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Gambaro S.E.,Italian Liver Foundation Fondazione Italiana Fegato | Gambaro S.E.,Institute Trasplante Multiorganico | Robert M.C.,Italian Liver Foundation Fondazione Italiana Fegato | Robert M.C.,Centro Binacional Argentina Italia Of Investigaciones En Criobiologia Clinica Y Aplicada Caic | And 3 more authors.
Archives of Toxicology | Year: 2016

In the Crigler–Najjar type I syndrome, the genetic absence of efficient hepatic glucuronidation of unconjugated bilirubin (UCB) by the uridine 5′-diphospho-glucuronosyltransferase1A1 (UGT1A1) enzyme produces the rise of UCB level in blood. Its entry to central nervous system could generate toxicity and neurological damage, and even death. In the past years, a compensatory mechanism to liver glucuronidation has been indicated in the hepatic cytochromes P450 enzymes (Cyps) which are able to oxidize bilirubin. Cyps are expressed also in the central nervous system, the target of bilirubin toxicity, thus making them theoretically important to confer a protective activity toward bilirubin accumulation and neurotoxicity. We therefore investigated the functional induction (mRNA, EROD/MROD) and the ability to oxidize bilirubin of Cyp1A1, 1A2, and 2A3 in primary astrocytes cultures obtained from two rat brain region (cortex: Cx and cerebellum: Cll). We observed that Cyp1A1 was the Cyp isoform more easily induced by beta-naphtoflavone (βNF) in both Cx and Cll astrocytes, but oxidized bilirubin only after uncoupling by 3, 4,3′,4′-tetrachlorobiphenyl (TCB). On the contrary, Cyp1A2 was the most active Cyp in bilirubin clearance without uncoupling, but its induction was confined only in Cx cells. Brain Cyp2A3 was not inducible. In conclusion, the exposure of astrocytes to βNF plus TCB significantly enhanced Cyp1A1 mediating bilirubin clearance, improving cell viability in both regions. These results may be a relevant groundwork for the manipulation of brain Cyps as a therapeutic approach in reducing bilirubin-induced neurological damage. © 2014, Springer-Verlag Berlin Heidelberg.

Robert M.C.,Centro Binacional Argentina Italia Of Investigaciones En Criobiologia Clinica Y Aplicada Caic | Robert M.C.,CONICET | Juan de Paz L.,Centro Binacional Argentina Italia Of Investigaciones En Criobiologia Clinica Y Aplicada Caic | Graf D.A.,Servicio de Electronica y Optica | And 6 more authors.
Cryobiology | Year: 2016

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. © 2016 Elsevier Inc.

Bessone V.,National University of Rosario | Pizarro M.D.,Centro Binacional Argentina Italia Of Investigaciones En Criobiologia Clinica Y Aplicada Caic | Izaguirre M.F.,National University of Entre Rios | Biancardi M.E.,National University of Rosario | And 5 more authors.
Cryo-Letters | Year: 2011

Human cardiac valve allografts (HVAs) suffer injuries during the cryopreservation period. Here, we described structural, ultrastructural and functional damages suffered by HVAs after an increment of their cryostorage temperature (100°C). Two experimental groups of pulmonary and aortic HVAs were compared: cryopreserved (HVAcryo) and cryopreserved with temperature changes (HVAT)- Transmission electron microscopy (TEM) was used to analyze valve fibroblasts and extracellular matrix morphology. Total collagen amount was estimated using two different methods and fibroblast viability was assessed measuring their oxygen consumption rate. Porcine heart grafts valves were used to set the techniques. Disorganized collagen network was seen in HVAT by TEM. Fibroblasts showed damages in the cellular membrane and many secretor vesicles. Mitochondria and chromatin were also altered. HVAT had less amount of collagen and fibroblasts showed an oxygen consumption rate markedly diminished compared to HVAcryo. The increment of 100°C suffered by HVAs caused damages that made them unsuitable for clinical purposes. © CryoLetters.

de Paz L.J.,Centro Binacional Argentina Italia Of Investigaciones En Criobiologia Clinica Y Aplicada Caic | Robert M.C.,National University of Rosario | Graf D.A.,Centro Binacional Argentina Italia Of Investigaciones En Criobiologia Clinica Y Aplicada Caic | Guibert E.E.,National University of Rosario | Rodriguez J.V.,National University of Rosario
Cryo letters | Year: 2015

BACKGROUND: Slow cooling is a cryopreservation methodology where samples are cooled to its storage temperature at controlled cooling rates.OBJECTIVE: Design, construction and evaluation of a simple and low cost device for slow cooling of small biological samples.MATERIALS AND METHODS: The device was constructed based on Pye's freezer idea. A Dewar flask filled with liquid nitrogen was used as heat sink and a methanol bath containing the sample was cooled at constant rates using copper bars as heat conductor.RESULTS: Sample temperature may be lowered at controlled cooling rate (ranging from 0.4°C/min to 6.0°C/min) down to ~-60°C, where it could be conserved at lower temperatures. An example involving the cryopreservation of Neuro-2A cell line showed a marked influence of cooling rate over post preservation cell viability with optimal values between 2.6 and 4.6°C/min.CONCLUSION: The cooling device proved to be a valuable alternative to more expensive systems allowing the assessment of different cooling rates to evaluate the optimal condition for cryopreservation of such samples.

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