A07 Session 2: Biopreservation of biologics: Translation from stem cells to cancer

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Abstracts / Cryobiology 69 (2014) 184–199

A07 Session 2: Biopreservation of biologics: Translation from stem cells to cancer Cryopreservation of alginate encapsulated monkey stem cells under controlled induced nucleation Birgit Glasmacher, O. Gryshkov, L. Lauterboeck, Denis Pogozhykh, T. Mueller, N. Hoffmann, Institute for Transfusion Medicine, Hannover Medical University, Hannover, Germany, Institute for Problems of Cryobiology and Cryomedicine of the NAS of Ukraine, Kharkiv, Ukraine, Institute for Multiphase Processes, Leibniz University Hannover, Callinstraße 36, D-30167 Hannover, Germany On-shelf availability of rare cell types requires their long-term preservation. The cryopreservation procedures which are commonly used for long-term preservation of living cells involve high concentrations of cryoprotective agents (CPAs), such as Me2SO, which protect cells at low temperatures from cryoinjury. Despite of this, the introduction of such CPA for cryopreservation does have pronounced toxic effects to the cells. To solve such a problem, a decrease in concentration of CPA is required. This may be achieved by encapsulation of living cells into gel-like alginate structures which mimic an extra-cellular matrix also providing 3D arrangement for cells. However, the efforts being made so far were mainly applied to control the cooling and warming temperatures during freezing but only a few publications are known being tried to control the nucleation during cryopreservation. Furthermore, there is no information about controlled nucleation during cryopreservation of encapsulated cells [1]. In the case of cryopreservation of encapsulated cells, the size of such constructs is the main property which restricts the diffusion of CPA as well as heat and mass transport to encapsulated cells [2]. In this work we introduce the electro-spraying encapsulation technology to produce small-sized (less then 200 lm) alginate 3D constructs with entrapped living cells under the high voltages. Alginate solution (1.6% (w/v))) is mixed with mesenchymal stem cells derived from a Common marmoset (cjMSCs) and pumped at 10 ml/h flow rate through the nozzle where the high voltage is applied (>15 kV). When the strength of electric field exceeds the surface tension, the spraying into cross-linking bath (100 mM CaCl2) is observed. After being gelled for 10 min, beads were washed with PBS and immediately cryopreserved using electro-freezing and seeding under controlled nucleation using optimal protocol (5% DMSO (v/v) with cooling rate 7.5 K/min down to 30 °C and 3 K/min to 80 °C). The commonly used freezing protocol (1 K/min, CM 2000, Carburos Metallicos) served as a control. Samples were kept at 80°C for a week followed by rapid warming at 37°C. Membrane integrity of cells was assessed using Trypan Blue assay. Proliferation activity of cells which undergone encapsulation and cryopreservation procedures was measured using MTT proliferation assay (ProNova, USA). High-voltage electro-spraying allows encapsulation of living cells into small alginate 3D constructs without significant influence on viability and proliferation of cells post-encapsulation. The MSCs encapsulated and frozen under controlled nucleation showed normal morphology, attached and proliferated the same as compared to control. The viability and proliferation activity of cells post-cryopreservation may be improved by controlling the nucleation temperature. These results may serve as a background for further development of cryopreservation of encapsulated cells with controlled induced nucleation for long-term storage. We will also describe different methods and devices for induction of ice nucleation. Acknowledgements: This work is supported by funding from the Cluster of Excellence REBIRTH (DFG EXC 62/1).

References [1] Murua A, Orive G, Hernandez RM, et al.. Biomaterials 2009;30:3495–501. [2] Sambu SK, Xu X, Ye H, et al.. J. Biotechnol. 2010;150(Suppl 1):446–7. http://dx.doi.org/10.1016/j.cryobiol.2014.06.016

A08 Session 2: Biopreservation of biologics: Translation from stem cells to cancer Differential activation of stress pathways in human mesenchymal stem cells following biopreservation. William L. Corwin 1,3, John M. Baust 1,3, John G. Baust 2,3, Robert G. Van Buskirk 1,2,3, CPSI Biotech, 2 Court St, Owego, NY 13827, United States, 2 Department of Biological Sciences, Binghamton University, Binghamton, NY 13902, United States, 3 Institute of Biomedical Technology, Binghamton University, Binghamton, NY 13902, United States 1

Research on human mesenchymal stem cells (hMSC) has expanded considerably in recent years as continued investigations have demonstrated their potential for reestablishment of in vivo functions. Further, as hMSC studies have increased our understanding of their utility, this has in turn driven the development of hMSC-based technologies. These technologies range from the exploita-

tion of innate in vivo functions to target and repair damaged tissue, to removal and purification of hMSC for use in numerous other settings such as tissue engineering, cell therapies, etc. Essential to these developments is the ability to process and maintain these unique biologics without the loss of viability and functionality. As such, bioprocessing has become a limiting factor in the downstream use of hMSC as non-uniform samples and variation in function impairs efficacy, outcome and application of these advancements. In an effort to improve hMSC bioprocessing protocols, this study focused on the analysis and modulation of hMSC stress response following biopreservation. Two biopreservation stress regimes (hypothermic storage and hypoxic normothermic storage) were utilized to examine cell stress pathway activation. Specifically the unfolded protein response (UPR) was examined as a mediator of post-storage stress response given its recent link to preservation failure in other cell systems. Additionally, the incorporation of two chemical agents (i.e. salubrinal and resveratrol) during storage was conducted to examine the effect of targeted molecular modulation. The results of these studies demonstrated a differential response of hMSC to targeted modulation dependent upon storage temperature and carrier medium. The addition of resveratrol to MSCGM (complete growth media), HBSS (balanced salt solution) or ViaSpan (UW solution) yielded marked improvement to immediate post-storage viability (increases of 53%, 50%, and 10%, respectively) and regrowth of hMSC following hypothermic storage. Interestingly, salubrinal addition yielded modest improvement to MSCGM and HBSS stored hMSC survival (16% and 8% increases) while its addition to ViaSpan resulted in decreased viability and impaired ability to repopulate. In contrast, use of these modulators during hypoxic normothermic storage in either MSCGM or HBSS yielded negative impacts as growth inhibition or cell loss was observed in the samples. Further in-depth analyses revealed changes to both apoptotic and necrotic populations post-storage through the use of these modulators. Western blot analysis confirmed changes in the apoptotic signaling pathways as well as implicated the involvement of the UPR pathway signaling following storage. These findings illustrate the potential for storage specific cell stress pathway modulation to improve hMSC biopreservation. Further, the differential response of pathway modulation demonstrates that a more in-depth understanding of hMSC response is needed to improve each step throughout the biopreservation process (pre-storage processing, preservation and post-preservation recovery) to enable the delivery of optimal hMSC samples. http://dx.doi.org/10.1016/j.cryobiol.2014.06.017

A09 Session 2: Biopreservation of biologics: Translation from stem cells to cancer Cryobiological biophysics and freeze–thaw viability of the HL-1 cardiac muscle cell line: Implications for cryoablation studies Jeunghwan Choi, Chunlan Jiang, Dushyant Mehra, John Bischof, University of Minnesota, 111 Church St., SE Minneapolis MN 55455, United States Atrial fibrillation (AF) is the irregular beating of the heart affecting an estimated 2.7 million Americans and responsible for 26 billion dollars of the nation’s annual healthcare cost. Cryoablation is a clinically approved technique for the treatment of paroxysmal AF in which an ablation catheter rapidly cools the cardiac tissue. Through freezing, discrete lesions are created in the region where the pulmonary veins join with the atria in order to terminate the conduction of abnormal electrical signals responsible for AF. While the efficacy of AF cryoablation is well known, the thermal thresholds and mechanisms of cryoinjury that determine efficacy are not known thereby hindering development of the next generation of AF cryoablation systems. This study begins to address this by improving understanding of the mechanisms and thermal thresholds of cryodestruction in a cardiac muscle cell line (HL1). Specifically, we determine biophysical parameters governing known mechanisms of injury (membrane permeability and likelihood of intracellular ice formation, IIF). Additionally we correlate these biophysics and thermal history (cooling rate and end = temperature) to post-thaw viability in vitro in HL-1s guide optimal lesion formation and thus cryoablation device design. HL-1 cells were cultured using previously published methods. Cells were harvested using trypsinization and kept as a suspension on ice until needed. A temperature stage was used in conjunction with a light microscope to observe freezing of the samples. The controlled thermal history included 3 variables including: cooling rate (0.5 to 130 °C/min), end temperature ( 5 to 60 °C), and hold time (0–5 min). These were varied in an experimental matrix to determine the importance of each. Before cooling, samples were pre-nucleated by applying a chilled needle on the outer edge of the sample. Change in water content within cells (cellular dehydration) was measured based on observed changes to projected cell area during freezing. IIF events were measured based on observing a sudden change in opacity (darkening) of the cell interior. Measured data were applied to water transport and nucleation models to determine membrane permeability and IIF nucleation parameters. Additionally, post-thaw viability of samples were determined after a rapid thaw and 15 min incubation period using a fluorescence membrane dye exclusion assay. For cases