Posters
Developing a Porcine Transdermal Absorption Model using Normothermic Machine Perfusion.
Pig skin closely mirrors human skin, with comparable stratum corneum and dermis layers. The vasculature and tissue architecture facilitate a drug absorption route from topical application through the skin layers to the blood, paralleling human physiological processes. However, there are inherent ethical and economic restrictions. To address this, we have developed ex-vivo perfusion models that replicate in-vivo physiology using surplus porcine tissue from the meat industry.
Entire forelimbs, livers and autologous blood were retrieved following clinical transplant protocols. Blood was cell-saved, producing packed red blood cells, and combined with a proprietary perfusate before recirculation through the vasculature using a normothermic perfusion circuit consisting of advanced life-support technology. After 1hr of perfusion a 25mg nicotine patch was placed on the skin of the limb, and blood samples were collected hourly. Mass spectrometry was used to determine nicotine and cotinine concentration.
All tissues perfused well, and physiological haemodynamics (based on mean arterial and portal vein blood flow, pressure and intravascular pressure) were restored within 60 minutes. Blood biochemistry and metabolic state, calculated via arterial and venous blood gas analysis, were normal. The liver actively engaged in gluconeogenesis and bicarbonate conversation, with stable blood pH and no metabolic acidosis. The pharmacokinetic profile of nicotine closely followed previously published levels reported in human clinical trials. Cotinine conversion by the liver was active, and again followed blood nicotine levels. An isolated limb-liver perfusion system can be used for absorption studies, with the potential to bypass in-vivo models in the future.
Development of a Physiological Whole Blood System for Drug Evaluation
Traditional in-vitro blood cultures cannot replicate in-vivo physiology due to their static nature. To address this, we developed a physiological system that maintains whole blood under arterial conditions, replicating the dynamic environment of the vasculature.
In this study, a blood recirculation system was built, consisting of a proprietary hollow fibre oxygenator, a levitating centrifugal pump, a tubing set and an arterial filter. Whole blood was collected from pigs via exsanguination, loaded into the circuit, and de-aired. The system was pressurised to 80mmHg to replicate physiological mean arterial pressure. Blood flow was initiated and maintained at 300ml/min. Following priming, a gas blend of 95% O2 and 5% CO2 was connected and PaO2 and PaCO2 was titred to ~150mmHg and <45mmHg respectively. Serial arterial blood gases were performed.
Blood co-oximetry was normal, with O2 saturation remaining above 99%. Haemoglobin parameters were physiological, with oxyhaemoglobin averaging 97.1%, and minimal methaemoglobin, carboxyhaemoglobin or deoxyhaemoglobin. Blood glucose and bicarbonate slowly declined over 6 hours, demonstrating active cellular metabolism. Blood potassium, total haemoglobin and haematocrit, which are surrogate markers of haemolysis, did not change throughout. pH was stable and within normal range, with no evidence of metabolic acidosis or alkalosis.
Using this approach, the in vivo vascular environment is closely replicated, where more than 4,000 components of blood are continually, dynamically and metabolically interacting. We are now developing a human blood system, which will be an invaluable model to evaluate immunotoxicity, immunogenicity, thrombotic events, safety and efficacy, in a ‘phase minus-one’ approach.
The Development of a Porcine Multi-Organ Liver-Kidney-Spleen Model to Advance Research in Tissue Engineering and Regenerative Medicine
The field of tissue engineering and regenerative medicine continues to evolve and offer significant promise in the delivery of medical and non-therapeutic applications. The roadmap for developing and testing new therapies is limited by preclinical models. Traditionally, live animal models act as a preclinical bridge to clinical incorporation, yet there is an ethical drive towards reducing live animal research. Small animal models rarely offer a route to clinical translation and microfluidic systems offer a poor substitute to in-vivo systems. Conversely, large animals offer better translational benefits but are associated with ethical, cost and time restraints. The development of alternative models are crucial to fast-track healthcare innovations into the clinic.
Our aim was to develop a porcine multi-organ Liver-Kidney-Spleen system that is capable of maintaining organs in perfect health outside of the body for at least 24 hours. This is done via the continuous infusion of an oxygenated blood-based perfusate through the vasculature of the organs, which all free drain into a central reservoir. This is recirculated at normothermia to support biological processes and physiology. The model design ensures that any effect an organ has on the perfusate is shared with the entirety of the closed system as it would in vivo.
We have demonstrated that our model is capable of maintaining organs in optimal health for 24 hours. In all organs, haemodynamics, co-oximetry and blood biochemistry remained stable and within physiological parameters. Bile and urine production began immediately and were maintained throughout, indicating restoration of organ function. Macroscopic tissue preservation following 24 hours of perfusion was excellent.
The model we have developed is closely comparable to the in-vivo environment, providing a highly translational, cost effective and ethical alternative to animal research. It is drug and medical agnostic, applicable from early proof of concept to new drug submission. Physiological evaluation of organs can be carried out, producing clinically relevant data such as drug distribution, tropism, uptake, organ specific toxicity, immunogenicity and bioavailability. Future work will consist of further refining the protocol to extend from 24 hours to several days, improving its application as a testing platform. Our aim is to integrate the model within the regular route for drug/therapy development.
The Development of an Improved 12-hour Porcine Kidney Preservation Protocol
Normothermic machine perfusion offers a superior alternative to existing hypothermic preservation strategies but is currently limited to 1-3 hours. Extending the time a kidney can be sustained using this technology could electivise transplantation, and enable physiological assessments of renal function. We aimed to develop a protocol that allows the safe preservation of donor kidneys for 12 hours using this technique.
Porcine kidneys (n=20) were retrieved and flushed with 1L 4°C Custodiol solution before being stored on ice for transport. Following a standardised cold ischaemic time of 3.5 hours, kidneys were placed onto a normothermic machine perfusion (NMP) circuit and perfused for 12 hours. Continual renal haemodynamics, biochemistry and urine output were recorded and analysed. At the end of perfusion, kidneys were scored based on the clinical assessment score and their suitability for transplant determined. Biopsies were collected at the end for histological assessment using the REMUZZI scoring system.
All kidneys were successfully reperfused with recordable renal blood flow (RBF) within 15 mins of perfusion. RBF continually improved over the course of the perfusions, peaking at 12 hours, and negatively correlated with intra-renal resistance. Perfusate sodium concentrations remained stable and within physiological parameters. Sodium bicarbonate increased over time with a corresponding decrease in lactate concentrations, demonstrating active renal gluconeogenesis. Urine production began in all kidneys upon connection to the perfusion circuit and was sustained, indicating a maintenance of functionality. Under the clinical perfusion assessment score, all kidneys received a score of 1 and would be considered suitable for transplantation. Histological assessment revealed all kidneys were injury free with a REMUZZI score of 0 for all categories.
We have developed an NMP protocol that can be used to safely preserve donor kidneys for 15.5 hours. In all kidneys, successful perfusion was achieved with stable haemodynamics, blood-perfusate biochemistry, and maintained urine output. Importantly, kidneys remained in optimal health, with no evidence of injury. This protocol may enable the electivisation of transplantation, while reducing ischaemic injury associated with static cold storage.
