X-Message-Number: 10840 Date: Thu, 26 Nov 1998 02:11:59 -0800 (PST) From: Doug Skrecky <> Subject: preserving tardigrades under pressure Nature 395: 853-854 October 28,1998 "Preserving Tardigrades Under Pressure" By Kunihiro Seki, Masato Toyoshima When an animal is exposed to high hydrostatic pressure, its membranes, proteins and DNA are damaged. At pressures of around 30 megapascals (MPa) proliferation and metabolism in micro-organisms stops; at 300 MPa, most bacteria and multicellular organisms die. But here we show that, in perfluorocarbon at pressures as high as 600 MPa, small terrestrial animals known as tardigrades can survive in a dehydrated state. Terrestrial tardigrades become immobile and shrink into a form known as the 'tun' state when the humidity decreases. In this state, they can survive extreme temperatures, as low as -253 C or as high as 151 C, as well as exposure to a vaccuum or to X-rays. We have now tested the ability of the tardigrades Macrobiotus occidentalis (order Eutardigrada) and Echiniscus japonicus (Heterotardigrada) to survive under extraordinarily high hydrostatic pressures. We sealed M. occidentalis tardigrades in a small plastic container (6 ml) placed inside a pressure capsule (R7K-3-10, Yamamoto Suiatu Kougyoucho) and compressed using water as the pressure medium. The outside temperature was 21 C and the water temperature inside the capsule was 25 C; stepped hydrostatic pressures were applied for 20 minutes at a time (100, 200, 300, 400, 500 and 600 MPa). Pressure was increased by 100 MPA per minute and then increased by 100 MPa per minute and then decreased at the same rate. After decompression, M occidentalis was removed from the capsule and examined under a light microscope, which revealed that all organisms died at pressures over 200 MPa. We then investigated whether tardigrades could acquire pressure resistance in the tun state (a process known as anhydrobiosis) by dehydrating them before applying pressure. M. occidentalis and E. japonicus were dehydrated on filter paper in Petri dishes for more than 24 hours, whem the relative humidity in the dishes dropped from 70-80% to 10-30%. To prevent the tardigrades from rehydrating during compression, we used an inert solvent, perfluorocarbon (C8F18 Fluorinate PC77, Sumitomo 3M), as the pressure medium instead of water. Tardigrades were then removed from the pressure capsule and soaked in water to rinse off the perfluorocarbon. One hour later, we confirmed that they had changed from the tun state to the active state. To test whether the perfluorocarbon increased the survival of tardigrades exposed to high hydrostatic pressure, we subjected tardigrades in perfluorocarbon, which were still in the active state, to the same hydrostatic pressure changes. All active-state tardigrades were dead at pressures above 200 MPa. We evaluated the data at 600 MPa for the group (n = 20) in the tun state in perfluorocarbon and in the active state in water and perfluorocarbon. The survival rate of M. occidentalis was 95% at 600 MPa, and there was a difference between active state and tun state animals. The survival rate of E. japonicus was 80%, as some animals had died and their fluid had leaked onto the filter paper, which we attributed to inadequate dehydration before the experiment. Tardigrades are composed of about 40,000 cells, which survive not only high speed compression under a hydrostatic pressure of 600 MPa (equivalent to six times the pressure of sea water at a depth of 10,000 meters), but also being maintained at this pressure, and high-speed decompression as well. With perfluorocarbon as the pressure medium, we have demonstrated the viability of tardigrades after keeping them in an anhydrobiotic state. This viability is influenced not just by pressure but by the absolute amount of water in the organism's body, enabling us to exploit its dehydration/water-absorption mechanism for preservation purposes. (the following article was previously posted on cryonet) New Scientist p 7 November 7, 1998 "Putting Life on Hold" Weird creatures have some cute lessons for organ tranplanters By Jon Copley Taking a tip from tiny animals that can live for more than a century, Japanese have invented a new technique for storing human organs for transplant. The team, which has successfully revived a rat's heart after 10 days in storage, says the work may lead to organ banks similar to blood banks. These would allow doctors to avoid the frantic dash to bring suitable organs straight from donors to the operating table. Organs can usually be stored for only 30 hours before they have to be used. For hearts and lungs, the time limit is even less - just 4 hours. The main problem with keeping organs in cold storage is that water damages cell membranes at low temperatures. Unfortunately, removing water from tissues usually causes at least as much damage. But Kunihiro Seki and his colleagues at Kanagawa University in Hiratsukashi, Japan, knew that animals called tardigrades can withstand extreme conditions by losing most of the water in their bodies (In Brief, 31 October, p 26). They can survive in this state for a century or more. When water was added to a dried-out moss kept in a museum for 120 years, tardigrades were later found crawling all over it. To achieve this feat, tardigrades use a sugar called trehalose to stabilise the structure of cell membranes. "This suggested that the physiological mechanism for preservation and resuscitation of tardigrades could be applied to preservation of mammalian organs," says Seki. To test this, the team flushed rat hearts with trehalose solution before packing them in silica gel to remove the water from their cells. The hearts were then immersed in perfluorocarbon, a biologically inert compound, and stored at 4 C in airtight jars. Ten days later, the team took the hearts out of the jars and resuscitated them. Within half an hour, they were beating again. Within half an hour, they were beating again. Measurements of their electrical activity suggested that the heart cells had survived intact. Seki believes that the trehalose and perfluorocarbon replace the water in the cells, preventing tissue damage. He plans to repeat the experiment with a complete autopsy to confirm that the tissues are preserved intact. The researchers also plan to demonstrate the procedure with other animal organs and prolong their storage for up to a year. Seki hopes that within a few years the technique could be used to preserve human organs. "The implications for transplant patients would be huge," comments Vanessa Morgan, who chairs the UK Transplant Co-Ordinators Association. "It could lead to planned, elective operations rather than emergency surgery." She adds that recipients could have a greater choice of donor organs, improving their chances of a good match. But she cautions that the quality of transplant organs must be maintained during long-term storage. "The condition of organs is crucial and I wouldn't want to compromise condition for time. Additional note by Doug Skrecky: Given the very poor permeation of disaccharides into tissue, I have some doubt that the trehalose flush had very much to do with preserving the rat hearts. Currently the only chemical preservation method which can prevent DNA and RNA from degrading quickly, is dehydration in ethanol. (Am J Clinical Pathology 96(1): p 144 July 1991) All attempts to preserve DNA and RNA in an aqueous environment have failed. Note that ethanol itself is not inert, and that it destroys cell membranes for example. What Kunihiro Seki may have discovered is the first fully reversible chemical preservation method - dehydration in inert perfluorocarbon. Rate This Message: http://www.cryonet.org/cgi-bin/rate.cgi?msg=10840