Supercooling technique improves preservation of human tissue

Researchers at UC Berkeley were able to revive human heart tissue after it was kept in a frozen and supercooled state for 1-3 days.

By keeping heart tissue at constant volume in a rigid isochoric chamber, the researchers were able to prevent the formation of ice crystals that could have damaged microcells in the heart muscle. Researchers examined the structural integrity of heart cells and tested whether the tissue retained normal functions, such as autonomic beats and responsiveness to drugs and external electrical stimuli.

The results, described in an article published September 22 in the journal Communications Biology, are a key proof of concept for cryopreservation of supercooled tissue.

The method they used, called isochoric supercooling, was developed in the lab of Boris Rubinsky, professor at UC Berkeley in the Graduate School of the Department of Mechanical Engineering, emeritus professor of bioengineering and senior author of study.

“To our knowledge, this is the very first report on supercooling and rebirth of an autonomously designed human heart muscle,” said Matt Powell-Palm, co-lead author of the study, postdoctoral researcher at Rubinsky’s laboratory.

Rubinsky noted that these findings have short-term implications for the preservation and transport of organ-on-a-chip platforms, expanding access beyond the few select labs that can manufacture them for research and industry. . “Such platforms are valuable tools for the design of new drugs for diseases of the heart and other organs,” he said.

The results also suggest that isochoric supercooling may be a viable technique in the future for preserving donor tissues and organs, a significant challenge facing the transplant community and the medical research field, the authors said. study. Researchers cited estimates that seven in ten organs from thoracic donors are discarded each year due to the inability to store them long enough to reach patients in need. The viability of a donor heart, for example, is measured in hours, which significantly limits the number of potential recipients who could benefit from a life-saving transplant.

“Currently, Florida patients cannot receive a heart or lung from California because the organ would not survive the trip across the country,” said Powell-Palm.

Meeting these challenges is a key goal of the Research Center in Engineering of Advanced Technologies for the Preservation of Biological Systems (ATP-Bio), funded last year by the National Science Foundation. Kevin E. Healy, professor of bioengineering and materials science and engineering, is the head of ATP-Bio at UC Berkeley and another lead author of this study.

Giving no space for ice

The constant volume state that is characteristic of isochoric freezing requires that the sample be stored in liquid in a rigid, sealed, air-free container. Such conditions leave no space for cell-damaging ice formation, even at temperatures below freezing.

This differs from conventional isobaric freezing, in which the material is in contact with the atmosphere at constant pressure. The samples are frozen solid under atmospheric pressure, a process that also takes more energy.

Rubinsky noted that in previous studies they were able to cool samples down to minus 22 degrees Celsius while keeping 40% of the material unfrozen. “It’s fundamental thermodynamics. When the material to be frozen is confined in a rigid box, then only part of the volume freezes, ”he said.

Rubinsky, Powell-Palm and scientists from the United States Department of Agriculture recently showed that isochoric freezing can be applied to the food industry, leading to better food preservation and lower energy consumption compared to conventional methods. freezers.

The researchers hold a UC patent on isochoric supercooling, and Powell-Palm is the CEO of BioChoric Inc., a startup working on the clinical translation of this technique.

Comforting results

For this study, the researchers used heart tissue cultured from adult stem cells, a heart-on-a-chip system developed in Healy’s lab in 2015. Heart tissue beats at a rate comparable to that of a human heart, and the microfluidic system channels mimic how cells are exposed to nutrients and drugs.

“It’s not enough to say that these biological samples survived supercooling,” Healy said. “We wanted to demonstrate that physiological and metabolic function remained largely intact. “

The researchers immersed the heart-on-a-chip in a rigid chamber filled with a common organ preservation solution that had been cooled to minus 3 degrees Celsius. They removed the heart cells from the solution after durations of 24, 48 or 72 hours and brought them back to a warm temperature of 37 degrees Celsius.

Examination of the heart tissue confirmed that isochoric supercooling did not alter the structural integrity of the heart tissue, nor did it significantly affect the beat rate or the beat waveform. The study noted that there was a slight upward trend in heart rate duration with longer periods of supercooling, but the physiological impact of this change was unclear.

They found that spontaneous beats resumed for 65% to 80% of human heart muscle chips that had been supercooled for three days. In addition, they found no significant difference resulting from supercooled shelf life.

“We consider these percentages relatively high for conservation recovery results, especially given the inherent variability of these core-on-chip platforms and the fact that no cryoprotectants were used,” said Powell-Palm. .

The researchers also found that after emerging from cryopreservation, heart tissue remained sensitive to isoproterenol, a drug that causes an increased heart rate.

The researchers stressed that more work is needed to extend these findings to whole organs.

“The technology used to cool fabrics is strong and robust, but now we need to develop techniques to warm things consistently,” Healy said. “It was easier with the mini heart muscles that we used for this study. Working on whole organs will take more work.

Reference: Powell-Palm MJ, Charwat V, Charrez B, Siemons B, Healy KE, Rubinsky B. Preservation and rebirth in isochoric supercooling of human cardiac microtissus. Common Biol. 2021; 4 (1): 1-7. do I: 10.1038 / s42003-021-02650-9

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