for the studied initial concentration of Me2SO. However, three regions are identified within which, the variation is almost similar; the three regions are: 0.1M⩽cgi⩽0.7, 0.7M⩽cgi⩽0.9, and 0.9M⩽cgi⩽1.3M. It has been shown that the predicted optimal cooling rate is in a very good agreement with the published experimental/numerical prediction.

Comparison of the permeability properties and post-thaw motility of ejaculated and epididymal bovine spermatozoa

There are very few experimental reports on the comparative water transport (membrane permeability) characteristics of ejaculated and epididymal mammalian spermatozoa during freezing. In the present study, we report the effects of cooling ejaculated and epididymal bovine sperm from the same males with and without the presence of a cryoprotective agent, glycerol. Water transport data during freezing of ejaculated and epididymal bovine sperm suspensions were obtained at a cooling rate of 20 degrees C/min under two different conditions: (1) in the absence of any cryoprotective agents, CPAs and, (2) in the presence of 0.7 M glycerol. Using values published in the literature, we modeled the spermatozoa as a cylinder of length 39.8 microm and a radius of 0.4 microm with an osmotically inactive cell volume, V(b), of 0.61 V(o), where V(o) is the isotonic cell volume. The subzero water transport response is analyzed to determine the variables governing the rate of water loss during cooling of bovine spermatozoa, i.e. the membrane permeability parameters (reference membrane permeability, L(pg) and activation energy, E(Lp)). The predicted best-fit permeability parameters ranged from, L(pg)=0.021-0.038 microm/min-atm and E(Lp)=27.8-41.1 kcal/mol. The subzero water transport response and consequently the subzero water transport parameters are not significantly different between the ejaculated and epididymal bovine spermatozoa under corresponding cooling conditions. If this observation is found to be more generally valid for other mammalian species as well, then in the future the sperm extracted from the testes of a postmortem male could be optimally cryopreserved using procedures similar to those derived for ejaculated sperm.

Water transport in epididymal and ejaculated rhesus monkey ( Macaca mulatta) sperm during freezing

In the present study, we report the effects of cooling ejaculated and epididymal rhesus monkey ( Macaca mulatta) sperm with and without the presence of a cryoprotective agent, glycerol. Water transport data during freezing of ejaculated and epididymal sperm cell suspensions were obtained at a cooling rate of 20 °C/min in the absence of any cryoprotective agents and in the presence of 0.7 M of glycerol, as well. Using previously published values, the macaque sperm cell was modeled as a cylinder of length 73.83 μm with a radius of 0.40 μm and an osmotically inactive cell volume, V b, of 0.772 V o, where V o is the isotonic cell volume. This translated to a surface area, SA to initial water volume, WV ratio of ∼22 μm −1. By fitting a model of water transport to the experimentally determined volumetric shrinkage data, the best-fit membrane permeability parameters (reference membrane permeability to water at 0 °C, L pg or L pg [ cpa] and the activation energy, E Lp or E Lp [ cpa]) were found to range from: L pg or L pg [ cpa] = 0.0020–0.0029 μm/min-atm; E Lp or E Lp [ cpa]) = 10.6–18.3 kcal/mole. By incorporating these membrane permeability parameters in a recently developed equation (optimal cooling rate, B opt = 1009.5 · exp ( - 0.0546 · E Lp ) · ( L pg ) · ( SA WV ) ; where the units of B opt are °C/min, E Lp or E Lp [ cpa] are kcal/mole, L pg or L pg [ cpa] are μm/min-atm and SA/WV are μm −1), we determined the optimal rates of freezing macaque sperm to be ∼23 °C/min (ejaculated sperm in the absence of CPAs), ∼29 °C/min (ejaculated sperm in the presence of glycerol), ∼24 °C/min (epididymal sperm in the absence of CPAs) and ∼24 °C/min (epididymal sperm in the presence of glycerol). In conclusion, the subzero water transport response and consequently the subzero water transport parameters are not significantly different between the ejaculated and epididymal macaque spermatozoa under corresponding cooling conditions.

Effect of glycerol and cholesterol-loaded cyclodextrin on freezing-induced water loss in bovine spermatozoa

Recent experimental data show that incubating bovine sperm with cholesterol-loaded cyclodextrin (CLC) before cryopreservation increases the percentages of motile and viable cells recovered after freezing and thawing, compared with control sperm. In the present study, we report the effect of incubating bovine sperm with CLC on the subzero water transport response and the membrane permeability parameters (reference membrane permeability (L(pg)) and activation energy (E(Lp))). Water transport data during freezing of bovine sperm cell suspensions were obtained at a cooling rate of 20 degrees C/min under three different conditions: 1. in the absence of cryoprotective agents (CPAs); 2. in the presence of 0.7 M glycerol; and 3. in the presence of 1.5 mg/ml CLC and 0.7 M glycerol. With previously published values, the bovine sperm cell was modeled as a cylinder of length 39.8 microm and radius 0.4 microm, with osmotically inactive cell volume (V(b)) of 0.61 V(o), where V(o) is the isotonic cell volume. By fitting a model of water transport to the experimentally obtained data, the best-fit water transport parameters (L(pg) and E(Lp)) were determined. The predicted best-fit permeability parameters ranged from L(pg) = 0.02 to 0.036 microm/min-atm and E(Lp) = 26.4 to 42.1 kcal/mol. These subzero water transport parameters are significantly different from the suprazero membrane permeability values (obtained in the absence of extracellular ice) reported in the literature. Calculations made of the theoretical response of bovine spermatozoa at subzero temperatures suggest that the optimal cooling rate to cryopreserve bovine spermatozoa is 45-60 degrees C/min, agreeing quite closely with experimentally determined rates of freezing bovine spermatozoa.

Freezing response and optimal cooling rates for cryopreserving sperm cells of striped bass, Morone saxatilis

This study explored the optimization of techniques for sperm cryopreservation of an economically important fish species, the striped bass Morone saxatilis. The volumetric shrinkage or the water transport response during freezing of sperm cells was obtained using a differential scanning calorimeter (DSC) technique. Water transport was obtained in the presence of extracellular ice at a cooling rate of 20°C/min in two different media: (1) without cryoprotective agents (CPAs), and (2) with 5% (v/v) dimethyl sulfoxide (DMSO). The sperm cell was modeled as a cylinder of length of 22.8μm and diameter 0.288μm and was assumed to have an osmotically inactive cell volume (Vb) of 0.6V0, where V0 is the isotonic or 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=0.011–0.001μm/min-atm, and ELp=40.2–9.2kcal/mol). The parameters obtained in this study suggested that the optimal rate of cooling for striped bass sperm cells in the presence and absence of DMSO range from 14 to 20°C/min. These theoretically predicted rates of optimally freezing M. saxatilis sperm compared quite closely with independent and experimentally determined optimal rates of cooling striped bass sperm.

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 °C/min in two different media: (1) without cryoprotective agents (CPAs), or (2) with 5% (v/v) dimethyl sulfoxide (Me 2SO). For calculations, the sperm cell was modeled as a cylinder of length 24.8 μm and diameter of 0.305 μm, while the osmotically inactive cell volume ( V b) was assumed to be 0.6 V o, where V o 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, L pg or L pg [ cpa] and the activation energy, E Lp or E Lp [ cpa]) were determined, and ranged from L pg = 0.51–1.7 × 10 −15 m 3/Ns (0.003–0.01 μm/min-atm), and E Lp = 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 ∼22 °C/min, a value that compares closely with experimentally determined optimal rates of cooling (∼16 °C/min).

Transport phenomena during freezing of adipose tissue derived adult stem cells

In the present study a well-established differential scanning calorimeter (DSC) technique is used to measure the water transport phenomena during freezing of stromal vascular fraction (SVF) and adipose tissue derived adult stem (ADAS) cells at different passages (Passages 0 and 2). Volumetric shrinkage during freezing of adipose derived cells was obtained at a cooling rate of 20 degrees C/min in the presence of extracellular ice and two different, commonly used, cryoprotective agents, CPAs (10% DMSO and 10% Glycerol). The adipose derived cells were modeled as spheres of 50 microm diameter with an osmotically inactive volume (Vb) of 0.6Vo, where Vo is the isotonic cell volume. By fitting a model of water transport to the experimentally obtained volumetric shrinkage 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. The "best-fit" membrane permeability parameters for adipose derived cells in the absence and presence of CPAs ranged from: Lpg=23.1-111.5x10(-15) m3/Ns (0.135-0.652 microm/min-atm) and ELp=43.1-168.8 kJ/mol (9.7-40.4 kcal/mol). Numerical simulations of water transport were then performed under a variety of cooling rates (5-100 degrees C/min) using the experimentally determined membrane permeability parameters. And finally, the simulation results were analyzed to predict the optimal rates of freezing adipose derived cells in the presence and absence of CPAs.

Subzero water permeability parameters and optimal freezing rates for sperm cells of the southern platyfish, Xiphophorus maculatus

This study reports the subzero water transport characteristics (and empirically determined optimal rates for freezing) of sperm cells of live-bearing fishes of the genus Xiphophorus, specifically those of the southern platyfish Xiphophorus maculatus. These fishes are valuable models for biomedical research and are commercially raised as ornamental fish for use in aquariums. Water transport during freezing of X. maculatus sperm cell suspensions was obtained using a shape-independent differential scanning calorimeter technique in the presence of extracellular ice at a cooling rate of 20 °C/min in three different media: (1) Hanks’ balanced salt solution (HBSS) without cryoprotective agents (CPAs); (2) HBSS with 14% (v/v) glycerol, and (3) HBSS with 10% (v/v) dimethyl sulfoxide (DMSO). The sperm cell was modeled as a cylinder with a length of 52.35 μm and a diameter of 0.66 μm with an osmotically inactive cell volume ( V b) of 0.6 V 0, where V 0 is the isotonic or initial cell volume. This translates to a surface area, SA to initial water volume, WV ratio of 15.15 μm −1. By fitting a model of water transport to the experimentally determined volumetric shrinkage data, the best fit membrane permeability parameters (reference membrane permeability to water at 0 °C, L pg or L pg [cpa] and the activation energy, E Lp or E Lp [cpa]) were found to range from: L pg or L pg [cpa] = 0.0053–0.0093 μm/min atm; E Lp or E Lp [cpa] = 9.79–29.00 kcal/mol. By incorporating these membrane permeability parameters in a recently developed generic optimal cooling rate equation (optimal cooling rate, B opt = 1009.5 exp ( - 0.0546 E Lp ) ( L pg ) ( SA WV ) where the units of B opt are °C/min, E Lp or E Lp [cpa] are kcal/mol, L pg or L pg [cpa] are μm/min atm and SA/WV are μm −1), we determined the optimal rates of freezing X. maculatus sperm cells to be 28 °C/min (in HBSS), 47 °C/min (in HBSS + 14% glycerol) and 36 ° C/min (in HBSS + 10% DMSO). Preliminary empirical experiments suggest that the optimal rate of freezing X. maculatus sperm in the presence of 14% glycerol to be ∼25 °C/min. Possible reasons for the observed discrepancy between the theoretically predicted and experimentally determined optimal rates of freezing X. maculatus sperm cells are discussed.

A theoretically estimated optimal cooling rate for the cryopreservation of sperm cells from a live-bearing fish, the green swordtail Xiphophorus helleri

Sperm cryopreservation of live-bearing fishes, such as those of the genus Xiphophorus is only beginning to be studied, although these fishes are valuable models for biomedical research and are commercially raised as ornamental fish for use in aquariums. To explore optimization of techniques for sperm cryopreservation of these fishes, this study measured the volumetric shrinkage response during freezing of sperm cells of Xiphophorus helleri by use of a shape-independent differential scanning calorimeter (DSC) technique. Volumetric shrinkage during freezing of X. helleri sperm cell suspensions was obtained in the presence of extracellular ice at a cooling rate of 20 °C/min in three different media: (1) Hanks’ balanced salt solution (HBSS) without cryoprotective agents (CPAs); (2) HBSS with 14% (v/v) glycerol; and (3) HBSS with 10% (v/v) dimethyl sulfoxide (DMSO). The sperm cell was modeled as a cylinder of 33.3 μm in length and 0.59 μm in diameter with an osmotically inactive cell volume ( V b) of 0.6 V o, where V o is the isotonic or initial cell volume. By fitting a model of water transport to the experimentally determined volumetric shrinkage data, the best-fit membrane permeability parameters (reference membrane permeability to water, L pg or L pg[cpa] and the activation energy, E Lp or E Lp[cpa]) of the Xiphophorus helleri sperm cell membrane were determined. The best-fit membrane permeability parameters at 20 °C/min in the absence of CPAs were: L pg = 0.776 × 10 −15 m 3/Ns (0.0046 μm/min atm), and E Lp = 50.1 kJ/mol (11.97 kcal/mol) ( R 2 = 0.997). The corresponding parameters in the presence of 14% glycerol were L pg[cpa] = 1.063 × 10 −15 m 3/Ns (0.0063 μm/min atm), and E Lp[cpa] = 83.81 kJ/mol (20.04 kcal/mol) ( R 2 = 0.997). The parameters in the presence of 10% DMSO were L pg[cpa] = 1.4 × 10 −15 m 3/Ns (0.0083 μm/min atm), and E Lp[cpa] = 90.96 kJ/mol (21.75 kcal/mol) ( R 2 = 0.996). Parameters obtained in this study suggested that the optimal rate of cooling for X. helleri sperm cells in the presence of CPAs ranged from 20 to 35 °C/min and were in close agreement with recently published, empirically determined optimal cooling rates.

Subzero water transport characteristics of boar spermatozoa confirm observed optimal cooling rates.

Incomplete understanding of the water transport parameters (reference membrane permeability, L(pg), and activation energy, E(Lp)) during freezing in the presence of extracellular ice and cryoprotective agents (CPAs) is one of the main limiting factors in reconciling the difference between the numerically predicted value and the experimentally determined optimal rates of freezing in boar (and in general mammalian) gametes. In the present study, a shape-independent differential scanning calorimeter (DSC) technique was used to measure the water transport during freezing of boar spermatozoa. Water transport data during freezing of boar sperm cell suspensions were obtained at cooling rates of 5 and 20 degrees C/min in the presence of extracellular ice and 6% (v/v) glycerol. Using previously published values, the boar sperm cell was modeled as a cylinder of length 80.1 microm and a radius of 0.31 microm with an osmotically inactive cell volume, V(b), of 0.6 V(o), where V(o) is the isotonic cell volume. By fitting a model of water transport to the experimentally obtained data, the best-fit water transport parameters (L(pg) and E(Lp)) were determined. The "combined-best-fit" parameters at 5 and 20 degrees C/min for boar spermatozoa in the presence of extracellular ice are: L(pg) = 3.6 x 10(-15) m(3)/N. s (0.02 microm/min-atm) and E(Lp) = 122.5 kJ/mole (29.3 kcal/mole) (R(2) = 0.99); and the corresponding parameters in the presence of extracellular ice and glycerol are: L(pg)[cpa] = 0.90 x 10(-15) m(3)/N. s (0.005 microm/min-atm) and E(Lp)[cpa] = 75.7 kJ/mole (18.1 kcal/mole) (R(2) = 0.99). The water transport parameters obtained in the present study are significantly different from previously published parameters for boar and other mammalian spermatozoa obtained at suprazero temperatures and at subzero temperatures in the absence of extracellular ice. The theoretically predicted optimal rates of freezing using the new parameters ( approximately 30 degrees C/min) are in close agreement with previously published but experimentally determined optimal cooling rates. This analysis reconciles a long-standing difference between theoretically predicted and experimentally determined optimal cooling rates for boar spermatozoa.