S092 EFFECTS OF CELL DENSITY ON VIABILITY POST CRYOPRESERVATION K. Mahbubani 1, * , N. Dolezalova 2 , P. Kilbride 3 , N. Georgakopoulos 2 , J. Morris 3 , N. Slater 1 , K. Saeb-Parsy 2 . 1 University of Cambridge, Department of Chemical Engineering and Biotechnology, United Kingdom; 2 Department of Surgery, Cambridge, United Kingdom; 3 Asymptote Ltd., Cambridge, United Kingdom * Corresponding author. Many factors are known to affect the viability of cryopreserved cells, ranging from growth conditions, physiological state of the cells, choice of cryoprotectant, handling techniques and cell density. While all of these are important factors and they each impact upon the post-thaw outcome; the effect of cell density is relatively understudied. Typically as seen both in literature and practice, the final concentration of cells for cryopreservation lies between 106 and 107cells/mL. There is no clear explanation as to why this is the desired cell density. Additionally, there is no clear understanding of the post-thaw outcomes that would be seen if there is a variation in cell density used. The cryopreservation of a range of cell types was investi- gated: an adherent osteosarcoma cell line (SAOS-2), a suspension cell line (CHO), and a mononuclear mixed culture of splenocytes (haematopoietic stem cells, T cells, B cells), harvested from the spleen of deceased human organ donors. Cells were frozen at densities between 104 up to 109 cells/ mL in dimethyl sulphoxide (with and without fetal calf serum) and at a consistent cooling rate of -1  C per minute from ambient down to -80  C. All frozen samples were thawed at 37  C and post-thaw effects were evaluated through a combination of enumeration and viability tests. Flow cytometry was used to enumerate cell numbers and evaluate viability using TruCount beads and a live/dead cell stain (Syto13/eFluor 780). Additionally, cell metabolic activity is characterised using the reduction of an MTS tetrazolium compound along with cell doubling times over a 72- hour window. Preliminary results from the splenocytes, SAOS-2, and CHO cells suggest that higher than typical concentrations of cells (>107 cells/ mL) give improved post-thaw viabilities and metabolic activity. Further work is ongoing to determine the significance of the improvement and to determine a suitable maximum concentration of cells for cryopreservation. Source of funding: Funding for this work was provided through a Medical Research Council (UK) Proximity to Discovery Fellowship grant. S093 USING MICROFLUIDICS TO STUDY BIOFILM FORMATION AND DISRUPTION WITH AN ICE-BINDING BACTERIUM C. Stevens 1, * , S. Yazdi 2 , S. Guo 1 , M. Bar-dolev 3 , C. Escobedo 2 , I. Braslavsky 3 , P. Davies 1 . 1 Queen's University, Department of Biomedical and Molecular Sciences, Kingston, Ontario, Canada; 2 Queen's University, Department of Chemical Engineering, Kingston, Ontario, Canada; 3 The Hebrew University of Jerusalem, Institute of Biochemistry, Food Science and Nutrition, The Robert H. Smith Faculty of Agriculture, Food and Environment, Rehovot, Israel * Corresponding author. Ice-binding proteins (IBPs) are produced by many overwintering organ- isms as an adaptation to surviving sub-zero temperatures. IBPs adsorb to ice surfaces to serve biological roles, including freezing-point depression and ice-recrystallization inhibition, thereby protecting their host from the damages of freezing. Typically, IBPs are small, single-domain proteins secreted into the medium or body fluids. In contrast, the IBP produced by the Antarctic bacterium Marinomonas primoryensis, is an extremely large 1.5-MDa multi-domain protein. The ice-binding region is located near the C terminus and accounts for ~2% of the protein mass. Bioinformatic com- parisons indicate this IBP is an adhesion, exploited by the bacterium to remain in the top of the water column for access to oxygen and nutrients. Using a cooling stage with temperature control it is possible to see the M. primoryensis form micro-colonies on the ice surface. Using an antibody raised against the ice-binding region, we can abolish bacterial ice adhe- sion. We are also using microfluidic devices for studying ice-binding and biofilm formation activity of M. primoryensis. One simple, robust microfluidic apparatus consists of a round middle channel with two curved adjacent channels that allows formation of a two-phase interface. The middle and adjacent channels are interconnected through 100-mm-gaps between trapezoidal posts. Ice is formed in the middle chamber, then the adjacent channel can be filled with bacterial culture. Using these ap- proaches, we show ice binding is specific to M. primoryensis and depen- dent on its motility and the ice-binding region. The system also allows for perfusion and replacement of the solution surrounding the bacteria to investigate disruption of the biofilm. Currently, M. primoryensis is the only known bacterium with the ability to adhere to ice. Using this approach, we can study biofilms at ice-liquid interfaces in real-time with important biomedical applications towards the development of new treatments that prevent bacterial infections. S094 CRYOPRESERVATION OF MICROENCAPSULATED CELLS WITH LOW MOLECULAR WEIGHT HYALURONAN H. Gurruchaga 1, * , J. Ciriza 2 , L. Saenz Del Burgo 1 , G. Orive 1 , R.M. Hernandez 1 , J.L. Pedraz 1 . 1 NanoBioCel Group, Laboratory of Pharmacy and Pharmaceutical Technology, Faculty of Pharmacy, University of Basque Country, UPV/EHU, Vitoria-Gasteiz, Spain; 2 Biomedical Research Networking Centre on Bioengineering, Biomaterials and Nanomedicine, CIBER-BBN, Vitoria-Gasteiz, Spain * Corresponding author. Cell encapsulation allows the immobilization of a desired cell type and its protection from the immune system. Moreover, it permits the exchange of nutrients between the environment and the core of the microcapsule containing the cells while allowing the release of therapeutic molecules by the entrapped cells. This technology is being studied as a drug and cell delivery system for the treatment of different diseases such as diabetes, anemia, cancer, and hepatic diseases. Cryopreservation would help to move this technology to the clinic since it would reduce cost and facilitate the “on demand” access of patients to the treatment in a clinic far from the manufacturer. However, optimized and standardized protocols for cryo- preservation of microencapsulated cells are necessary and must be developed. Dimethyl sulfoxide is the most used cryoprotectant for the cryopreservation of microencapsulated cells by slow freezing protocol. However, as with almost all cryoprotectants, it shows cytotoxic effects. Alternatively, hyaluronan, a natural product present in diverse regions of the body, has been used in the cryopreservation of some cell lines, such as fibroblast. In this study we investigated the cryoprotective effect of low molecular weight hyaluronan in the cryopreservation of micro- encapsulated cells compared to 10% dimethyl sulfoxide cryoprotectant solution. S095 EFFECT OF LIPOSOME-TREATED HUMAN RBCS ON ENDOTHELIAL CELL ADHESION MOLECULE EXPRESSION L. da Silveira Cavalcante * , J.L. Holovati, J.P. Acker. University of Alberta and Canadian Blood Services, Edmonton, Alberta, Canada * Corresponding author. Liposomes have been shown to minimize RBC membrane damage occur- ring during 42-day hypothermic storage (1e6  C). This study aimed to assess the impact of these membrane changes on the expression of adhesion molecules by endothelial cells. Unilamellar liposomes were synthesized to contain unsaturated phospholipids (DOPC:cholesterol, 7:3 mol%). RBCs were incubated with HEPES-NaCl solution (control) or 2 mM DOPC liposomes, and assessed at day 2 and 42 of hypothermic storage. Adhesion molecules (VCAM-1 and E-selectin) expression on pre-activated (LPS-stimulated) and non-activated HUVECs was measured by flow cytometry after a 24 hour incubation with control and liposome-treated RBCs. Hemolysis was measured using Drabkin’s method. Expression of VCAM-1 at day 2 was comparable in control and liposome-treated RBCs on pre-activated HUVECs, while E-selectin expression was increased in lipo- some-treated RBCs (13.4 ± 0.9% vs. 9.0 ± 1.7%, p ¼ 0.005). On non-activated Abstracts / Cryobiology 73 (2016) 399e443 424