The Determination of Temperature-Dependent CPA Diffusion Properties in Feline Testicular Tissue

Fertility preservation would benefit young males who must undergo treatments that can result in sterilization, such as radiation treatments for cancer. This can be achieved by removing some testicular tissue before treatment and putting it into frozen storage for later use, a process known as cryopreservation. Cryopreservation requires the use of cryoprotective agents (CPAs), such as dimethyl sulfoxide (DMSO), to reduce injury from ice crystal formation. Because DMSO can be toxic at high exposure levels, it is important to determine the exposure time that is necessary to achieve adequate concentrations for freeze protection, without over-exposing the tissue. Mass diffusion models can be used to predict this loading time, but these models depend on property parameters that are often unknown, such as the mass diffusion coefficient for a given CPA in a specific tissue.
To facilitate the development of cryopreservation protocols for testicular tissue, we determined the mass diffusion coefficient for DMSO in thin (~1 mm) tissue sections that were precision cut from feline testes that were discarded from veterinary sterilization procedures. Samples were placed in a custom tube that was mounted on the surface of an Attenuated Total Reflectance Fourier Transform Infrared (ATR-FTIR) and then exposed to DMSO on the opposite side. Spectra were recorded for 60 minutes, and the area of peak centered at 950 cm-1 was determined as a function of time. This time course absorbance data was then fit to an equation developed by Barbari and Fieldson (1993) that considers both mass diffusion and tissue absorption properties. By minimizing the sum of squares, estimates for the DMSO diffusion coefficient were obtained from each time sequence. Samples were analyzed at 22°C and 4°C.
Because of the inherent variability in biological tissues, alginate-gelatin and agarose were also evaluated for their potential as reference standard materials, to facilitate methodology development and training. Alginate compacted to thicknesses of 1.7 ± 0.2 mm resulting in an effective DMSO diffusion coefficient of 4.3 ± 0.3 x 10-6 cm2/s (n=4). Agarose compacted to thicknesses of 1.1 ± 0.1 mm. The effective diffusion coefficient of DMSO in agarose was 9.2 ± 0.2 x 10-6 cm2/s at 22°C (n=9) and 5.6 ± 0.2 x 10-6 cm2/s (n=9) at 4°C. Although alginate and agarose had similar variability in their thicknesses, agarose had much lower within batch and between batch variability than alginate-gelatin for the effective diffusion coefficients and thus is the preferred reference material for ATR-FTIR diffusion studies. Testicular tissue samples compacted to 2.1 ± 0.7 mm. The effective diffusion coefficient was 10.3 ± 4 x 10-6 cm2/s at 22°C and 7.1 ± 5 x 10-6 cm2/s at 4°C. The high variability is likely due to native variability in the testicular tissue samples. However, these nominal values can be used to inform preservation procedure planning.