Cryopreservation is a technology with wide ranging applications including helping to prevent specis extinction (Hopkins and Herr, 2010). However, the current applicability of cryopreservation protocols is limited. This limitation warranted experimentation that could contribute to the optimization of current protocols for cryopreservation. The experiments presented herein were designed to contribute to the plunge-cooling method of cryopreservation. The importance for further development of current plunge-cooling methods is for the cryopreservation of cell-types sensitive to the other method of cryopreservation, slow-equilibrium freezing. The purpose for plunge-cooling is to cool a sample from ambient temperature to liquid nitrogen temperature at a fast rate. If cooling is sufficiently fast, ice-formation is inhibited. This creates a glass-like vitrified state. To optimize the probability of vitrification, current approaches were modified. Different cooling methods were investigated. Containers called cryostraws were crafted from six different materials, materials with different thermal conductivity properties; all straws were of similar size. The minimum concentrations of several ice-inhibitors (cryoprotectants) needed to achieve vitrification were studied. Novel cooling methods, which involved the use of spinning coolant and/or semi-solid nitrogen, called slush nitrogen (SN), as the cooling medium were invented and then tested. The novel methods enabled rates of cooling that were far faster than previously possible. A chronic problem that plagued the progress with plunge cooling was the difficulty in making SN repeatable. The cause of this problem was researched and identified as contamination of the LN by condensed oxygen. The quantified cooling rates of similar sized containers constructed from different materials yielded unexpected results. We determined that a material\u27s thermal conductivity was a poor predictor of cooling rates. In fact, implementing a material that was porous to LN, such as polyvinyl chloride, yielded the fastest rate of cooling, indicating that a material\u27s porosity is a much better predictor of cooling rate. --Document
