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.

Organ Preservation into the 2020s: The Era of Dynamic Intervention.

Organ preservation has been of major importance ever since transplantation developed into a global clinical activity. The relatively simple procedures were developed on a basic comprehension of low-temperature biology as related to organs outside the body. In the past decade, there has been a significant increase in knowledge of the sequelae of effects in preserved organs, and how dynamic intervention by perfusion can be used to mitigate injury and improve the quality of the donated organs. The present review focuses on (1) new information about the cell and molecular events impacting on ischemia/reperfusion injury during organ preservation, (2) strategies which use varied compositions and additives in organ preservation solutions to deal with these, (3) clear definitions of the developing protocols for dynamic organ perfusion preservation, (4) information on how the choice of perfusion solutions can impact on desired attributes of dynamic organ perfusion, and (5) summary and future horizons.

The Novel N,N-bis-2-Hydroxyethyl-2-Aminoethanesulfonic Acid-Gluconate-Polyethylene Glycol-Hypothermic Machine Perfusion Solution Improves Static Cold Storage and Reduces Ischemia/Reperfusion Injury in Rat Liver Transplant.

Organ transplantation is the treatment of choice against terminal and irreversible organ failure. Optimal preservation of the graft is crucial to counteract cold ischemia effects. As we developed an N,N-bis-2-hydroxyethyl-2-aminoethanesulfonic acid-gluconate-polyethylene glycol (BGP)-based solution (hypothermic machine perfusion [HMP]), we aimed to analyze the use of this solution on static cold storage (SCS) of rat livers for transplantation as compared with the histidine tryptophan ketoglutarate (HTK) preservation solution. Livers procured from adult male Sprague Dawley rats were preserved with BGP-HMP or HTK solutions. Liver total water content and metabolites were measured during the SCS at 0°C for 24 hours. The function and viability of the preserved rat livers were first assessed ex vivo after rewarming (90 minutes at 37°C) and in vivo using the experimental model of reduced-size heterotopic liver transplantation. After SCS, the water and glycogen content in both groups remained unchanged as well as the tissue glutathione concentration. In the ex vivo studies, livers preserved with the BGP-HMP solution were hemodynamically more efficient and the O2 consumption rate was higher than in livers from the HTK group. Bile production and glycogen content after 90 minutes of normothermic reperfusion was diminished in both groups compared with the control group. Cellular integrity of the BGP-HMP group was better, and the histological damage was reversible. In the in vivo model, HTK-preserved livers showed a greater degree of histological injury and higher apoptosis compared with the BGP-HMP group. In conclusion, our results suggest a better role of the BGP-HMP solution compared with HTK in preventing ischemia/reperfusion injury in the rat liver model.

Proteome variation of the rat liver after static cold storage assayed in an ex vivo model.

Cold storage is a common procedure for liver preservation in a transplant setting. However, during cold ischemia, the liver suffers molecular alterations that can affect its performance. Also, deleterious mechanisms set forth in the storage phase are exacerbated during reperfusion. This study aimed to identify liver proteins associated with injury during cold storage and/or normothermic reperfusion using the isolated perfused rat liver model. Livers from male rats were subjected to either (1) cold storage for 24 h, (2) ex vivo normothermic reperfusion for 90 min or (3) cold storage for 24 h followed by ex vivo normothermic reperfusion for 90 min. Then, the livers were homogenized and proteins were extracted. Protein expression between each experimental group and the control (freshly resected livers) was compared by two-dimensional (2D) gel electrophoresis. Protein identification was carried out by matrix-assisted laser desorption/ionization time-of-flight spectrometry (MALDI-TOF/TOF) using MASCOT as the search engine. 23 proteins were detected with significantly altered levels of expression among the different treatments, including molecular chaperones, antioxidant enzymes, and proteins involved in energy metabolism. Some of them have been postulated as biomarkers for liver damage while others had been identified in other organs subjected to ischemia and reperfusion injury. The whole data set will be a useful resource for studying deleterious molecular mechanisms that result in diminished liver function during storage and subsequent reperfusion.

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°C. After 24 h 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 24 h in HTK (BCD); II--Livers procured after cardiac death (45 min) and cold stored 24 h in HTK (ACD); III--Livers procured after cardiac death (45 min) and cold stored 24 h in HTK+10 µM Sodium Sulfide (Na2S) (ACD-SS). Data suggest that after 45 min 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-α 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.

DESIGN OF A SIMPLE 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°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.

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 efficiency 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.

Delivery of the bioactive gas hydrogen sulfide during cold preservation of rat liver: effects on hepatic function in an ex vivo model.

The insults sustained by transplanted livers (hepatectomy, hypothermic preservation, and normothermic reperfusion) could compromise hepatic function. Hydrogen sulfide (H2S) is a physiologic gaseous signaling molecule, like nitric oxide (NO) and carbon monoxide (CO). We examined the effect of diallyl disulfide as a H2S donor during hypothermic preservation and reperfusion on intrahepatic resistance (IVR), lactate dehydrogenase (LDH) release, bile production, oxygen consumption, bromosulfophthalein (BSP) depuration and histology in an isolated perfused rat liver model (IPRL), after 48 h of hypothermic storage (4 °C) in University of Wisconsin solution (UW, Viaspan). Livers were retrieved from male Wistar rats. Three experimental groups were analyzed: Control group (CON): IPRL was performed after surgery; UW: IPRL was performed in livers preserved (48 h-4 °C) in UW; and UWS: IPRL was performed in livers preserved (48 h-4 °C) in UW in the presence of 3.4 mM diallyl disulfide. Hypothermic preservation injuries were manifested at reperfusion by a slight increment in IHR and LDH release compared with the control group. Also, bile production for the control group (1.32 µL/min/g of liver) seemed to be diminished after preservation by 73% in UW and 69% in UW H2S group at the end of normothermic reperfusion. Liver samples analyzed by hematoxylin/eosin clearly showed the deleterious effect of cold storage process, partially reversed (dilated sinusoids and vacuolization attenuation) by the addition of a H2S delivery compound to the preservation solution. Hepatic clearance (HC) of BSP was affected by cold storage of livers, but there were no noticeable differences between livers preserved with or without diallyl disulfide. Meanwhile, livers preserved in the presence of H2S donor showed an enhanced capacity for BSP uptake (k(A) CON = 0.29 min⁻¹; k(A) UW = 0.29 min⁻¹ ; k(A) UWS = 0.36 min ⁻¹). In summary, our animal model suggests that hepatic hypothermic preservation for transplantation affects liver function and hepatic depuration of BSP, and implies that the inclusion of an H2S donor during hypothermic preservation could improve standard methods of preparing livers for transplant.

A modular perfused chamber for low- and normal-temperature microscopy of living cells.

An inexpensive modular perfused chamber (MPC) designed for low- and normal-temperature live-cell imaging is presented. The device consists of four lathed pieces of stainless steel assembled as a cylindrical open chamber that can hold either round or square glass coverslips. The chamber is connected to a thermal-bath operating with recirculation. For image acquisition at 4°C, cooled air is blown toward the coverslip surface to prevent condensation. Principal advantages of this device are thermal stability in the sample environment, rapid response to changes in temperature set point, and easy sample insertion. The device enables the study of dynamic processes in cells governed by large temperature differences such as those imposed by hypothermic preservation of cells (0-4°C) followed by rewarming to normothermia (37°C). The capabilities of the MPC were demonstrated by monitoring the internalization of fluorescent quantum dots (QDs) in rat hepatocytes after hypothermic storage and during rewarming with an inverted microscope.

Organ Preservation: Current Concepts and New Strategies for the Next Decade.

SUMMARY: Organ transplantation has developed over the past 50 years to reach the sophisticated and integrated clinical service of today through several advances in science. One of the most important of these has been the ability to apply organ preservation protocols to deliver donor organs of high quality, via a network of organ exchange to match the most suitable recipient patient to the best available organ, capable of rapid resumption of life-sustaining function in the recipient patient. This has only been possible by amassing a good understanding of the potential effects of hypoxic injury on donated organs, and how to prevent these by applying organ preservation. This review sets out the history of organ preservation, how applications of hypothermia have become central to the process, and what the current status is for the range of solid organs commonly transplanted. The science of organ preservation is constantly being updated with new knowledge and ideas, and the review also discusses what innovations are coming close to clinical reality to meet the growing demands for high quality organs in transplantation over the next few years.

Heterologous ferredoxin reductase and flavodoxin protect Cos-7 cells from oxidative stress.

BACKGROUND: Ferredoxin-NADP(H) reductase (FNR) from Pisum sativum and Flavodoxin (Fld) from Anabaena PCC 7119 have been reported to protect a variety of cells and organisms from oxidative insults. In this work, these two proteins were expressed in mitochondria of Cos-7 cells and tested for their efficacy to protect these cells from oxidative stress in vitro. PRINCIPAL FINDINGS: Cos-7/pFNR and Cos-7/pFld cell lines expressing FNR and Fld, respectively, showed a significantly higher resistance to 24 h exposure to 300-600 µM hydrogen peroxide measured by LDH retention, MTT reduction, malondialdehyde (MDA) levels and lipid peroxide (LPO; FOX assay) levels. However, FNR and Fld did not exhibit any protection at shorter incubation times (2 h and 4 h) to 4 mM hydrogen peroxide or to a 48 h exposure to 300 µM methyl viologen. We found enhanced methyl viologen damage exerted by FNR that may be due to depletion of NADPH pools through NADPH-MV diaphorase activity as previously observed for other overexpressed enzymes. SIGNIFICANCE: The results presented are a first report of antioxidant function of these heterologous enzymes of vegetal and cyanobacterial origin in mammalian cells.

A device to measure oxygen consumption during the hypothermic perfusion of the liver.

This work deals with the construction and performance of a device designed to measure the oxygen consumption by the liver during hypothermic perfusion in the rat model. Due to its simple design and the utilization of standard materials, it could serve to determine the role of oxygenation during hypothermic perfusion of the liver. The system consists of a reservoir containing the preservation solution, a peristaltic pump and an internal oxygenator made of silicone tube. A five ports manifold connects the circulation to the liver (inflow), to a hydrostatic manometer and to two sample ports; the liver outflow and temperature sensor or gas calibration. Finally the exit port connects the circulation fluid with an oxygen electrode. The preservation solution is pumped through the liver at a constant pressure (77 i 15 mm H2O) and a perfusion flow of 0.39 - 0.49 mL per min per g liver. To test the system, two to four hours perfusion experiments were performed, at temperatures of 5 and 10 degree C. Two preservation solutions were evaluated: Custodiol and Bes-Gluconate-Sucrose. The solubility of oxygen in the preservation solutions was determined, and the oxygen consumption by preserved rat livers was measured.

Subzero nonfreezing storage of rat hepatocytes using UW solution and 1,4-butanediol. II- functional testing on rewarming and gene expression of urea cycle enzymes.

UNLABELLED: In the present study we have analyzed the viability and metabolic competence of isolated rat hepatocytes subjected first, to subzero nonfreezing storage (up to 120 h at -4 degrees C) in modified University of Wisconsin (UW) solution with 8% 1,4-butanediol, and then to a normothermic rewarming step (KHR media, 37 degrees C, up to 120 min, carbogen atmosphere). Results were compared with hepatocytes stored up to 120 h at 0 degrees C in modified UW solution and with freshly isolated hepatic cells. We have found that only cell suspensions stored in subzero nonfreezing conditions were able to finish the rewarming period with a viability comparable with the control group. Also, we have investigated the enzyme activities and the relative expression at messenger RNAs levels of two of the Urea cycle (UC) enzymes: Carbamyl phosphate synthetase I (CPSI) and ornithine transcarbamylase (OTC), during 60 min of rewarming. Results were compared with the ammonium removal efficiency of the three groups. IN CONCLUSION: These data indicated that hepatocytes preserved under cold or subzero conditions up to 120 h followed by 60 min of rewarming, maintain UC enzymes at levels similar to freshly isolated hepatocytes, allowing their use in bioartificial liver devices.

Morphological and biochemical analysis of human cardiac valve allografts after an increment of the cryostorage temperature.

Cryopreserved human cardiac valve allografts could suffer lethal damages if the temperature is elevated during cryostorage. This work describes the functional and morphological alterations suffered by human cardiac valve allografts after a gradual increment of the cryostorage temperature from -147 degrees C to -47 degrees C due to a technical failure. Three experimental groups of human pulmonary and aortic allografts were compared: fresh, cryopreserved (-147 degrees C) and cryopreserved with temperature changes from -147 degrees C up to -47 degrees C and back to -147 degrees C. Fibroblast functionality was studied to asses the degree of valvular damages. Collagen network was also analyzed with bright light field and polarized microscopy; an immunohistochemistry for procollagen I was performed and the MTT colorimetric assay was used to evaluate fibroblast mitochondrial enzymatic activity. Porcine heart grafts valves were used to set the MTT colorimetric assay. With bright light field microscopy, disorganized collagen network was seen together with interstitial edema in cryopreserved groups. Polarized microscopy showed that fresh allografts had abundant collagen type I and III, cryopreserved group had less amount of collagen type I and in allografts that suffered cryopreservation temperature elevation collagen type I synthesis could not been demonstrated. Procollagen I was present in fibroblast cytoplasm of fresh group, but it was diminished in cryopreserved group and was absent in the group that suffered temperature elevation. Temperature changes during the cryopreservation period of human cardiac valve allografts induced fibroblast activity reduction. When the cryopreservation temperature is elevated during cryostorage, fibroblasts lost their functionality and the allografts may be not suitable for transplant.

Subzero nonfreezing storage of rat hepatocytes using modified University of Wisconsin solution (mUW) and 1,4-butanediol. I- effects on cellular metabolites during cold storage.

Various cryopreservation techniques have been investigated to extend the storage of isolated hepatocytes; however, most have a reduced viability after rewarming due to ice crystal formation. Subzero nonfreezing conditions could theoretically reduce organ metabolism without damage due to ice crystal formation. In the present work we evaluated the viability and metabolic parameters of isolated rat hepatocytes preserved in subzero nonfreezing condition. Cell suspensions were maintained in modified University of Wisconsin (mUW) solution using 8% - ,4-butanediol as cryoprotectant, up to 120 h at -4 masculineC. The time course evolution of hepatocytes viability were measured by LDH release and propidium iodide assay. The cellular concentrations of glutathione, ATP, glycogen and the lactate production during cold storage were also determined. Finally, results were compared with conventional hypothermic storage at 0 masculineC in mUW solution without cryoprotectant. After 5 days of subzero storage, we found an improvement in the ability of rat hepatocytes to maintain the metabolic resources in comparison with the cold preserved group.

A novel BES-gluconate-sucrose (BGS) solution for cold storage of isolated hepatocytes.

We have used hepatocyte suspensions to study how hypothermic storage in a modified University of Wisconsin (mUW) solution affects liver cell metabolism and cell membrane properties. At present the UW solution is the gold standard of organ preservation. However it contains several ingredients which either are expensive or cannot easily be obtained worldwide. The aim of the present study was the development of a novel preservation solution for rat hepatocytes effective and comparable with the mUW, with enhanced buffer capacity and less expensive. In particular, we investigated the effects of the buffering agent BES, a derivate of aminosulfonic acid, on liver cells metabolism during cold storage and rewarming. In the development of this novel preservation solution we have included three key components: gluconate as impermeant anion, sucrose to give additional osmotic support and the aminosulfonic acid BES for its buffer capacity. Our results shown that BGS solution was equally effective to mUW to protect rat hepatocytes against cold preservation injury due to ischemia and reoxigenation. Also, BGS solution is a good alternative with its high buffer capacity, best indices of respiration activity and it is considerably less expensive than the mUW solution. The use of this solution for the storage of isolated hepatocytes may facilitate hepatocyte research in situations in which the more complex and expensive mUW solution is not available since the cost of one liter of BGS is about 1/3 that an equal volume of mUW solution.

Construction and performance of a minibioreactor suitable as experimental bioartificial liver.

This work deals with the construction and performance of a hollow fiber-based minibioreactor (MBR). Due to its simple design and the utilization of standard materials, it could serve as a suitable tool to evaluate the behavior and performance of cold preserved or cultured hepatocytes in bioartificial liver devices. The system consists of 140 fiber capillaries through which goat blood is pumped at a flow of 9 mL/min. The cell compartment contains 90 x 10(6) rat hepatocytes (volume 10 mL) and an internal oxygenator made of silicone tubing. To test the in vitro function of the system, 2-h perfusion experiments were performed, the evolution of hematocrit, plasma and extra-fiber fluid osmolality, and plasma urea and creatinine concentrations were evaluated. The detoxication efficiency of an ammonia overload was tested, showing that the system has enough capacity to remove ammonium. Also, the MBR oxygen transfer capacity to hepatocytes was tested, showing that the cells received an adequate oxygen supply.

Cold preservation of isolated hepatocytes in UW solution: experimental studies on the respiratory activity at 0 degrees C.

To date, little attention has been paid to the role of the gas milieu in preservation solutions and its effect on cell viability. Dissolved O2 in the preservation media may be an important parameter to consider. In this study we polarographically measured the O2 concentration in air-equilibrated UW solution at 0 degrees C, as well as the respiratory activity of isolated hepatocytes cold-preserved in this solution up to 72 hours. To perform measurements at 0 degrees C, it was first necessary to characterize the sensor behavior at low temperatures. We verified that the sensor response is still linear at this temperature but the rate of response is significantly slower. The O2 solubility in UW-air solution at 0 degrees C was determined using a modified physical method and it was 410 microM O2, which, as expected, is lower than the solubility in water at the same temperature (453 microM O2). Isolated hepatocytes cold-stored in UW-air solution retained a measurable respiratory activity during a period of 72 hours. The O2 consumption rate was 0.48 +/- 0.13 nmol/O2/min/10(6) cells, which represents 1% of the control value at 36 degrees C (61.46 +/- 14.61 nmol/O2/min/10(6) cells). The respiratory activity and cell viability were well maintained during the preservation period. At present, preservation conditions need to be improved for cells to remain functionally active. Dissolved O2 may be required for energy re-synthesis but it also leads to an increment in reactive oxygen species. The O2 concentration in the preservation solution should be carefully controlled, reaching a compromise between cell requirement and toxicity.