X-Message-Number: 12592 Date: Mon, 18 Oct 1999 21:35:02 -0700 (PDT) From: Doug Skrecky <> Subject: a major breakthrough in the understanding of DMSO toxicity? Effects of Electrolyte Composition and pH on the Structure and Function of Smooth Muscle Cooled to -79 C in Unfrozen Media Cryobiology 9: 82-100 B.C. Elford and C.A. Walter Clinical Research Center, Harrow, Middlesex, HA1 3UJ and the National Institute for Medical Research, Mill Hill, London NW7, England Summary: The function and structure of isolated strips of taenia coli were assessed by isometric contractile responses to histamine and by electron microscopy, respectively, before and after the muscles were progressively cooled to -79 C in various solutions in which the concentration of the cryoprotective nonelectrolyte dimethyl sulfoxide was gradually increased to 60% (w/v) in order to prevent freezing. When the subzero incubation media had a composition based on that of Krebs solution, the muscles were slow to recover and were severely damaged both functionally and structurally. When potassium-rich media containing Na+, K+, and Cl- at concentrations similar to those normally present in the intracellular fluid were used, the muscles recovered more promptly after rewarming, but the eventual degree of functional recovery depended on the size of the anion that was used to partially replace chloride in the bathing medium. Contractibility of the muscles improved siginificantly when either glycerophosphate, TES, or PIPES (N-tris(hydroxymethyl)-methyl-2 amino or piperazine-N N'-bis-2-ethanesulfonates) were present in the bathing media, but not when the media contained either sulfate or ethylsulfate anions. The recovery of muscles cooled in K-rich PIPES-based media was dependant on the pH of the solutions both in the presence and absence of calcium, recovery being weaker as the pH was reduced. The contractibility and morphology of muscles cooled in the "standard" PIPES-buffered solution were comparible to those of muscle that had been incubated in Krebs solution for about 4 hr at +37 C. Additional note by poster: DMSO 60% (w/v) is a vitrifiable concentration of this cryoprotectant. Here DMSO toxicity was found not to be due to the cryoprotectant itself, but instead was caused by the nature of the buffer solution, which was mixed with the DMSO. IMHO, this report constitutes a major advance in the understanding of the DMSO toxicity, and seemingly virtually eliminates the only remaining significant barrier to vitrification of entire organs. Based on this assessment one could suppose that non-toxic vitrification solutions would soon be available. However there is one item I have neglected to mention thus far - the year of publication of the above report. The year was nineteen seventy-two. Rate This Message: http://www.cryonet.org/cgi-bin/rate.cgi?msg=12592