A new system for restoring cell function and tissues in mammals after death could expand the availability of organs for transplantation. The research also opens up a previously unexplored field of research in the molecular and cellular mechanisms triggered after death, an area of potential significance, since it covers different biological processes with multiple applications.

The project was led by Nenad Sestan, professor of Neuroscience at Yale School of Medicine, who previously was responsible for a groundbreaking experiment in which the cellular activity of pig brains was restored 4 hours after death.

In a new study published on August 3, 2022, in Nature, Sestan reports improving this BrainEx technique and applying it to whole organisms.

By perfusing them with neuroprotective factors, nutrients, neuronal activity antagonists and several drugs, the researchers succeeded in restoring blood circulation and cellular functionality in pigs that had been anesthetized and then killed with an induced heart attack.

Sestan said the procedure, called OrganEx, was developed as a response to the brain restoration research. "When the first study came out, it created a lot of interest. We are neuroscientists, but many of our colleagues who are working in transplantation surgery started knocking on our doors and we started to collaborate."

Initially, the transplant specialists were interested in applying the brain restoration protocol to isolated organs. But bioethicists and scientists from other fields all felt the whole-organism approach was more relevant. "This way, we could test in one study the effect of this technology, that needed to be scaled up and adapted to multiple organs," Sestan told BioWorld Science.

Zvonimir Vrselja, coauthor of the study, who is associate research scientist at the Department of Neuroscience of Yale School of Medicine, said the decision to study the brain first was because of its high level of complexity, and also because it is the organ that is the most susceptible to ischemic injury. "We found out that certain cell functions can be restored in a dead brain. That was a key takeaway from that study. But it also got us thinking if it could be done in the brain, it should be done in all other organs as well," Vrselja said.

After scaling up and adapting the procedure for a whole-body experiment, "basically we found that things are not as previously presumed," Vrselja said. "The second most important thing [about] this study is that we have demonstrated that we can initiate cell repair on a molecular level. We were able now to show that we can persuade cells not to die."

Cells are highly sensitive to a lack of oxygen. With death or ischemia, intracellular acidosis and injury result in cell death. On a larger scale, a molecular response is triggered in the body, led by the release of cytokines and the activation of the nervous, immune and coagulation systems that end up damaging organs.

Cell recovery is possible from ex vivo perfusion of isolated whole organs, as demonstrated with the BrainEx procedure. OrganEx uses a cytoprotective acellular synthetic solution that is applied to the whole body for 6 hours after 1 hour of induced warm ischemia. A perfusion device simulates both heart and lung function, pushing a combination of blood and perfusate throughout the body. A second perfusion system is used for the rest of the organs.

The OrganEx procedure was shown to preserve tissue integrity, decrease cell death and restore molecular and cellular processes in the heart, brain, liver and kidneys. In these organs, hemorrhage or tissue swelling was reduced, compared with control animals that received conventional extracorporeal membrane oxygenation (ECMO) treatment.

Gene expression analysis revealed expression patterns that are hallmarks of cell repair.

In the case of both the BrainEx and OrganEx experiments, electrophysiological monitoring did not detect any kind of electrical activity. The bioethics committee required the OrganEx perfusion to be stopped after 6 hours, so it is not known how long the cellular revival could be maintained.

This technology is far from being ready for implementation in humans, with the next step being further studies to assess the functionality of treated pig organs.

Reviewing intensive care procedures

In a comment piece published in the same issue of Nature, Brendan Parent, assistant professor and director of transplant ethics and policy research at NYU Grossman School of Medicine, New York University, examines the implications of the OrganEx study for intensive care procedures.

ECMO is used to save the lives of patients whose hearts, lungs or both have stopped working. It delays cell death, and can also be used to preserve organs if the patient dies.

For organ donors, doctors can use normothermic regional perfusion (NRP) to preserve organs once a patient has died. Both NRP, which provides some cell recovery, and OrganEx, which improves cell architecture and cell repair in multiple organs, raise ethical questions around decisions on whether a patient could recover, or if organs can be donated.

OrganEx promises to have several advantages in comparison to ECMO. The key to this lies in its components, a cocktail of 13 drugs that reduce cellular stress, have a cytoprotective function, prevent cell death, improve metabolism, and modulate the nervous system, the immune system and coagulation.

"We have added several other things that together make it work," said David Andrijevic, co-author of the study, who is associate research scientist in the Department of Neuroscience, Yale School of Medicine. "It has like an artificial kidney system inside the circuit. The perfusate is supplemented with various sensors that can tell us in real-time the perfusion parameters, so we can intervene at the same time. Also, we have constructed a custom-made pulse generator that can mimic the pulsatility of the heart. At the end of the day, it is a combination of all these things that have made this work," Andrijevic told BioWorld Science.

"If you think about a surgical operation, you just do not use one tool to achieve your goal. So, it is like multiple tools that are combined to get to this point," Vrselja added.

This highly provocative work poses more questions than it answers, as good research often does, said James Markmann, chief of the Division of Transplant Surgery and Director of Clinical Operations at the Transplant Center at Massachusetts General Hospital. "More work is needed to determine if this approach can yield additional organs for life-saving transplants," he told BioWorld Science.

Markmann, who was not involved in Sestan's study, is co-author of an article, published in the January 5, 2022, issue of JAMA Surgery, which is cited by Sestan. In it, Markmann reports measuring the impact of portable normothermic blood-based machine perfusion on liver transplant, restoring some cell viability after shorter ischemia from cardiac arrest in a clinical trial for liver transplantation. The normothermic machine perfusion led to a decrease in early liver graft injury or ischemic biliary complications and greater organ utilization.

Advancing in two directions

The next steps in cell restoration after death can go in two directions, both to increase the availability of organs for transplantation and to treat local ischemic damage. Sestan noted in the press conference held to discuss the OrganEx research that these are very expensive projects requiring the participation of multiple departments of the medical schools.