Development of Advanced Tools for the Monitoring of Cell Cryopreservation Conditions - Applications for Marine Cell Lines

 

Dennis McDaniel*, John T. Elliott*, Kurt Langenbach*, Alexandro Tona*, Paul Becker^, Henry Rodriguez*

*Biotechnology Division National Institute of Standards and Technology Gaithersburg, MD 20899, USA, ^NOAA Hollings Marine Laboratory, Charleston, SC 29412, USA

 

Marine mammals are an important part of both the economy and ecology of coastal environments and a greater understanding of their physiology could have enormous implications for the improvement of human health. Despite their importance, experimentation with living marine mammals is difficult for both practical and political reasons. Thus, it is clear that in vitro experiments using cells derived from marine mammals will play a critical role in understanding marine mammalian physiology as well as the effects of environmental conditions. In addition, development of specific marine cell lines will pave the way for enhancing basic insights into fundamental cellular processes in marine model organisms as well as allowing marine biopharmaceutical production. Today’s cryopreservation protocols, developed in the 1950s and 60s, do not support the functional requirements necessary for modern cell-preservation needs in the pharmaceutical, drug discovery, basic science and toxicological studies. In the current study, a number of assays were performed on a terrestrial mammalian cell line (NIH 3T3) and baseline values were determined for parameters such as cellular morphology as well as rates of apoptosis, necrosis, adherence, metabolism and proliferation under highly reproducible culture conditions. These baseline values will then be compared to assay results obtained post-thaw in order to ensure that the freeze/thaw process has not resulted in the inadvertent selection of a subset of cells that are dissimilar to the original population. Knowledge of the expected results of these assays will help identify tools that aid in determining the optimal storage, preservation and propagation conditions that ensure retention of selected phenotypic properties in a marine mammal cell line. All of the assays employed are applicable to most mammalian cell types and will next be performed on a marine cell line currently being generated by the laboratory of Dr. John Wise and the National Marine Cell Line Library.