The Determination of Temperature-dependent CPA Diffusion Properties in Feline Testicular Tissue
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Abstract
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, a process known as cryopreservation. Cryopreservation has been successfully performed for many types of mammalian testicular tissue, but with only limited success for human tissue [1-4]. In general, immature spermatozoa are more vulnerable to toxicity damage caused by cryo-protective agents, or CPAs, which are necessary to prevent freezing damage [5]. To determine the optimal concentrations of CPAs to use during preservation procedures while minimizing the risk of damage due to CPA toxicity, toxicity cost models have been used to predict the CPA diffusion time into tissues, with the goal of avoiding overexposure [6-8]. The accuracy of these predictions is limited in part by the lack of tissue property data such as CPA diffusion coefficients. The goal of the current work is to determine the effective diffusion coefficient for DMSO in testicular tissue at 22°C and 4°C, to support the planning of CPA loading protocols that minimize toxicity damage during preservation procedures. Testicular tissue consists of Leydig and myoid cells and seminiferous tubules containing various other cell types [9]. The arrangement of cells within tissues can vary spatially and from testis to testis. Sectioning, even within the same sample, can thus yield different values of the same property, which complicates the identification of sources of error in diffusion testing. The creation of a reference standard is pursued in this work, to support the identification of experimental errors in the development of diffusion testing methodology and to provide a means of standardizing measurements between different labs and investigators. Sodium alginate cross-linked with gelatin and low melting point agarose were evaluated for their potential as reference standards. Sample thickness, which is an essential input when estimating diffusivity, was determined before and after sample placement using ATOS Scanbox 4105 employing the triple scan principle. A Frontier (PerkinElmer, MA, USA) Fourier transform infrared spectroscopy (FTIR) with a Gladi-ATR attachment (Pike Technologies, WI, USA) was used to obtain absorbance values as DMSO diffused into SA-gels (n=4), agarose (n=9), or testicular tissue (n=9) at room temperature. These absorbance values were fit to a model developed by Barbari and Fieldson (1993) to determine diffusion coefficients for each sample [10, 11]. A 2-parameter estimation program was created in Excel to allow simultaneous estimation of the effective diffusivity, Deff, values and the equilibrium absorbance, A∞, values. For samples that equilibrated within a 2-hour period, the absorbance values were normalized to the final equilibrium values and fit using a 1-parameter estimation Excel program for only the diffusion coefficient. The pooled average Deff values were determined to be 4.3 ± 0.3 x 10-6 cm2/s, 9.2 ± 0.2 x 10-6 cm2/s, and 10± 4 x 10-6 cm2/s for SA-gels, agarose, and testes, respectively. The variation in effective diffusion coefficients between batches and between replicates within the same batch for agarose was much lower than for alginate-gelatin. It was also easier to slice into prescribed thicknesses and thus was the preferred reference material for diffusion studies utilizing FTIR. The good repeatability of diffusion coefficient estimates in agarose established the validity of the set-up and methodology. The variability in the diffusion coefficients determined for testicular tissue could thus be attributed to inherent differences between tissue samples. A thermal control system was assembled which held temperatures within a range of 4°C ± 4°C. The pooled average Deff values at 4°C were determined to be 5.6 ± 0.2 x 10-6 cm2/s and 7 ± 5 x 10-6 cm2/s for agarose and testicular tissue, respectively. As expected, the diffusion coefficients decreased with temperature. The standard deviation between testis samples increased compared to room temperature samples. Programming of CPA loading procedures will need to consider the variability that is inherent in testicular tissue.