Since the early days of transplant medicine, the parallel practice of organ preservation branched off into a separate science and field of research. The primary goal of mechanical perfusion and oxygenation is to protect organs from the consequences of hypoxia for as long as possible until transplantation can occur.

Cooling is an obvious method to reduce metabolic demand. The ability to apply appropriate cold temperature ex vivo, in ways that could be readily reversed, has led to considerable research and development. Now, flush cooling and ice storage with synthetic preservation solutions have become the most widely used method and have allowed for cost-effective, multiple retrievals from a single donor. 

For years, static cold storage has dominated clinical preservation practices, but currently, there is growing interest in more dynamic strategies that implement perfusion. Although this method is by no means new, current interest is driven by changing donor demographics, new potential clinical applications, and the need to utilize all available organs for transplantation to meet rising demand.

Perfusion Based Organ Preservation Research

Organ preservation, using continuous mechanical perfusion with blood-based or bloodless perfusates has been an active field of research for years. Beyond simply extending its viability beyond 24 hours (the standard for cold-storage techniques), the research reveals other benefits that perfusion methods might offer.

One recent study out of the Department of Surgery and Transplantation, Swiss Hepato-Pancreato-Biliary (HPB) Center, University Hospital Zurich, Zurich, Switzerland reveals that integrated mechanical perfusion and oxygenation can preserve injured human livers for one week.

Study Design 

Given that organs can usually be delivered within 24 hours to the operating table, what are the advantages of preserving them for longer time frames? The authors of the Zurich study highlight there is a high reject rate due to damage or injury. However, if the body parts could be kept viable and nursed to recovery, they could potentially increase the pool of donors.

The study included the development of specialized perfusion and oxygenation equipment equipped with intravascular pressure and blood gas tension monitoring in addition to a dialysis unit for physiologic electrolyte balance and removal of metabolic waste products from the blood. 

An algorithm was used to automatically adjust the dialysate flow, controlling the concentration of red blood cells (hematocrit) based on continuous measurements. Automated insulin and glucagon administration was used to maintain physiological blood glucose levels (targeted range of 3.5–6.5 mmol l−1).

To simulate diaphragmatic liver movement in the ex vivo system, they were placed on a silicone mat. An inflatable balloon was positioned beneath the mat and connected to an air oscillator to induce movement that mimics diaphragm motion which in turn helps prevent pressure necrosis.

Mechanical perfusion: Study Results & Potential Clinical Applications

After seven-day perfusion, six of the human livers showed preserved function as indicated by bile production, coagulation factor synthesis, maintained cellular energy (ATP), and an intact structure.

As the authors state, with additional research, multiple potential benefits could be realized with extended organ perfusion technology, such as:

  • Repair of damaged or diseased organs to be transplanted later.
  • Graft immunogenicity modification to induce tolerance. 
  • Treatment of tumors with chemotherapeutic agents that are too toxic for systemic use.
  • Safe transport over long distances to improve global sharing.
  • Liver regeneration, divided into segments to regrow ex vivo. Such an approach could double or even triple availability. 
  • Partial grafts for auto-transplantation in patients with liver cancer, obviating the need for immune suppression.

Article References:

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

https://www.karger.com/Article/FullText/499610#ref19

An integrated perfusion machine preserves injured human livers for 1 week

https://www.nature.com/articles/s41587-019-0374-x

Kevin McCusker Ph.D., MSc, CCP, Chief Research Scientist Professor Serdar Gunaydin M.D, Ph.D.

Fundamentally, science is not a competition or a zero-sum game; it is neither a chess match nor a wrestling contest. It is an infinite-sum game from which we all benefit. The outcome of the scientific process is not up to the practitioners since reality is set by nature itself –no negotiating. Scientists aim to figure out what reality is by collecting as much evidence as we can. We argue with each other about possible interpretations when the evidence is limited, but we do so only as spectators of a show that we were never responsible for in the first place. The scientific process may remind us of Michelangelo. When asked how he produced beautiful sculptures from a featureless block of marble, he replied: “It is already there, I just have to chisel away the superfluous material.” Similarly, science uses evidence to “chisel away” the superfluous hypotheses which are ruled out by the data we acquire. When we focus our view this way, it is a work of art.

I wish to reintroduce ourselves, I am Kevin McCusker Ph.D., CCP, I have been a clinical perfusionist for 32 years, and an Assistant Professor of Surgery at New York Medical College in Valhalla, New York. My research partner for the past 25 years has been Professor Serdar Gunaydin M.D., Ph.D. who is a Cardio-Thoracic Surgeon in Ankara, Turkey. Over the past two decades, we have researched and published our findings in many areas, such as biocompatible coatings, leukocyte filtration, condensed cardiopulmonary bypass circuitry, membrane oxygenators, polymer coatings, cardioplegia solutions, fibrin sealants, and blood management techniques.

From time to time, we would like to highlight and share our recent research findings here on this webpage. This will allow us to have a forum to share our studies with everyone and also hopefully stimulate others to join us in any of our new laboratory and clinical studies.

Our latest study covers “Long Term Protective Effects of Single/Multi-dose Cardioplegic Solutions in Cell Culture Models”

Objective: Despite the popularity of single-dose cardioplegic techniques, the time window and targeted population for successful reperfusion remain unclear. We tested currently available techniques based on cell viability and integrity to demonstrate long-term cardioprotection and clarify whether these solutions were acting better on neonatal/adult endothelium or myocardium examining different cell lines.

Methods: 
Cell viability with MTT proliferation assay until 48 h and membrane integrity (LDH test) until 24 h were documented in a cell culture setting and microscopy on adult cells (HUVEC-human umbilical vein endothelium), neonatal (H9C2-cardiomyocytes) and myofibroblasts (L929). After 24-hour seeding, cells were incubated in control (Dulbecco’s Modified Eagle), St. Thomas, blood cardioplegia (4:1), Histidine-Tryptophan-Ketoglutarate (HTK) and Del Nido solutions at 34 °C for 2 h followed by an additional 6, 24, and 48 h in standard conditions (37 °C, 5% CO2). All wells were filled with related cardioplegic solutions (200 microliters). Experiments were repeated eight times.

Results: 
Cultured L929 (2×104 cells/ml), HUVEC (2×10cells/ml), and H9C2 (5×103 cells/ml) were seeded in 96-well plates for MTT analysis and in 24-well plates for Acridine-Orange (AO) and Propidium Iodide (PI) double staining microscopy. For LDH evaluation, 5×10cells/ml from each group were allowed to attach for 24 h, then exposed to cardioplegic solutions at 34°C for 2 hours, followed by 24 h incubation. When reporting viability results, levels better than control were accepted as significant since the control solution is a media for cell culture. For LDH, higher levels noted higher disintegration (Fig 1)(*: p<0.05 with respect to control).

Conclusions: 
Experimental works on cardioplegia aiming to assess cardioprotection use time-consuming and often expensive approaches that are unrealistic in clinical practice. We have focused on the direct cellular effects of different cardioplegia solutions and documented long-term effects that we believe are the most underestimated facts about cardioplegia topics in the literature.

Please feel free to reach out to Professor Gunaydin MD, Ph.D., and me, Kevin McCusker Ph.D., MSc., CCP, with any questions or ideas towards research projects.

Kevin McCusker Ph.D., MSc., CCP
Assistant Professor of Surgery
New York Medical College
Valhalla, New York 10575

Serdar Gunaydin M.D., Ph.D.
Professor of Surgery
University of Health Sciences
Ankara, Turkey