using the metabolic indicator alamarBlue TM . To determine the level of apoptotic and necrotic involvement, the Vibrant Ò apoptosis assay was performed and visualized via fluorescent microscopy at 1, 4, 8 and 24 hour time points. Analysis of cryopreserved samples revealed that apoptosis is responsible for about 15% of the cell death in the M5 condition, peaking four to eight hours after thawing. Samples preserved in CS2 experienced a marked decrease in apoptotic events with only about 6% of the total pop- ulation involved, again peaking four to eight hours post-thaw. Upon via- bility assessment at 24 hours post-thaw, a significant separation was seen between the M5 sample, with 53% (±4%) survival, as opposed to CS2, which showed 84% (±3%) survival. When the pan caspase inhibitor VI was added (pre-freeze) to the different preservation solutions, no signifi- cant increase in the 24 hour post-thaw survival was observed. However, when the inhibitor was added to the post-thaw media during the 24 hour recovery a significant increase (20%) was seen in the viability of the M5 sample and a less significant increase (5%) with CS2. These results show that the initial 24 hours of recovery is a crucial period for overall cell sur- vival and an area of preservation where molecular advancements could help overcome cryopreservation failure. Our data supports an integrated approach, which combines post-freeze conditioning of cells, to achieve optimal cell survival and efficacy of cryopreservation. (Conflicts of inter- est: None declared. Source of funding: None declared). doi:10.1016/j.cryobiol.2007.10.138 136. An improved cryopreservation method for the R1 embryonic stem cell line. Corinna M. Kashuba Benson, James D. Benson, John K. Critser, University of Missouri-Columbia, Columbia, MO, USA The percent post-thaw recovery of viable mouse embryonic stem (ES) cells varies greatly among genetic backgrounds and between individual cell lines within a genetic background. There is a need to improve the efficiency and reduce the variability of current mouse ES cell cryopreservation meth- ods. To address this need, we explored the fundamental cryobiological parameters of the R1 ES cell line. The R1 ES cell line is remarkable for its highly efficient germ line transmissibility, a vitally important quality for the generation of mutant mouse strains that serve as human disease models in biomedical research. Using fundamental cryobiological princi- ples, we characterized membrane permeability characteristics of the R1 cell line in order to predict optimal cooling rates, plunge temperature, and type of cryoprotectant. The resulting protocol was verified experimen- tally and resulted in a 1.8 fold increase in percent post-thaw recovery of viable ES cells as compared to the standard freezing protocol. (Conflicts of interest: None declared. Source of funding: None declared). doi:10.1016/j.cryobiol.2007.10.139 137. Hepatocyte preservation as liver slices. Lia H. Campbell, Simona C. Baicu, Elizabeth D. Greene, Zhen Chen, Brian Leman, Michael J. Taylor, Kelvin G. M. Brockbank, Cell & Tissue Systems, Inc., North Charleston, SC, USA Each year there is an increased need for high-throughput drug testing platforms for the rapid development of pharmaceutical drugs Along with this, is the continued push to reduce the number of animals used in research. Thus, the development of biologically relevant cell and tissue- based assay systems has become more important. Hepatocytes are partic- ularly relevant because of their important role in drug metabolism; how- ever, their specific functions are lost in culture relatively quickly. An alternative strategy is the use of precision-cut liver slices (PCLS). Liver slices have been recognized as the only high-throughput in vitro tool, which strongly imitates in vivo conditions for xenobiotic metabolism and biotransformation activities in drug screening. In vitro studies on liver slices can accelerate the drug development process by using human liver tissues obtained from viable and non-transplantable donor livers. In spite of major advances in tissue preservation and engineering, utilization of PCLS is mostly limited by imperfect preservation techniques and short incubation periods. Tissue banking of PCLS by successful cryopreserva- tion and improved metabolic functions for extended incubation periods can provide an unlimited supply of liver slices for drug discoveries. Cryo- preservation involving freezing in the presence of dimethyl sulfoxide (Me 2 SO) is a preferred method for storing cells and tissue. There is evi- dence that ice formation within the extracellular matrix of multicellular tissues is the principal event that limits the survival of cryopreserved tis- sues using conventional freezing techniques. This mode of injury can be circumvented by using a new cryopreservation approach, known as vitri- fication or the amorphous solidification of a supercooled liquid. The goal of this study was to develop a cryopreservation strategy using vitrification that produced better viability and function in preserved rat liver slices over conventionally cryopreserved slices using Me 2 SO. Seven cryopreservation solutions were assayed and included 6 vitrification formulations and one cryopreservation formulation consisting of 18% Me 2 SO. Several parame- ters were measured for comparison and included cytochrome p450 activ- ity, albumin secretion and ATP levels. Measurement of ATP levels after rewarming demonstrated that ATP levels after preservation with 2 vitrifi- cation solutions, VS55 (8.4m combination of Me 2 SO, formamide, 1,2-pro- panediol) and DP6-1,4CHD (6.0M combination of Me 2 SO, 1,2- propanediol, 1,4-cyclohexanediol) were significantly better than with 18% Me 2 SO (p < 0.05). Based on these assays, metabolic and functional differences between vitrified and frozen liver slices were observed suggest- ing that vitrification may provide a better preservation strategy for liver slices. (Conflicts of interest: None declared. Source of funding: None declared). doi:10.1016/j.cryobiol.2007.10.140 Vitrification 138. Ultrastructural observation of plasma membrane of plant shoot apices in vitrification-based protocols. D. Tanaka a , T. Niino a , M. Uemura b , S. Fujikawa c , a National Institute Agrobiological Sciences, 2-1-2 Kan- nondai, Tsukuba, 305-8602, Japan; b Cryobiosystem Research Center, Iwate University, Morioka 020-8550, Japan; c United Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589, Japan Although there are several long-term preservation methods for plant germplasm, cryopreservation of shoot tips has been recognized as the most reliable, cost- and space-efficient method. However, it is not yet widely employed as a reliable long-term preservation protocol due to the lack of basic knowledge on the cellular and water behavior in tissues when immersed in liquid nitrogen. In this investigation, survival and ultrastruc- tural characteristics of cells following rapid cooling are presented. doi:10.1016/j.cryobiol.2007.10.141 139. Cryopreservation of hairy roots of medicinal plants by vitrification and droplet-vitrification procedure. Haeng-Hoon Kim, Hae-Sung Hwang, Yoon-Geol Lee, Taesan Kim, National Institute of Agricultural Bio- technology, Suwon, Republic of Korea Though hairy roots are important sources of secondary metabolites with commercial value, there are only a limited number of studies report- ing on the cryopreservation of hairy root cultures, which is produced by infection with Agrobacterium rhizogens. Previous studies mainly used an encapsulation-dehydration protocol, due to difficulties in handling the free fine roots in viscous vitrification solutions. However, vitrification, with or without encapsulation, has been considered to be a more successful proto- col than encapsulation-dehydration. Moreover, the droplet-vitrification protocol, a combination of droplet-freezing and solution-based vitrifica- tion, produced higher recovery than droplet freezing or vitrification, since ultra-rapid cooling and warming are facilitated in this protocol. We inves- tigate the effect of the source material, preculture, loading, dehydration, 370 Abstracts / Cryobiology 55 (2007) 324–378