somewhat inconclusive; and the current study argued that analysing the DNA dam-age response (DDR) from the cryopreserved cells would help characterise the nat-ure of the putative DNA damage resulting from cryopreservation. To that end, thecurrent study set out to investigate the effects of different cryopreservation param-eters on the genome in the context of the event of double strand breaks (DSBs), sin-gle strand breaks (SSBs), and various forms of sequence alteration using 51/4 h postfertilisation (hpf) zebrafish (Danio rerio) embryos as the model organism. The cryo-preservation parameters under which the investigation was carried out were chill-ing of the embryos at 0 �C for up to 120 min, treatment of the embryos with twocryoprotective additives (CPA), MeOH and Me2SO, for 30 min, and cooling of theembryos to �35 �C and subsequent warming. Assays for DSB-activated DDR pro-teins and SSB-activated DDR proteins in 51/4 hpf zebrafish (Danio rerio) weredeveloped under the current study. The assays were then utilised to investigatethe event of DSBs and SSBs in the genome of the embryos treated with differentcryopreservation parameters. The study also analysed the expression profiles of aset of genes unique to the base excision repair (BER), nucleotide excision repair(NER) and mismatch repair (MMR) as indicators of the occurrence of various formsof sequence alteration in the genome of the embryos treated with the cryopreser-vation parameters. The study found that chilling and CPA treatment did not induceDSBs or SSBs but up-regulated the MMR and BER mechanisms, respectively. CPAtreatment also down-regulated NER and MMR mechanisms. Cooling and warming,on the contrary, did not induce DSBs but induced SSBs in the genome, which wererepaired when the embryos were provided with a recovery time. Cooling andwarming also up-regulated the NER and BER mechanisms in the embryos. The find-ings of the study provided important insights into how eukaryotic cells respond todifferent cryopreservation parameters, which will significantly enhance the knowl-edge regarding the effects of cryopreservation on the genome of biological objects.

Source of funding: Overseas Research Students Awards Scheme (ORSAS) and Uni-versity of Bedfordshire

Conflict of interest: None declared.Email address: raju.ahmed@beds.ac.uk

http://dx.doi.org/10.1016/j.cryobiol.2013.09.131

126Establishment of a live young mouse production system using unfertilizedoocytes cryopreserved via a simple vitrification method. Naomi Nakagata, ToruTakeo, Kiyoko Fukumoto, Tomoko Kondo, Yumi Takeshita, Yuko Nakamuta, ShujiTsuchiyama, Eri Kohagura, Mari Iwamoto, Fumi Takahashi, Yuta Ishizuka, Division ofReproductive Engineering, Center for Animal Resources and Development (CARD),Kumamoto University, Kumamoto, Japan

Since the first successful reports into oocyte freezing, many papers concerningthe cryopreservation of mouse oocytes have been published. However, a simple andpractical cryopreservation method for unfertilized C57BL/6 mouse oocytes, and anIVF system using these cryopreserved oocytes have yet to be established, in spite ofthe fact that C57BL/6 is the prevalent inbred strain and is used for large-scaleknockout programs. In this study, unfertilized C57BL/6 mouse oocytes were cryo-preserved via a simple vitrification method. After warming, IVF was performedusing cryopreserved unfertilized oocytes and fresh sperm, cryopreserved unfertil-ized oocytes and cold-stored sperm, cryopreserved unfertilized oocytes and frozensperm (C57BL/6 strain sperm), and cryopreserved unfertilized oocytes and frozensperm derived from GEM strains (C57BL/6 background GEM strains). Nearly all ofthe cryopreserved oocytes were recovered, of which over 90% were morphologicallynormal. Those oocytes were then used for in vitro fertilization, resulting in 72–97 %of oocytes developing into 2-cell embryos. A portion of the 2-cell embryos weretransferred to recipients, resulting in live young being produced from 32% to 49 %of the embryos.

Source of funding: This study was supported by a Grant-in-aid for National Bio-Resource Project (NBRP) Fundamental Technologies Upgrading Program. No addi-tional external funding was received for this study. The funders had no role in thestudy design, data collection and analysis, decision to publish, or preparation of themanuscript.

Conflict of interest: None declared.Email address: nakagata@gpo.kumamoto-u.ac.jp

http://dx.doi.org/10.1016/j.cryobiol.2013.09.132

127Measuring of Sf21 cell membrane biophysical parameters using a new nondilutesolute transport equation. Jian Ren, Jianye Wang, Gang Zhao, School of InformationScience and Technology, University of Science and Technology of China, Hefei, PRChina

In cryobiology, Kedem-Katchalsky model (KK) and two-parameter model (2P)has been used to predict cell volume changes. However, the KK model might leadto conceptual errors when used for cell membranes and the 2P model is based onideal and dilute solution. Recently, Heidi Y. Elmoazzen et al. (Heidi Y. Elmoazzen,Janet A. W. Elliott, and Locksley E. McGann, ‘‘Osmotic Transport across Cell Mem-branes in Nondilute Solutions: A New Nondilute Solute Transport Equation,” Bio-physical Journal, vol. 96, pp. 2559–571, 2009) had proposed a new nondilutesolute transport equation which could fit or predict cell osmotic volume changesmore appropriate. The purpose of the article is to verify the validity of the newequation. We used SF21 cell volume changes in different concentrations of Glyceroland different temperatures to fit the new model to water permeability coefficient(L) and solute permeability coefficient (P) of cell membrane. The experiment wascarried out by using microperfusion system designed by our lab. What’s more,the extracellular concentration of CPA was simulated by using a universal self-adaptive time-varying function, which is derived by Gang Zhao (Gang Zhao, ‘‘A Uni-versal Self-Adaptive Time-Varying Function for Extracellular Concentration duringOsmotic Shift for Curve-Fitting Permeability Coefficients of Cell Membrane,” Cryo-letters, vol. 31, pp. 279–90, 2010). Mean (means ± SD) L values in the presence of2.0 M Glycerol at 25, 20, 15, and 10 �C were determined to be 0.68 ± 0.29,0.78 ± 0.33, 0.77 ± 0.20, and 0.67 ± 0.16 10-28mol2/ (minatmlm5). The P valueswere 4.6 ± 0.1.69, 1.11 ± 0.99, 3.84 ± 0.65, and 2.96 ± 2.81 10–32 ml2/ (min-atmlm5). L values in the presence of 1.5 M Glycerol at 25, 20, 15, and 10 �C weredetermined to be 0.97 ± 0.61, 1.27 ± 0.64, 0.818 ± 0.29, and 0.533 ± 0.17 10–28mol2/ (minatmlm5). The P values were 4.18 ± 2.51, 3.00 ± 1.62, 1.14 ± 0.58, and2.3 ± 1.53 10–32 mol2/ (minatmlm5). L values in the presence of 1.0 M Glycerolat 25, 20, 15, and 10 �C were determined to be 1.78 ± 0.65, 1.24 ± 0.66,1.02 ± 0.26, and 1.15 ± 0.39 10–28 mol2/ (minatmlm5). The P values were2.67 ± 0.94, 2.70 ± 1.53, 1.45 ± 0.96, and 0.79 ± 0.40 10–32 mol2/ (minatmlm5).From fitting lines of the experiment data, we could find out the new nondiluteequation is very similar to 2P model, which demonstrates, in our opinion, thenew nondilute equation can be used to fit and predict cell volume changes. There-fore, more experiments should be done to prove which one might lead to a betterresult.

Source of funding: This work was supported by the National Natural ScienceFoundation of China (Nos. 51076149, 51276179)

Conflict of interest: None declared.Email address: zhaog@ustc.edu.cn

http://dx.doi.org/10.1016/j.cryobiol.2013.09.133

128Development of a low cost sensor system for determination of glass transitiontemperatures during cooling and warming. J. Jahangir 1, R. Andres 2, J. Morris 3, B.Haggett 1, D.M. Rawson 1, 1 iBEST Institute, University of Bedfordshire, Luton, UK,2 Smart Holograms Ltd, Cambridge, UK, 3 Asymptote Ltd, Cambridge, UK

Ice nucleation and glass transition (Tg) temperatures influence cell viabilityduring cryopreservation. Knowledge of these phase changes for cryoprotectantmixtures is an essential step in optimising cryopreservation protocols for cell sur-vival. Differential scanning calorimetry (DSC) is the conventional device used todetermine Tg, but the expensive nature of such instrumentation limits its wide-spread use. A cost-effective sensor system has been designed to monitor ice nucle-ation and Tg events in cryoprotectant solutions. Glycerol was used a test solutionand Tg values were measured (with and without NaCl supplement) in cryotubesand cryostraws, using RTD temperature and screen-printed impedance sensors.The effect of changes to ice-nucleation temperature on Tg was also investigatedusing ice nucleators. Cooling rates of (a) 5 �C min�1, (b) 0.1 �C min�1 and (c) directplunge into liquid nitrogen were set using a Grant Asymptote (EF600) controlledrate freezer. Protocols (a) and (b) were run from 0 �C to �90 �C followed by rapidplunge into liquid nitrogen. The warming event of the three protocols was moni-tored during slow warming of the samples in air at room temperature. The Tg val-ues during warming events compared favourably with the values obtained by DSCreported in the literature. The reported DSC derived Tg values (Morris et al., 2006)for 10% glycerol +0.15 M NaCl (�55 �C) are close to the values obtained in the cur-rent work (�52 to �57 �C). The availability of low cost sensor systems to monitorchanges in impedance allows the determination of Tg of cryopreservation mediaduring both cooling and warming cycles, and should enable such measurementsto become routine in laboratories.

Source of funding: University of Bedfordshire Research Development funds.Conflict of interest: None declared.Email address: jahanbeen.jahangir@beds.ac.uk

http://dx.doi.org/10.1016/j.cryobiol.2013.09.134

434 Abstracts / Cryobiology 67 (2013) 398–442