X-Message-Number: 7521 From: Brian Wowk <> Date: Fri, 17 Jan 1997 01:33:02 -0600 Subject: Cryoprotectant Toxicity Tim Freeman <> writes on CryoNet: >>Moreover, the putative Visser agent, dimethylformamide >>(DMF) appears to be highly toxic to kidneys at 40% v/v concentration >>(which is well below the minimum 52% concentration needed to vitrify). >>While DMF has reduced toxicity at lower concentrations, such as 25%, >>the quantities of ice that form with such concentrations are fatal to >>kidneys. So claims of perfected kidney preservation appear >>particularly implausible. >How long does the toxicity take to happen? Minutes, at any temperature near 0'C-- too fast to load and unload such high concentrations even at sub-zero temperatures. In short, there does not appear to be any way to load and unload DMF as a monoagent in concentrations sufficient to provide robust cryoprotection of ice-sensitive organs and still be non-toxic. >I understand the people who know the answer to this may still be under >non-disclosure, so I will understand if there is no answer. The results to be presented below were obtained independent of any collaboration with Visser or other organizations, so we are not bound by non-disclosure at this time. Recently, a focused effort has been made by 21st Century Medicine and BioPreservation, Inc. to find new cryoprotective agents that are more permeant to the CNS, less toxic, and better glass forming agents than are currently available. Our familiarity with DMF springs from this effort. The first step in evaluating putative cryoprotectants is to determine to what extent they are able to inhibit ice formation in aqueous solutions and, of course, to determine if they are likely to be suitably nontoxic. This first phase of evaluation consists of looking at the physical chemistry, biochemistry and toxicology of the agent. If the agent is, for instance, insoluble in a pH range compatible with the target system, this would be a strong contraindication to proceeding further with evaluation. Thus, the very first step is to examine the literature and learn as much as possible about what is already known about the proposed agent. If an agent presents no obvious contraindications on first appraisal, the next step is to determine its phase diagram and its concentration needed to vitrify and glass transition temperature. If this profile is favorable, the next step is to evaluate the membrane toxicity and membrane cryoprotection of the agent in an appropriate model. In the 21st Century Medicine lab we use the human red blood cell as the "membrane test system" for determining if the candidate cryoprotectant should be further evaluated. RBCs are equilibrated with a range of concentrations of the test agent as suggested by the toxiciological profile, the phase diagram and other theoretical considerations. Varying incubation times and temperatures (determined by likely perfusion times which are in turn determined by the permeability constant of the agent) are then used to evaluate its membrane toxicity in the absence of freezing or vitrification. The next step is to subject RBCs loaded with concentrations of the agent deemed cryoprotective to cooling to and rewarming from Tg or below at a family of cooling rates determined again by complex considerations such as the toxicity of the agent, cooling rate required to achieve vitrification and warming rate to avoid devitrication, and so on. Once the effectiveness (or lack thereof) of the agent as a cryoprotectant in a simple and economical systems such as the human RBC is established, the next phase is to evaluate the agent toxicologically and cryobiologically in a relevant tissue slice model. This basic approach to screening new cryoprotectant agents is leading to advances in our understanding of the mechanisms of ice inhibition and in the identification of several new classes of cryoprotective molecules. DMF is a member of the alkylamide class of cryoprotectants (alkylated versions of amides such as formamide and acetamide). Alkylamides such as MF, DMF DMA, etc. are attractive in part because of the success that Greg Fahy has had with formamide and acetamide in his kidney vitrification solutions. Like formamide, they are highly penetrating molecules, but also better glass formers than pure amides. Unfortunately, better glass forming ability tends to correlate with toxicity, so the value of such tradeoffs must be determined experimentally. Below are the results of human RBC freezing experiments with several different cryoprotectants, including DMF. VS4 is the DMSO/formamide/PG solution used by Greg Fahy to vitrify kidneys (tested here in non-vitrifiable concentration). RC1 is one of a new proprietary class of CPAs being studied by 21st Century Medicine. RC1/DMF/PG is a 2:2:1 volume ratio mixture of RC1, DMF, and propylene glycol. Conc. Agent Cooling rate: 6'C/minute 2'C/minute 0.3'C/minute ------------------------------------------------------------------------- 25% v/v VS4 0% hemolysis 20% 0% 25% RC1 0% 0% 40% 25% RC1/DMF/PG 0% 15% 80% 25% DMF 20% >80% 100% RBCs were suspended in normal saline at +4'C, and the concentration of these highly penetrating cryoprotectants was ramped to 25% in a single step. Samples were cooled to approximately -130'C at the indicated rates, and then rewarmed to 0'C approximately 5x faster. Pre and post hematocrits (intact RBC densities) were measured to determine the percent of cells hemolyzed (obliterated) during freezing and thawing. DMF was disappointing because cell membranes apparently only survive exposure to this agent at very high cooling rates. Indeed, these results suggest that at the cooling rates typical of cryonics (0.3'C/minute) a 25% DMF solution will completely dissolve all cell membranes during freezing. Of these agents, only Greg Fahy's VS4 appears sufficiently non-toxic to cell membranes for use in cryonics. We are continuing to investigate RC1 and related compounds for use in cryonics for reasons that are perhaps most dramatically illustrated by the following results obtained at a cooling rate of 2'C per minute: Agent Concentration: 25% v/v 30% 35% ------------------------------------------------------------------------- VS4 20% hemolysis 15% 15% RC1 0% 0% 10% RC1/DMF/PG 15% 25% 20% DMF >80% >80% 55% RC1 is a highly effective cryoprotectant at low concentrations and moderate cooling rates (even better than VS4). It is a good glass former, and most importantly, perhaps the most potent inhibitor of ice crystal growth any of us have ever seen. RC1 (and its relatives) are shaping up to be valuable components in cryoprotectant mixtures now being developed for vitrification of cryonics patients. Of course, the fact remains that Olga Visser is the first person to ever recover an intact beating mammalian heart from liquid nitrogen. And the fact that she did so with what is in many ways a suboptimal cryoprotectant is all the more remarkable. We have not made any attempt to replicate the Visser experiment as of yet (our primary interest at this time being brains, not hearts), so we can only speculate on why her experiment works. Our data suggest that the success of her experiment is intimately related to the rapid cooling rate, and that slower cooling rates (particularly cryonics cooling rates) will not be successful. Even with rapid cooling, it is unlikely that even the tough muscle fibers of the heart can survive long-term after exposure to the quanities and size of ice crystals that form in non-toxic DMF solutions. Confirmation or refutation of these speculations will have to await further study or disclosures from Visser and collaborators. *************************************************************************** Brian Wowk CryoCare Foundation 1-800-TOP-CARE President Human Cryopreservation Services http://www.cryocare.org/cryocare/ Rate This Message: http://www.cryonet.org/cgi-bin/rate.cgi?msg=7521