The present proposal relates to a method of inducing ice formation (e.g. ice nucleation or """"""""seeding"""""""") in aqueous solutions surrounding cells and tissues during low temperature preservation (cryopreservation). Typically, seeding is performed by touching the sample containers with a precooled instrument. This process exposes the samples to potential damage due to warming (if the samples are removed from the cooling device to perform seeding) or extensive cooling and intracellular ice formation if the induction of extracellular ice cools the sample too quickly to very tow temperatures. This shortcoming is addressed by providing a general method and specific devices that chemically induce ice formation without interruption of the cooling process or application of external cooling. This project utilizes the ice nucleating properties of sterol compounds, specifically cholesterol, bound to solid state matrices to automatically induce ice. The overall goal of this research relates to refining these novel methods and devices currently under development, and test the effectiveness of them to increase post-thaw recovery and viability, especially as it relates to retention of normal structure and function, of several specific cell types. To achieve this goal, the following specific aims will be performed:
Specific Aim 1. Test the hypothesis that the solid-state nucleating system will result in a reduction of supercooling and therefore an increased recovery of frozen-thawed human semen using standard, commercial sperm banking methods.
Specific Aim 2. Test the hypothesis that the solid-state nucleating system will result in a reduction of supercooling and therefore an increased recovery of frozen thawed umbilical cord blood hematopoietic stem cells.
Specific Aim 3. Test the hypothesis that the solid-state nucleating system will result in a reduction of supercooling and therefore an increased recovery of frozen thawed bull semen using standard commercial agriculture methods of cryopreservation.
