Integrating Continuous Renal Replacement Therapy into Ex-situ Normothermic Liver Machine Perfusion

Summary

This protocol describes a method for integrating continuous renal replacement therapy (CRRT) into exsitu normothermic liver perfusion by connecting it to the perfusion reservoir. This configuration supports hemodynamic stability, helps prevent excessive intravascular pressures, and may reduce the risk of tissue edema, in particular during extended perfusion.

Abstract

Ex situ normothermic machine perfusion (NMP) of the liver has emerged as a dynamic preservation strategy, enabling the maintenance of metabolic activity and assessment of grafts prior to transplantation. Prolonged perfusions, however, are limited by the accumulation of metabolic waste, electrolyte imbalances, and inflammatory mediators that can compromise graft function. Continuous renal replacement therapies (CRRT), especially continuous venovenous hemodiafiltration (CVVHDF), offer a potential means to support solute clearance and homeostatic regulation during extended perfusion periods.

In this protocol, we describe a reproducible and safe method for integrating CVVHDF into an ex situ liver NMP system by connecting the filtration circuit independently from the main organ perfusion circuit. This configuration supports hemodynamic stability, helps prevent excessive intravascular pressures, and may reduce the risk of tissue edema. The physical connection, priming, and monitoring of the CRRT circuit are explained in detail, along with instructions on how to utilize a Hoffman clamp to simulate physiological venous pressure. When compared to direct in-line circuit integration, representative results suggest that the out-of-circuit approach is associated with reduced graft edema, while maintaining stable circuit pressure and solute clearance. This approach can be easily integrated into other similar perfusion devices in order to improve solute handling while maintaining stable perfusion dynamics.

Introduction

Ex situ normothermic machine perfusion (NMP) is a dynamic preservation strategy that aims to maintain the metabolic activity of the organ, providing oxygen and metabolic substrates at physiological temperatures^1,2,3. Under these conditions, it is possible to assess injury in and to some degree the functionality of organs prior to their transplantation. As well, ex situ NMP offers a potential platform for the reconditioning, treatment, and evaluation of organs, for both transplant and non-transplant purposes^3,

Protocol

Animal care and experimental protocols were approved by the institutional and regional ethics committees (Division of Agriculture, Livestock, and Food of the Community of Madrid and the Animal Welfare Body [OEBA] of the Autonomous University of Madrid). All animals were housed and procedures conducted at the Experimental Surgery Service of Hospital Universitario La Paz, in accordance with current national and European regulations, accredited by the International Standards Organization (ISO9001:2015) and authorized by the regional authority (ES280790001941). Seventeen livers were obtained from healthy porcine donors and subjected to a minimal-injury model.

Results

The aim of this protocol is to enable the integration of CRRT during ex situ liver NMP in a manner that preserves perfusion stability. CRRT operates under negative pressure and flow conditions that, when directly connected to a perfusion circuit within the system, can disrupt circuit hemodynamics (Figure 2A). To overcome this, the protocol introduces a configuration in which the CRRT circuit interfaces with the graft/perfusate reservoir, maintaining functional independence between t.......

Discussion

This protocol presents a detailed and reproducible method for integrating a CRRT device during ex situ liver NMP by connecting the filtration circuit to the perfusion reservoir. The key advantage of this approach is the preservation of perfusion circuit hemodynamics and prevention of the deleterious pressure alterations typically observed with direct in-circuit CRRT integration. Critical steps include the correct placement of CRRT lines within the reservoir to prevent air entrapment, the use of a Hoffman clamp t.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
20 mL syringe Luer slip	Nipro Medical Spain SL	SY3-10ESC-GEC
Accusol 35 K+ 2 (Potassium 2 mmol/L)	NIKKISO Europe GmbH	PPE02	Replacement fluid for CRRT
AqualineS Bloodline	NIKKISO Europe GmbH	AQUASET03LV	Pediatric bloodline set for CRRT
Aquamax Filter HF03	NIKKISO Europe GmbH	AQUASET03LV	Pediatric hemofilter (up to 30 kg donors)
AquariusTM System	NIKKISO Europe GmbH		Continuous renal replacement therapy machine
Ex situ liver perfusion system	Guangdong Devocean Medical Instrument Co., Ltd.	Devocean-Liver&Kidney 3000	Main organ perfusion platform composed by two centrifugal pumps with pulsatile/continual pressure perfusion, control panel (sensors for pressure, flow and temperature) and heater unit
Fresenius isotonic saline solution 500 mL	Fresenius Kabi	138900
Hoffman clamp	Guangdong Devocean Medical Instrument Co., Ltd.	Custom made	Regulation of return line pressure
Male Luer-lock connectors	Provided by the Hospital	N/A	For the connection between the perfusion system tubing and the bloodline of the CRRT machine
Sodium Heparin 25.000 UI	Laboratorios Farmacéuticos Rovi S.A	641639.6
Sterile tubing set	Guangdong Devocean Medical Instrument Co., Ltd.	Custom made	For reservoir access and return connections
Universal hose clamp	Achim SchuLz-Lauterbach Vertrieb medizinischer Producte GmbH	KL 20