plants of wild type St. John's Wort and cryopreserved using a droplet- vitrification method with variations in preculture, vitrification solution treatment, and unloading. The effect of mother plant age (from 2 to 8 weeks) and explant type were also investigated. The developed cryo- preservation protocol was applied to five lines of St. John's Wort from GRIPP’s in vitro collection. The results of this study indicate that: 1) the age of mother plants is one of the most important factors that affect regrowth of root explants after cryopreservation; 2) the duration of explant culture after excision affects post-cryopreservation regrowth, with an optimal duration of 10e-15 days; 3) vitrification solution treatment is critical for regrowth, and highest regrowth of cryopreserved roots (80%) was achieved following treatment with vitrification solution B5 (40% glycerol + 40% sucrose, w/v) for 30 mins; 4) the middle sections of roots had higher regrowth ability than root tips and basal sections; 5) roots with pre- existing buds showed enhanced regrowth rates after cryopreservation; and 6) regrowth of cryopreserved roots was genotype specific and varied from 45 to 80%. This study provides insights into root-based regeneration and the response to cryopreservation in Saint John’s Wort, which could help in developing similar technologies for other medicinal species. Source of funding: This work was supported by the Gosling Research Institute for Plant Preservation (GRIPP, Guelph, Canada). P31 ASSESSMENT OF SMARTTHAW, A NOVEL DRY THAWING SYSTEM FOR CRYOPRESERVED CELL PRODUCTS Kimberly L. Santucci 1,2, 3 , John M. Baust 2, 3 , Kristi K. Snyder 2, 3 , Robert G. Van Buskirk 1, 2, 3 , John G. Baust 1, 2 . 1 Department of Biological Sciences, SUNY Binghamton, Binghamton, NY, USA; 2 Institute for Biomedical Technology, SUNY Binghamton, Binghamton, NY, USA; 3 CPSI Biotech, Owego, NY, USA Thawing cryopreserved cell products in a 37  C water bath is standard practice in most laboratories and clinical settings. Water baths are also a major source of contamination given their access to open air and warm, humid environment. The utilization of a device that provides a clean, safe, and reproducible thaw for a variety of container types would be optimal. We compared the viability of multiple cell types thawed in either a stan- dard water bath or novel dry thawing system (SmartThaw TM ) and found that this system yielded similar or better results upon post-thaw assess- ments in standard cryovials as well as 25 ml cryostore (CS25) bags. Mul- tiple cell types were tested, including Chinese hamster ovary (CHO) cells, umbilical artery smooth muscle (UASMC) cells, and human umbilical vein endothelial (HUVEC) cells. The metabolic activity indicator alamarBlue was used to assess cell viability and repopulation following thawing. Phase microscopy was conducted for morphological and structural assessment for three days post thaw. We observed that post-thaw viability obtained with the SmartThaw TM device set to 40  C for bags and 45  C for vials was very similar to that of the water bath at 37  C. In UASMC cells thawed in cryovials, SmartThaw and water bath post-freeze viability was not significantly different (65.6% vs 67.8% on day 1 post freeze, respectively). Similarly, HUVEC cell viability in vials was 85.4% in SmartThaw TM condi- tions vs 82.7% in water bath thawed samples. HUVEC and CHO cells cry- opreserved in CS25 bags yielded viabilities within 3% between the two thawing methods. These results support the use of the SmartThaw TM system as an alternative to water baths. P32 JC-1: A NEW METHOD TO EVALUATE FRESH AND CRYOPRESERVED RABBIT EMBRYO FUNCTIONALITY M. Teixeira * , L. Commin, L. Gavin-plagne, P. Bruyere, A. Philibert, T. Joly, S. Buff. University of Lyon, Vetagro Sup, Marcy-l'  Etoile, France * Corresponding author. One of the main challenges in embryo biotechnology research is to develop in vitro evaluation methods of embryo quality to detect early alterations correlated with embryo development competence. JC-1 (5,5 0 ,6,6 0 -tetra- chloro-1,1 0 ,3,3 0 -tetraethylbenzimidazolylcarbocyanine iodide) is a lipo- philic cationic dye, which accumulates within mitochondria according to its mitochondrial membrane potential (MMP) emitting different fluores- cent properties. High MMP mitochondria accumulate more cationic dye (J aggregates) and exhibit red fluorescence, while low MMP mitochondria accumulate J monomers, showing green fluorescence. Disruption of MMP has been associated with metabolic stress and early cellular apoptosis. Our aim was to adapt a JC-1 staining method to fresh and cryopreserved rabbit embryos in order to evaluate their quality.To this end, fresh (n ¼ 39) and slow frozen (n ¼ 25; Me 2 SO, 1.5 M) embryos were analyzed according to morphological quality and classified as normal or damaged embryos. Embryos were pretreated with pronase, stained with JC-1 (1.5 mM) for 75 min (38.5  C, 5% CO 2 ) and observed under an epifluorescence micro- scope. CCCP, a MMP disruptor, was used as a control to confirm the JC-1 sensitivity to changes in MMP. The staining intensity (pixel) was deter- mined in two randomly defined areas on each embryo, and the red/green ratio was measured. Significant differences were found between fresh (R ¼ 3.55 ± 0.94) and normal cryopreserved embryos (R ¼ 2.55 ± 0.78; p<0.01), as well as between normal and damaged (R ¼ 1.03 ± 0.50; p<0.05) cryopreserved embryos. We conclude embryo morphological defects are associated with MMP disruption, and cryopreservation seems to impair the mitochondrial metabolism even in the absence of identifiable alter- ations of the embryo. This study is the first to describe a JC-1 staining protocol for rabbit embryo evaluation, showing it can be used as a valuable indicator of embryo health and functionality for this species. Source of funding: National Network of Biological Resources Centers CRB- ANIM (ANR11-INSB-0003) P33 ESTIMATING PRESERVATION POTENTIAL OF A DISACCHARIDE SOLUTION USING RAMAN MICROSCOPY N. Chakraborty, M. Wang, Q. Osgood * . University of Michigan, Dearborn, Michigan, United States * Corresponding author. Disaccharides are known for their ability to protect cells and other bio- molecules during processing for lyo(dry)-preservation. While several theories exist regarding the underlying molecular mechanism, alteration in hydrogen bonding characteristics play an important role in exerting the protective effect of disaccharides. In this study an attempt to understand the effect of hydrogen bonding was undertaken by observing the effect of disaccharides on OH stretching peaks using Raman spectroscopy. The effective change in OH stretching peaks (symmetric and asymmetric) can be correlated to the concentration of disaccharide molecules present in the solution state. The symmetric portion of the OH stretching peak can be associated with the water molecules that incorporate fully developed hydrogen bonding, whereas the asymmetric portion of OH stretching peak can be associated with the water molecules which lack fully developed (unbound) hydrogen bonding. Herein we report that disaccharides influ- ence both the symmetric and asymmetric portions of the OH stretching peaks and quantification of this change in characteristics of the OH stretching peaks can be correlated to the extent of protection offered by the disaccharide. Source of funding: National Science Foundation: Award No. 1510072, University of Michigan’s research seed fund: UM Project No. U038726 P34 MICROFLUIDIC COLD-FINGER DEVICE FOR THE INVESTIGATION OF ANTIFREEZE PROTEINS L. Haleva 1, * , Y. Celik 2 , M. Bar-dolev 1 , A. Kaner 1 , N. Pertaya 2 , P. Davies 3 , I. Braslavsky 1 . 1 The Hebrew University of Jerusalem, Institute of Biochemistry, Food Science and Nutrition, The Robert H. Smith Faculty of Agriculture, Food and Environment, Rehovot, Israel; 2 Ohio University, Department of Physics, Athens, Ohio, United States; 3 Queen’s University, Department of Biomedical and Molecular Sciences, Kingston, Ontario, Canada * Corresponding author. Antifreeze Proteins (AFPs) are a subset of ice-binding proteins that serve to prevent freezing, control recrystallization, and adhere to ice in cold- Abstracts / Cryobiology 73 (2016) 399e443 440