Biopreservation of hepatocytes: current concepts on hypothermic preservation, cryopreservation, and vitrification

Isolated liver cells (primarily isolated hepatocytes) have found important applications in science and medicine over the past 40 years in a wide range of areas, including physiological studies, investigations on liver metabolism, organ preservation and drug de-toxification, experimental and clinical transplantation. An integral component of many of these works is the need to store the isolated cells, either for short or long-term periods. This review covers the biopreservation of liver cells, with a focus on the history of liver cell biopreservation, the application of hypothermia for short-term storage, standard cryopreservation methods for isolated hepatocytes, the biopreservation of other types of liver cells, and recent developments such as vitrification of hepatocytes. By understanding the basis for the different approaches, it will be possible to select the best options for liver cell biopreservation in different applications, and identify ways to improve preservation protocols for the future.Fil: Fuller, Barry J.. University College London; Estados UnidosFil: Petrenko, Alexander Y.. Ukraine Academy of Sciences; UcraniaFil: Rodriguez, Joaquin Valentin. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; Argentina. Universidad Nacional de Rosario. Secretaria de Ciencia y Técnica. Centro Binacional de Investigación en Criobiología Clínica y Aplicada; ArgentinaFil: Somov, Alexander Y.. Ukraine Academy of Sciences; UcraniaFil: Balaban, Cecilia Lucía. Universidad Nacional de Rosario. Secretaria de Ciencia y Técnica. Centro Binacional de Investigación en Criobiología Clínica y Aplicada; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; ArgentinaFil: Guibert, Edgardo Elvio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; Argentina. Universidad Nacional de Rosario. Secretaria de Ciencia y Técnica. Centro Binacional de Investigación en Criobiología Clínica y Aplicada; Argentin

Extended cold storage of cultured hepatocytes impairs endocytic uptake during normothermic rewarming

During hypothermic preservation of cells (0-4°C), metab¬olism is diminished and energy-dependent transport processes are arrested. The effect of hypothermic preservation of hepatocytes in endocytic transport following rewarming has not been previously reported. We evaluated the uptake of EGF (Epidermal Growth Factor) ligand conjugated to fluorescent Quantum Dots (QDs) probes in rat hepatocytes after 24 and 72 h cold storage in University of Wisconsin (UW) solution at 4°C. QDs uptake was visualized during rewarming to 37°C under air or, in a second approach, at the end of rewarming under 5% CO2. After 24 h in UW solution, QDs were internalized under both rewarming conditions similar to non-preserved hepatocytes and cells maintained a normal cytoskeleton distribution. However, in hepatocytes preserved 72 h none of the cells internalized QDs, which remained bound to the membranes. After rewarming, this group showed diminished actin staining while viability was maintained at ~70%. Our results present evidence that, hypothermic preservation for 72 h in UW solution at 4°C does not prevent EGFR (Epidermal Growth Factor Receptor) activation but irreversibly impairs endocytic uptake upon EGF stimulation; presumably due to actin cytoskeleton disassembling. Our approach can be applied on other membrane receptor systems and with other hypothermic preservation solutions to understand the effect of cooling in endocytic transport and to determine the optimal cold storage period.Fil: Hovanyecz, P.. Universidad Nacional de Rosario. Secretaria de Ciencia y Tecnica. Centro Binacional de Investigación en Criobiologia Clinica y Aplicada; ArgentinaFil: Guibert, Edgardo Elvio. Universidad Nacional de Rosario. Secretaria de Ciencia y Tecnica. Centro Binacional de Investigación en Criobiologia Clinica y Aplicada; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; ArgentinaFil: Pellegrino, Jose Manuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Fisiología Experimental (i); ArgentinaFil: Rodriguez, Joaquin Valentin. Universidad Nacional de Rosario. Secretaria de Ciencia y Tecnica. Centro Binacional de Investigación en Criobiologia Clinica y Aplicada; ArgentinaFil: Sigot, Valeria. Universidad Nacional de Rosario. Secretaria de Ciencia y Tecnica. Centro Binacional de Investigación en Criobiologia Clinica y Aplicada; Argentin

A simple and reliable refractometric method to determine the total solids concentration of the cervico-vaginal bovine mucus samples

Cervico-vaginal mucus (CVM) is a viscoelastic substance continuously produced by secretory cells of the endocervix and the vagina of cows. Its physicochemical composition varies depending on the hormonal status of the estrous cycle. In veterinary medicine refractometry is a widely diffused technique to determine total solids (TS) content of biological samples, but there are not published data of CVM total solids from refractometric measures. Refractometric TS determination contributes to the qualitative constituents analysis of CVM, additionally it is an easier and more inexpensive technique than gravimetric TS determination. The main goal of the present paper was to validate a refractometric method to estimate TS concentration of the soluble fraction of CVM samples. Samples were collected from seventy-three Holando Argentino cows of Santa Fe province farms in Argentina. Cows were classified in three experimental groups: healthy, subclinical (SE) and clinical endometritis (CE) group. To achieve a solubilisation protocol for CVM samples, four Triton™ X-100 concentrations were tested. Refractive index (RI) and gravimetric total solid (gTS) concentration of solubilised samples were determined for the three experimental groups. A mathematical equation was determined with the experimental data from the healthy group, in order to obtain calculated total solid concentration (cTS) from refractivity (R) values. To validate the RI method for CVM samples, cTS concentrations were compared with gTS concentrations from endometritis group samples. Triton™ X-100 0.01% (V/V) improved CVM samples handling and did not change physicochemical parameters (gTS, Na+ and K+ concentration, and RI values). The linear regression equation obtained was: cTS (g/dL) = (R – 0.67)/16.2, r2 = 0.91. Correlation between gTS and cTS concentration was: r = 0.97 for SE group and r = 0.97 for CE group. The homogenization protocol allowed the measurement of physicochemical parameters without altering their values. A high correlation coefficient between cTS and gTS postulates refractometry as an accurate method to determine TS concentration for solubilised CVM samples.Fil: Savia, Caren Luciana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Veterinarias; ArgentinaFil: Osorio, Juliana Soledad. Universidad Nacional de Rosario. Secretaria de Ciencia y Técnica. Centro Binacional de Investigación en Criobiología Clínica y Aplicada; ArgentinaFil: Rodriguez, Joaquin Valentin. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; Argentina. Universidad Nacional de Rosario. Secretaria de Ciencia y Técnica. Centro Binacional de Investigación en Criobiología Clínica y Aplicada; ArgentinaFil: Guibert, Edgardo Elvio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; Argentina. Universidad Nacional de Rosario. Secretaria de Ciencia y Técnica. Centro Binacional de Investigación en Criobiología Clínica y Aplicada; ArgentinaFil: Rinaudo, Agustín. Universidad Nacional de Rosario. Facultad de Ciencias Veterinarias; Argentin

Human organs come out of the deep cold

Over the past 50 years, organ transplantation has developed into a life-saving treatment for many severe diseases of the kidney, liver, heart, pancreas and other organs. Underpinning this achievement are two remarkable scientific advances: the ability to manipulate patients’ immune systems to accept an organ donated from an unrelated individual and the possibility of preserving organs outside the body for at least several hours. But while immunosuppression strategies have improved substantially, progress in organ preservation has been modest.Fil: Fuller, Barry J.. University College London; Reino UnidoFil: Petrenko, Alexander. Ukraine Academy of Sciences; UcraniaFil: Guibert, Edgardo Elvio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; Argentina. Universidad Nacional de Rosario. Secretaria de Ciencia y Técnica. Centro Binacional de Investigación en Criobiología Clínica y Aplicada; Argentin

Hypothermic Machine Perfusion Versus Cold Storage in the Rescuing of Livers From Non-Heart-Beating Donor Rats

The aim of this work was to compare the effi- ciency of cold storage (CS) and hypothermic machine perfusion (HMP) methods of preserving grafts excised from non-heart-beating donors that had suffered 45 minutes of warm ischemia. We developed a new solution for HMP to use in liver transplantation, based on BES, gluconate, and polyethylene glycol (BGP-HMP solution). After 24 h of HMP or CS, livers were reperfused at 37°C with Krebs–Henseleit solution with added dextran. For both procedures, portal pressure and flow were measured and the intrahepatic resistance (IR) was calculated. The pH oscillations and enzyme activities (LDH, AST, and ALT) were evaluated for the perfusion buffer during normothermic reperfusion. O2 consumption of the liver, glycogen production, and bile flow were also measured during the normothermic reperfusion period. Portal flow and IR showed statistical differences (P < 0.05) between the two groups (n = 5). HMP with BGP-HMP solution resulted in higher values of portal flow and lower IR than CS with HTK solution. Enzyme release after 90 min of reperfusion did not show statistical differences between groups. With regard to bile flow and O2 consumption, livers preserved by both processes were able to produce bile, but livers preserved with HMP were able to take up more O2 than livers preserved by CS.Fil: Carnevale, Matías Emanuel. Universidad Nacional de Rosario. Secretaria de Ciencia y Tecnica. Centro Binacional de Investigación en Criobiologia Clinica y Aplicada; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Balaban, Cecilia Lucía. Universidad Nacional de Rosario. Secretaria de Ciencia y Tecnica. Centro Binacional de Investigación en Criobiologia Clinica y Aplicada; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Guibert, Edgardo Elvio. Universidad Nacional de Rosario. Secretaria de Ciencia y Tecnica. Centro Binacional de Investigación en Criobiologia Clinica y Aplicada; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Bottai, Hebe. Universidad Nacional de Rosario; ArgentinaFil: Rodriguez, Joaquin Valentin. Universidad Nacional de Rosario. Secretaria de Ciencia y Tecnica. Centro Binacional de Investigación en Criobiologia Clinica y Aplicada; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Design of a slow cooling device for cryopreservation of small biological samples

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 ºC/min to 6.0 ºC/min) down to ~-60 ºC, 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.Fil: Juan de Paz, Leonardo. Universidad Nacional de Rosario. Secretaria de Ciencia y Técnica. Centro Binacional de Investigación en Criobiología Clínica y Aplicada; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; ArgentinaFil: Robert, María Celeste. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Rosario. Secretaria de Ciencia y Técnica. Centro Binacional de Investigación en Criobiología Clínica y Aplicada; ArgentinaFil: Graf, Daniel Adolfo. Universidad Nacional de Rosario. Secretaria de Ciencia y Técnica. Centro Binacional de Investigación en Criobiología Clínica y Aplicada; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; ArgentinaFil: Guibert, Edgardo Elvio. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Rosario. Secretaria de Ciencia y Técnica. Centro Binacional de Investigación en Criobiología Clínica y Aplicada; ArgentinaFil: Rodriguez, Joaquin Valentin. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Rosario. Secretaria de Ciencia y Técnica. Centro Binacional de Investigación en Criobiología Clínica y Aplicada; Argentin

The effect of a hydrogen sulfide releasing molecule (Na2S) on the cold storage of livers from cardiac dead donor rats. A study in an ex vivo model.

Liver transplantation is currently the preferred treatment option for end-stage liver disease. Donation after cardiac death was a common practice in the early years of organ donation before brain death criteria were established. Those organs were subjected to variable periods of warm ischemia that might intensify cold ischemia/reperfusion injuries. In the present, shortage of brain dead donors has led to the reassessment of organ donation after cardiac death. Since many cytoprotective roles have been describe for H2S during ischemia/reperfusion on a variety of tissues, we hypothesized that graft exposure to this bioactive gas might improve preservation of non-heart beating donated organs. Therefore, to establish a rat model of donation post-cardiac arrest and using this approach to judge H2S delivery effects on graft hypothermic preservation, were the main objectives of this investigation. Cardiopulmonary arrest was induced in sedated rats by overload of potassium (K(+)). Livers were surgically removed and subsequently stored in HTK Solution (Histidine-tryptophan-ketoglutarate) at 0-4\ub0C. After 24h of hypothermic preservation, livers were rewarmed in an ex vivo model. Three experimental groups were established as follows: I - Livers procured before cardiac death and cold stored 24h in HTK (BCD); II - Livers procured after cardiac death (45min) and cold stored 24h in HTK (ACD); III - Livers procured after cardiac death (45min) and cold stored 24h in HTK+10\u3bcM Sodium Sulfide (Na2S) (ACD-SS). Data suggest that after 45min of warm ischemia, viability parameters assessed during reperfusion in the ex vivo model were significantly impaired. Real time PCR revealed that after ex vivo reperfusion there is an increased expression of HO-1 and TNF-\u3b1 and a modest drop in Bcl-2 mRNA, which could be interpreted as the cellular response to the hypoxic insult sustained during warm ischemia. On the other hand, warm ischemic livers exposed to H2S during cold storage, improved microcirculation, morphology and viability parameters during ex vivo reperfusion and showed significant modulation of HO-1 mRNA expression. In conclusion, HTK supplementation with Na2S arose as a potential treatment to recover non-heart beating harvested organs. Furthermore, an appropriate model of cardiac dead liver donors was successfully developed

A device to record ultra-rapid cooling profiles

This work deals with the construction and performance of a measuring system capable of estimating temperature at sufficiently high speed (up to 1000 samples/sec). Due to its simple design and the utilization of standard materials, it could serve to recording the cooling profile of ultra-rapid procedures. An immersion device was also developed with the purpose of normalize the penetration speed of the sample in the LN2. The device allows also the comparative analysis of different cooling profiles. The system consists of an immersion device of the sample in the cooling agent, a temperature measurement system developed by Kleihans F and a laptop computer. To test the system, we recorded the cooling profiles of 10 L of distilled water and 6 M glycerol solution, obtaining a cooling rate of 8732 °C.min-1 and 4441 °C.min-1 respectively. Also we determine a cooling rate of 204.012 °C.min-1 during the immersion of the thermocouple assembly in LN2. Although, the same device, with small technical modifications related to the handling of the sample, could be used to evaluate the recovery from LN2 temperature to room temperature (re-warming).Fil: Juan de Paz, Leornardo. Universidad Nacional de Rosario. Secretaria de Ciencia y Tecnica. Centro Binacional de Investigación en Criobiologia Clinica y Aplicada; ArgentinaFil: Graf, Daniel Adolfo. Universidad Nacional de Rosario. Secretaria de Ciencia y Tecnica. Centro Binacional de Investigación en Criobiologia Clinica y Aplicada; ArgentinaFil: Scandizzi, Angel Luis. Universidad Nacional de Rosario. Secretaria de Ciencia y Tecnica. Centro Binacional de Investigación en Criobiologia Clinica y Aplicada; ArgentinaFil: Guibert, Edgardo Elvio. Universidad Nacional de Rosario. Secretaria de Ciencia y Tecnica. Centro Binacional de Investigación en Criobiologia Clinica y Aplicada; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Rodriguez, Joaquin Valentin. Universidad Nacional de Rosario. Secretaria de Ciencia y Tecnica. Centro Binacional de Investigación en Criobiologia Clinica y Aplicada; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin