Cryopreservation by slow cooling of rat neuronal cells |
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| Institution: | 1. Centro Binacional (Argentina-Italia) de Investigaciones en Criobiología Clínica y Aplicada (CAIC), UNR, Avda. Arijon 28 bis, Rosario, 2000, Argentina;2. Servicio de Electrónica y Óptica, FCByF, UNR, Suipacha 531, 2000, Rosario, Argentina;3. Fondazione Italiana Fegato (Italian Liver Foundation), AREA Science Park, BldgQ, SS14, Km 163.5, Basovizza, 34139, Trieste, Italy;4. Liver Center, Department of Medical, Surgical, and Health Sciences, University of Trieste, Trieste, Italy;5. Area Estadística y Procesamiento de Datos, FCByF, UNR, Suipacha 531, 2000, Rosario, Argentina;6. Concejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Argentina;1. Floating Hospital for Children at Tufts Medical Center, 800 Washington Street, Boston, MA 02111, USA;2. Mathematical, Computational and Modeling Sciences Center, Arizona State University, Tempe, AZ 85287, USA;1. Graduate School of Mechanical Engineering, Kanagawa Institute of Technology, 1030 Shimo-ogino, Atsugi, Kanagawa 243-0292, Japan;2. Cryobiofrontier Research Center, Faculty of Agriculture, Iwate University, Ueda 3-18-8, Morioka, Iwate 020-8550, Japan;3. Department of Mechanical Engineering, Oita National College of Technology, 1666 Maki, Oita, 870-0152, Japan;1. USDA, ARS, NLGRP, National Animal Germplasm Program, 1111 S. Mason St., Fort Collins, CO 80521-4500, USA;2. University of Brasilia, Brasilia, 70910-900, Brazil;3. Embrapa Genetic Resources and Biotechnology, Parque Estação Biológica, PqEB s/n°., Brasília, DF, Brasil, CEP 70770-901, Brazil;4. Colorado Parks and Wildlife, Fish Research Hatchery, 5500 WCR 50C, Bellvue, CO 80512, USA;5. Colorado Parks and Wildlife, 317 West Prospect Street, Fort Collins, CO 80526, USA;1. Institute of Biotechnology, National Tsing-Hua University, Hsinchu, Taiwan, ROC;2. Department of Urology, National Taiwan University Hospital and National Taiwan University College of Medicine, Taipei, Taiwan, ROC;1. Department of Pediatrics, Université de Sherbrooke, J1H 5N4, QC, Canada;2. Department of Mechanical Engineering, Université de Sherbrooke, J1K 2R1, QC, Canada;3. Inserm, U955, Equipe 3, Créteil, 94000, France;4. Université Paris Est, Ecole Nationale Vétérinaire d’Alfort, Maisons-Alfort, 94700, France;5. Hôpital d’enfant Armand Trousseau, Groupe Hospitalier Universitaire Est Parisien, Université Pierre et Marie Curie, Paris 6, Paris, France |
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| Abstract: | Although primary neuronal cells are routinely used for neuroscience research, with potential clinical applications such as neuronal transplantation and tissue engineering, a gold standard protocol for preservation has not been yet developed. In the present work, a slow cooling methodology without ice seeding was studied and optimized for cryopreservation of rat cerebellar granular cells. Parameters such as cooling rate, plunge temperature and cryoprotective agent concentration were assessed using a custom built device based on Pye's freezer idea. Cryopreservation outcome was evaluated by post thawing cell viability/viable cell yield and in culture viability over a period of 14 days. The best outcome was achieved when 10% of Me2SO as cryoprotective agent, a cooling rate of 3.1 ± 0.2 °C/min and a plunge temperature of −48.2 ± 1.5 °C were applied. The granular cells cryopreserved under these conditions exhibited a cell viability of 82.7 ± 2.7% and a viable cell yield of 28.6 ± 2.2%. Moreover, cell viability in culture remained above 50%, very similar to not cryopreserved cells (control). Our results also suggest that post-thaw viability (based on membrane integrity assays) not necessarily reflects the quality of the cryopreservation procedure and proper functionality tests must be carried out in order to optimize both post thaw viability/cell yield and in culture performance. |
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| Keywords: | Neuronal cells Slow cooling Cryopreservation |
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