title: Cryopreservation by slow cooling of rat neuronal cells.
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.
authors.affiliation: Centro Binacional (Argentina-Italia) de Investigaciones en Criobiología Clínica y Aplicada (CAIC), UNR, Avda. Arijon 28 bis, Rosario, 2000, Argentina; Fondazione Italiana Fegato (Italian Liver Foundation), AREA Science Park, BldgQ, SS14, Km 163.5, Basovizza, 34139, Trieste, Italy; Concejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Argentina. Electronic address: celeste_robert@hotmail.com.
authors.forename: M Celeste
authors.initials: MC
authors.lastname: Robert
authors.affiliation: Centro Binacional (Argentina-Italia) de Investigaciones en Criobiología Clínica y Aplicada (CAIC), UNR, Avda. Arijon 28 bis, Rosario, 2000, Argentina; Servicio de Electrónica y Óptica, FCByF, UNR, Suipacha 531, 2000, Rosario, Argentina. Electronic address: ljuandep@fbioyf.unr.edu.ar.
authors.forename: Leonardo
authors.initials: L
authors.lastname: Juan de Paz
authors.affiliation: Servicio de Electrónica y Óptica, FCByF, UNR, Suipacha 531, 2000, Rosario, Argentina. Electronic address: agraf@fbioyf.unr.edu.ar.
authors.forename: Daniel A
authors.initials: DA
authors.lastname: Graf
authors.affiliation: Fondazione Italiana Fegato (Italian Liver Foundation), AREA Science Park, BldgQ, SS14, Km 163.5, Basovizza, 34139, Trieste, Italy. Electronic address: silvia.gazzin@csf.units.it.
authors.forename: Silvia
authors.initials: S
authors.lastname: Gazzin
authors.affiliation: Fondazione Italiana Fegato (Italian Liver Foundation), AREA Science Park, BldgQ, SS14, Km 163.5, Basovizza, 34139, Trieste, Italy; Liver Center, Department of Medical, Surgical, and Health Sciences, University of Trieste, Trieste, Italy. Electronic address: ctliver@csf.units.it.
authors.forename: Claudio
authors.initials: C
authors.lastname: Tiribelli
authors.affiliation: Area Estadística y Procesamiento de Datos, FCByF, UNR, Suipacha 531, 2000, Rosario, Argentina. Electronic address: hbottai@fbioyf.unr.edu.ar.
authors.forename: Hebe
authors.initials: H
authors.lastname: Bottai
authors.affiliation: Centro Binacional (Argentina-Italia) de Investigaciones en Criobiología Clínica y Aplicada (CAIC), UNR, Avda. Arijon 28 bis, Rosario, 2000, Argentina; Concejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Argentina. Electronic address: jrodrig@fbioyf.unr.edu.ar.
authors.forename: Joaquín V
authors.initials: JV
authors.lastname: Rodriguez
mesh_terms: D000818:Animals; D002470:Cell Survival / Q000187:drug effects; D002531:Cerebellum / Q000166:cytology; D015925:Cryopreservation / Q000379:methods*; D003451:Cryoprotective Agents / Q000494:pharmacology; D005260:Female; D008297:Male; D009474:Neurons; D017207:Rats, Sprague-Dawley; D013696:Temperature
chemical_list: D003451:Cryoprotective Agents
keywords: Cryopreservation; Neuronal cells; Slow cooling