Steps for the Autologous Ex vivo Perfused Porcine Liver-kidney Experiment

Summary

Study of the animal organ physiology can be achieved by performing an experimental ex vivo perfusion system. The addition of a porcine kidney, as a homeostatic organ to our previously developed ex vivo liver perfusion model can be a principal step to achieve a better physiological environment.

Abstract

The use of ex vivo perfused models can mimic the physiological conditions of the liver for short periods, but to maintain normal homeostasis for an extended perfusion period is challenging. We have added the kidney to our previous ex vivo perfused liver experiment model to reproduce a more accurate physiological state for prolonged experiments without using live animals. Five intact livers and kidneys were retrieved post-mortem from sacrificed pigs on different days and perfused for a minimum of 6 hr. Hourly arterial blood gases were obtained to analyze pH, lactate, glucose and renal parameters. The primary endpoint was to investigate the effect of adding one kidney to the model on the acid base balance, glucose, and electrolyte levels. The result of this liver-kidney experiment was compared to the results of five previous liver only perfusion models. In summary, with the addition of one kidney to the ex vivo liver circuit, hyperglycemia and metabolic acidosis were improved. In addition this model reproduces the physiological and metabolic responses of the liver sufficiently accurately to obviate the need for the use of live animals. The ex vivo liver-kidney perfusion model can be used as an alternative method in organ specific studies. It provides a disconnection from numerous systemic influences and allows specific and accurate adjustments of arterial and venous pressures and flow.

Introduction

Studying a large animal organ physiology in an artificial environment is challenging. Furthermore, maintaining normal physiological and biochemical parameters for a prolonged period requires a closely monitored perfusion method and model. We have previously described our ex vivo autologous perfused porcine liver model for the study of hepatic physiology and significant progress has been achieved, since we began our liver reperfusion ex vivo model in 2005, in areas such as harvesting technique, understanding hepatic artery anatomical variations¹, inflammatory response^2,3, histological changes following liver electrolytic ablation (EA)⁴, and changes in acid base balance during EA⁵. Unfortunately hyperglycemia and metabolic acidosis require external supplements of insulin and bicarbonate to maintain an accurate physiological environment. Stable blood sugar levels and acid base balance are crucial when studying islet auto-transplantation, especially in the early implantation phase. We have developed a new model incorporating the kidney to clear excess metabolites and improve the environment for the islet transplantation^6,7.

Extracorporeal perfusion experiments lasting longer than 24 hr can also be performed, but these require a larger team to perform the experiments⁸.

Animals used in this study received human care and study protocols were in accordance with the United Kingdom laws. They were euthanized with final exsanguinations during the blood-harvesting procedure according to United Kingdom regulations. Porcine liver and a left kidney were retrieved from animals in the food chain and that would otherwise be discarded. In this way important ethical implications can be overcome and the economic burden is lower than more common in vivo studies. The short duration of liver and kidney perfusion time in this model, lasting from 6-24 hr, has been a major limitation of our studies; this can be improved by minimizing the exposure of the organs to the ischemic injury (cold and warm ischemia).

Protocol

1. Preparation of the Procedure Equipment, Solutions, and Experiment Setup Equipment: The following equipment is required: Dissecting instruments: scissors, scalpels, artery forceps, 6x artery clips. 4x 12-14F catheters (for portal vein, hepatic artery, bile duct and renal artery), 1x 12F catheter (ureter), suture material (20x Vicryl stitches and Vicryl ties), stitch scissors, 3x infusion sets, ice in large insulated transport container, gloves, a large sterile …

Representative Results

Liver Function The liver function tests remained stable for the first 6 hr. However, there were significant changes present only from the 19th hour of the 24 hr experiment (P<0.001). Factor V and X were progressively diminished during the whole experiment and significant changes were evident from the 5th hour of perfusion for factor V and from the 3rd hour for the factor X onward, (P<0.05; Figure 2)20. <p class…

Discussion

In our group the experience and the results achieved with the ex vivo perfusion of the liver have been paralleled over the years by concomitant modifications in the retrieval technique adopted. In the last few years our group has achieved considerable experience using an ex vivo porcine model to study the hepatic physiology^5,11, new liver ablation techniques^2,5, and liver immunology³. Despite the technical challenges derived from an additional organ, corrections of …

Materials

Epoprostenol sodium (Flolan)	Glaxo Smith Kline	PL10949/0310
Sodium Bicarbonate 8.4%	Polyfusor, Fresenius Kabi Ltd	PL8828/0043
Cefuroxime sodium (Zynacef)	Flynn Pharma Ltd	PL13621/0018
Calcium Chloride 14.7%	Martindale Pharmaceuticals	PL1883/6174R
Heparin	LEO Laboratories Limited	PL0043/0038R
Sodium taurocholate	Sigma-Aldrich	T4009-25G
Insulin (Actrapid)	Novo Nordisk	EU/1/02/230/003
Soltran (preservation solution)	Baxter Health Care	FKB4708G	preservation solution
Atraumatic centrifugal pump (Bio-console 560)	Medtronic Inc., Minneapolis, Minnesota- United States; custom pack, cardiac surgery division Europe