Cellular biophysics during freezing of rat and mouse sperm predicts post-thaw motility.

Though cryopreservation of mouse sperm yields good survival and motility after thawing, cryopreservation of rat sperm remains a challenge. This study was designed to evaluate the biophysics (membrane permeability) of rat in comparison to mouse to better understand the cooling rate response that contributes to cryopreservation success or failure in these two sperm types. In order to extract subzero membrane hydraulic permeability in the presence of ice, a differential scanning calorimeter (DSC) method was used. By analyzing rat and mouse sperm frozen at 5 degrees C/min and 20 degrees C/min, heat release signatures characteristic of each sperm type were obtained and correlated to cellular dehydration. The dehydration response was then fit to a model of cellular water transport (dehydration) by adjusting cell-specific biophysical (membrane hydraulic permeability) parameters L(pg) and E(Lp). A "combined fit" (to 5 degrees C/min and 20 degrees C/min data) for rat sperm in Biggers-Whitten-Whittingham media yielded L(pg) = 0.007 microm min(-1) atm(-1) and E(Lp) = 17.8 kcal/mol, and in egg yolk cryopreservation media yielded L(pg) = 0.005 microm min(-1) atm(-1) and E(Lp) = 14.3 kcal/mol. These parameters, especially the activation energy, were found to be lower than previously published parameters for mouse sperm. In addition, the biophysical responses in mouse and rat sperm were shown to depend on the constituents of the cryopreservation media, in particular egg yolk and glycerol. Using these parameters, optimal cooling rates for cryopreservation were predicted for each sperm based on a criteria of 5%-15% normalized cell water at -30 degrees C during freezing in cryopreservation media. These predicted rates range from 53 degrees C/min to 70 degrees C/min and from 28 degrees C/min to 36 degrees C/min in rat and mouse, respectively. These predictions were validated by comparison to experimentally determined cryopreservation outcomes, in this case based on motility. Maximum motility was obtained with freezing rates between 50 degrees C/min and 80 degrees C/min for rat and at 20 degrees C/min with a sharp drop at 50 degrees C/min for mouse. In summary, DSC experiments on mouse and rat sperm yielded a difference in membrane permeability parameters in the two sperm types that, when implemented in a biophysical model of water transport, reasonably predict different optimal cooling rate outcomes for each sperm after cryopreservation.

Cryopreservation of collagen-based tissue equivalents. II. Improved freezing in the presence of cryoprotective agents.

In Part I of this study we determined an optimal cooling rate for cryopreservation of collagen-based tissue equivalents (TEs) that preserves both the postthaw cell viability and mechanical properties, but results in tissue contraction and an overall loss of opacity. The empirically determined optimal cooling rate (5 degrees C/min) was obtained in a freezing medium consisting solely of phosphate-buffered saline (PBS) at physiological concentration (1x). In the present study we report the effect of freezing on TEs in the presence of PBS and two cryoprotective agents (CPAs) (glycerol and dimethyl sulfoxide [Me(2)SO]), at two different concentrations (0.5 and 1.0 M), to two different end temperatures (-80 and -160 degrees C), at a cooling rate of 5 degrees C/min. The controlled rate freezing experiments, postthaw cell viability, and mechanical property measurements were performed as described in Part I of this study. In addition to studying the effect of CPAs on the postthaw properties of TEs, we also investigated (1). the effect of freezing TEs attached to the substrate (as opposed to detached and floating in medium) to determine differences when freezing TEs subject to static mechanical stress via a mechanical constraint to contraction; (2). the effect of freezing glutaraldehyde-fixed TEs to determine differences in freezing-mediated damage to the microstructure; and (3). the effect of freezing more mature TEs that were incubated for 4 weeks in growth factor-supplemented medium as opposed to 2 weeks in basal medium. All TEs frozen at 5 degrees C/min to -80 degrees C in the presence of 0.5 M glycerol or Me(2)SO in PBS were found to be optimally cryopreserved in terms of maintaining opacity and structure as well as cell viability and mechanical properties as compared with unfrozen TEs. The postthaw mechanical properties were adversely affected by freezing to the lower end temperature of -160 degrees C in the presence of CPAs, with the samples frozen in the 1.0 M concentration of CPAs exhibiting a total loss of structural integrity on thawing. Furthermore, TEs frozen attached to the substrate showed decreased opacity and significant contraction as compared with TEs frozen detached from the substrate, as did cross-linked samples frozen without CPA.