Charles Y. Lee

The process of freezing and the mechanism of damage during hepatic cryosurgery

Experiments were performed to correlate the structures of liver tissue frozen during cryosurgery, liver frozen at various constant cooling rates, and unfrozen, dried normal liver. The results show that during freezing of tissue ice forms and propagates along the vascular system, expanding during freezing at low cooling rates. This expansion occurs over most of the region frozen during cryosurgery and may be one of the mechanisms of damage to tissue during cryosurgery.

Survival transplantation of preserved non???heart-beating donor rat livers: preservation by hypothermic machine perfusion1

Background. Non–heart-beating donor (NHBD) livers are an untapped source with the potential to provide relief to the current donor shortage problem. Hypothermic machine perfusion (MP) has the potential to reclaim and preserve these marginal donor organs. Methods. This study compared 5-day survival in a rat NHBD liver transplantation model with simple cold storage (SCS) and MP-preserved tissues that had experienced 30 min of warm ischemia followed by a 5-hr preservation period with the University of Wisconsin solution. Total release of lactate dehydrogenase (LDH) and alanine aminotransferase (ALT) were determined at major time points. Bilirubin levels and histology were examined after 5-day survival. Results. Six of seven control livers and five of six MP livers survived, whereas SCS tissues had survival in zero of seven. The results showed that MP livers had reduced release of LDH and ALT after 5 hr of storage, 5.07±1.42 and 2.02±0.69 U (mean±SE), respectively, compared with SCS, 15.54±0.81 and 3.41.3±0.73 U, respectively. Bilirubin values after 5-day survival of MP livers (1.17±0.49 mg/dL) were comparable to controls (0.91±0.36 mg/dL). Histology confirms that SCS displayed increased necrosis and MP tissue showed regions of near normal hepatic structure. Conclusions. These results suggest that MP for 5 hr improves survival and reduces cellular damage of liver tissue that has experienced 30 min of warm ischemia when compared with SCS tissues. Further studies need to be conducted, but this study suggests that MP preservation has the potential to reclaim and preserve NHBD liver tissues.

Preservation methods for kidney and liver

With the successful testing of the immunosuppressive effects of cyclosporine in transplant patients in 1978, the field of organ transplants began an exponential growth. With that, the field of organ preservation became increasingly important as the need to increase preservation time and improve graft function became paramount. However, for every patient that receives a transplanted organ, there are 4 more on the waiting list. In addition, a patient dies from the lack of a transplant almost every 1½ hour. To alleviate this donor crisis, there is a need to expand the donor pool to marginal donor organs. The main reason these organs are underutilized is because the current method of static preservation, simple cold storage, is ineffective. This article will provide a general review of the methods of preservation including simple cold storage, hypothermic machine perfusion, normothermic machine perfusion, and oxygen persufflation. In addition, the article will provide a review of how these dynamic preservation methods have improved the recovery and preservation of marginal donor organs including donation after cardiac death and fatty livers.

Heterogeneous flow patterns during hypothermic machine perfusion preservation of livers.

Background. Hypothermic machine perfusion preservation (MPP) has the potential to relieve the current donor shortage problem by providing superior preserved tissue and viable non-heart-beating donor tissue. For the liver, MPP has not improved preservation. Currently, the major cause of damage associated with MPP of livers is unknown. Methods. An intravital microscopy study was conducted to investigate the state of sinusoidal perfusion during MPP and the effect of a high potassium perfusion solution on liver microcirculation. Results. The results showed that there was an increase in vascular resistance (>200%) when the liver was perfused with a high K solution at either 37° or 5°C and with a flow rate of 14 or 5 ml/min. Heterogeneous flow perfusion at the microcirculatory level was evident with regions of no-flow, which after a 10 hr MPP protocol remained unperfused during rewarming. These areas also displayed increased cellular injury as determined by propidium iodide staining (245.4 vs. 12.3 cells) compared with regions that were perfused during MPP. Conclusion. These results suggest that a heterogeneous flow pattern occurs during MPP of the liver as a result of the high K perfusion solution. Regions of no-flow during extended MPP remained unperfused during rewarming resulting in tissue damage and may represent a cause of liver damage associated with MPP.

Improved Preservation of Warm Ischemic Livers by Hypothermic Machine Perfusion with Supplemented University of Wisconsin Solution

Hypothermic machine perfusion (HMP) has the potential to improve recovery and preservation of Donation after Cardiac Death (DCD) livers, including uncontrolled DCD livers. However, current perfusion solutions lack the needed substrates to improve energy recovery and minimize hepatic injury, if warm ischemic time (WIT) is extended. This proof-of-concept study tested the hypothesis that the University of Wisconsin (UW) solution supplemented with anaplerotic substrates, calcium chloride, thromboxane A2 inhibitor, and antioxidants could improve HMP preservation and minimize reperfusion injury of warm ischemic livers. Preflushed rat livers subjected to 60 min WIT were preserved for 5 h with standard UW or supplemented UW (SUW) solution. Post preservation hepatic functions and viability were assessed during isolated perfusion with Krebs–Henseleit solution. Livers preserved with SUW showed significantly (p <. 001) improved recovery of tissue ATP levels (μ mol/g liver), 2.06 ± 0.10 (mean ± SE), as compared to the UW group, 0.70 ± 0.10, and the level was 80% of that of fresh control livers (2.60 ± 0.13). At the end of 1 h of rewarming, lactate dehydrogenase (U/L) in the perfusate was significantly (p <. 05) lower in the SUW group (429 ± 58) as compared to ischemia–reperfusion (IR) (781 ± 12) and the UW group (1151 ± 83). Bile production (μ g/min/g liver) was significantly (p <. 05) higher in the SUW group (280 ± 13) as compared to the IR (224 ± 24) and the UW group (114 ± 14). The tissue edema formation assessed by tissue wet–dry ratio was significantly (p <. 05) higher in UW group. Histology showed well-preserved hepatic structure in the SUW group. In conclusion, this study suggests that HMP with SUW solution has the potential to restore and preserve livers with extended WIT.

Analysis of the introduction and removal of glycerol in rabbit kidneys using a Krogh cylinder model

In 1982, Rubinsky and Cravalho described a Krogh cylinder model for the analysis of cryoprotectant transport in a perfused organ. By application of the Kedem-Katchalsky equations, changes in tissue volume caused by movements of water and solute were used to predict changes in capillary radius (Cryobiology 19, 70-82, 1982). We have now measured the changes in vascular resistance that are produced when sucrose or glycerol is introduced into the perfusate flowing through rabbit kidneys at 10 degrees C, and have analyzed these data by means of the Rubinsky-Cravalho semiempirical model. The sucrose data provided an estimate of hydraulic conductivity and the dimensions of the Krogh tissue units. Three rates of addition of glycerol, 10, 30, and 90 mM/min to a final concentration of 3 M, were studied. The vascular resistance fell to approximately 40% of its initial value (radius approximately 128% of initial value) with all three rates of addition, and then returned toward its normal value while the glycerol concentration was still increasing. This behavior could be explained either by a sudden change in solute permeability at that capillary radius, or by an inverse dependence of reflection coefficient upon solute concentration. Evidence is presented that favors the latter interpretation. The best fits for the apparent hydraulic conductivity and apparent solute permeability for glycerol are 1 X 10(-6) cm/sec atm and 6 X 10(-8) cm/sec, respectively, with the reflection coefficient falling from 1.0 when the glycerol concentration is zero to 0.1 when it is 3 M. The model is used to predict tissue concentrations of glycerol throughout each experiment.

A multi-dimensional model of momentum and mass transfer in the liver

Abstract A new concept in modeling multi-dimensional fluid flow and mass transfer in the liver utilizing principles from porous media theory is introduced. From this model, a distribution of the pressure and velocity values is obtained for fluid in the microvasculature. The velocities for the fluid are then incorporated into a multi-dimensional conservation of chemical species equation. A distribution of the chemical species concentration in the fluid and the tissue is then determined using an equation which describes the mass transfer process across the cell membrane. Several parametric studies which included the effects of the vascular architecture on pressure and flow distribution, and the response to a step change in the inlet chemical species concentration are conducted to determine the sensitivity of the model.

Inhibition of TXA2 synthesis with OKY‐046 improves liver preservation by prolonged hypothermic machine perfusion in rats

Background and Aim:  We previously reported that hypothermic machine perfusion (HMP) for liver preservation is feasible, but hepatic microcirculatory dysfunction and significant liver damage remain major obstacles in its application when the preservation is extended to 24 h. The underlying injury mechanism is not well understood. The present study sought to investigate the role of thromboxane A2 (TXA2) in the pathogenesis of liver injury after prolonged HMP.

The effects of over expressing aquaporins on the cryopreservation of hepatocytes

During cryopreservation, aquaporins are critical in regulating water transport across cellular membranes and preventing osmotic damages. Hepatocytes express aquaporin (AQP) 0, 8, 9, 11, and 12; this study investigates whether increasing the localization of AQP8 on the cellular membrane would improve cell viability by increasing water transport during cryopreservation. Primary rat hepatocytes were cultured and treated with dibutyryl cAMP (Bt(2)cAMP) or glucagon to increase the expression of AQP8 at the cellular membrane via translocation. This phenomenon is verified through two experiments - confocal immunofluorescence microscopy and cell shrinkage analysis. The immunofluorescence results showed increase in AQP8 on the cellular membrane of treated cells, and cell shrinkage analysis showed an increase in water transport of treated cells compared to controls. Primary rat hepatocytes were treated with Bt(2)cAMP or glucagon and cryopreserved using standard protocols in a controlled rate freezer. This resulted in a significant increase in the cell viability on warming. These results indicate that Bt(2)cAMP or glucagon treated hepatocytes had increased expression of aquaporin in the cellular membrane, increased water transport during cryopreservation, and increased post-thaw viability.

Ex vivo evaluation of porcine livers post-hypothermic machine perfusion at 4-6ºC and 12-14ºC

Background: The goal of our research is the development of a clinical hypothermic machine perfusion method and device for liver preservation during storage and transport prior to transplantation. The purpose of this study was comparison of two hypothermic temperature ranges. Methods: Heart beating donor pig livers with 2h of static cold storage on ice were employed. Thirteen experimental livers were perfused, at either 4-6oC or 12-14oC with oxygenation, employing a prototype device. They were compared with six control 2h static cold stored livers during normothermic ex vivo blood perfusion. Physiological measurements and biochemical perfusate analyses were performed to assess the impact of storage conditions on liver functions. Statistics were assessed by one way analysis of variance, p<0.05 was regarded as significant. Results: During hypothermic perfusion perfusate solutes (Na + , K + and Ca ++ ) varied little with time. A single 12-14oC perfused liver exhibited a Ca ++ spike at 22 hours. Temperature dependent metabolic activity was observed in both groups. Lactate levels were ≤4.5mmol/L for 22h. During normothermic ex vivo testing most controls and perfusion-treated experimental livers produced bile within an hour. 10h perfusion liver lactate dehydrogenase, hyaluronic acid uptake, and total bile production were not significantly different from controls. There were trends for albumin, glucose and lactate. Significantly different Factor V, indocyanine green clearance, and blood urinary nitrogen concentrations were observed in both 10h perfusion groups. Bilirubin kinetics was perturbed in all perfusion groups, however the peak concentrations in the 10h perfusion groups were not significantly different, while the 22h 12-14oC perfusion group was significantly less than controls. A significant difference in alanine aminotransferase values between the 2 perfusion groups, was observed, however this may be due to washout during 12-14°C perfusion. Lactate dehydrogenase was doubled in both perfusion groups at 22h and individual 22h livers exhibited other assay values outside control and 10h perfusion ranges. Conclusions: Significant differences were observed between controls and both perfused groups during post hypothermic storage ex vivo assessment. These results suggest that both temperature ranges tested, with further optimization, may be suitable for liver support for up to 10h of oxygenated hypothermic perfusion. Since there were no clear advantages to 12-14°C perfusion we will continue development of a 4-6°C perfusion device for storage and transport of livers for transplantation.