X-Message-Number: 4793 Date: Fri, 18 Aug 1995 18:14:24 +0200 (MET DST) From: Eugen Leitl <> Subject: basic Fahy papers abstracts Cryonics/cryobiology Fahy et al. paper abstract/conclusion digest (Thanks, C.P.!) - basic bibliography crossection in ASCII form. Excuse numerous typos. -- Eugene =-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-= [1] Cryoprotectant Toxicity and Cryoprotectant Toxicity Reduction: In Search of Molecular Mechanisms. (Gregory M. Fahy, Terence H. Lilley, Helen Linsdell, Mary St. John Douglas, Harold T. Meryman) CRYOBIOLOGY 27, 247-8 (1990). ABSTRACT Cryoprotectant toxicity is a fundamental obstacle to the full potential of artificial cryopreservation, yet it remains in general a poorly understood phenomenon. Unfortunately, most relevant biochemical studies to date have not met the basic criteria for demonstrating mechanisms for toxicity. A model biochemical study of cryoprotectant toxicity was that of Baxter and Lathe, which demonstrated that alteration of a specific enzyme (fructose diphosphatase, or FDPase) was the cause of impaired glycolysis after treatment with and removal of dimethyl sulfoxide (D). FDPase alteration by D was reported to be preventable by the simultaneous presence of amides. This protection could be due to a "counteracting solute" effect similiar to that employed by nature, but we find no meaningful correlation between the general protein stabilizing or destabilizing tendency of the cryoprotectant medium and its toxicity. Baxter and Lathe postulated that the effect of D arises from hydrogen bonding between D and the epsilon amino groups of surface lysine residues on FDPase, and it was found that molecule which resembled this group could block the alteration induced by D, presumably by competing with lysine residues for assotiation with D. However, we find that the interaction between D and and lysine in the presence of water is actually thermodynamically repulsive, and that the presence of formamide does not affect the interaction beteen D and lysine, implying no useful complex formation between formamide and D. We were also unable to demontrate that the locking compounds consistently reduce toxicity when added to D rather than substituting for D, contrary to predictions based on complex formation between blocking compounds and D. In summary, it seem that present concepts of cryoprotectant toxicity are in need of serious revision. CONCLUSION Despite the critical relevance of cryoprotectant toxicity to cryobiology, the mechanisms of toxicity pertinent to the different cryoprotectants remain elusive. The biochemical tools needing for determining mechanisms of toxicity as well as many suggestive leads are available, but as of 1989, fully 18 years after the first comprehensive study by Baxter and Lathe, almost nothing has been done. We hope that this review will encourage others to develop answers to the many open questions presented by the observations considered here and by the overall enigma of cryoprotectant toxicity. =-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-= [2] Vitrification as an Approach to Cryopreservation (G. M. Fahy, D. R. MacFarlane, C. A. Angell, H. T. Meryman) CRYOBIOLOGY 21, 407-4 (1984) ABSTRACT Recent developments have opened the possibility that the problems of freezing and thawing organs might eventually be overcome by an alternative approach to organ cryopreservation, namely, vitrification. Here we will review some of the principles of vitrification, describe the current state of the art, consider how a practical vitrification scheme might work, and conclude by noting how the principles of vitrification relate to and illuminate the principles and practices of freezing. CONCLUSION In conclusion, although many formidable problems remain to be solved or even addressed, vitrification is an intriguing possibility for indefinite preservation of complex biological systems in general. It has the advantage of presenting problems that are well-defined and limited in number. It also seems to us to be closer to fruition than is organ cryopreservation by freezing. But we would also like to emphasize that the pursuit of vitrification may lead to improved freezing techniques as well. For example, the problems of cryoprotectant toxicity we must face with this approach may also be at the heart of an explanation for "solution effects" injury in many systems. In fact, the use of cryoprotectant toxicity neutralization has already improved the freeze-thaw recovery of kidney tissue (17). The problem of introducing high concentrations of cryoprotectant must be faced in any event in organ freezing procedures in order to prevent mechanical damage from ice (57), so the problems of cryoprotectant toxicity are immediate and practical ones for both procedures. Additional connections between vitrification and freezing are described in Fig. 17. The T_h/T_g intersection point (Fig. 17A) should be relevant for defining the temperature from which a slowly frozen cell or a cell cooled by a step procedure can best survive a plunge into liquid nitrogen, subject, of course, to several secondary consideration. Using this guide, it has in fact recently been possible to document the first substantial recovery of kidney tissue frozen to liquid nitrogen temperature (17). It also appears that the principles of vitrification may, as indicated here, provide a deeper understanding of the optimal cooling rate, which, as suggested here (Fig. 17B), may be that cooling rate which comes closest to bringing the cell's temperature to the cell's T_g at an intracellular concentration just high enough to avoid homogenous nucleation. =-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-= [3] Vitrification as an Approach to Organ Cryopreservation: Past, Present, and Future. (G. M. Fahy), Cryopreservation and Low Temperature Biology in Blood Transfusion (C. Th. Smit Sibinga, P. C. Das, H. T. Meryman, Eds.) Kluwer Academic Publishers, Boston 1990: pp. 255-8. ABSTRACT The concept of preserving organs in a viable condition outside of the human body dates at least from 1812, when LeGallois first proposed that after death of the body the human head (and therefore the individual) could be kept alive by removing it and supporting it by normothermic artificial machine perfusion [1]. History, however, has gone instead in the direction of hypothermic organ preservation, which seems to be a better option for both biological and economic reasons. The subject of this paper still lies in the future, and that is "cryothermic" preservation, i.e., preservation of organs at temperatures below -100 deg C. The primary advantage of this approach, should it prove to be possible, is that preservation times should become indefinite at such temperatures, thereby opening up many new and significant opportunities in transplantation medicine. This paper reviews the many steps towards the goal of organ cryopreservation by vitrification which have been taken since the beginning of this field in 1980, and also provides a very brief sketch of the rather ironic history of this area of research. CONCLUSION Although vitrification and successful reimplantation of an animal kidney has yet to be achieved, success with single cells or small multicellular specimens has already been demonstrated and reports of success with increasingly complex tissue can be anticipated in the near future. There is a large variety of potentially transplantable tissues what awaits only improved techniques for preventing graft rejection to become clinically useful. Some of the most promising advances in transplantation immunology involve the induction of donor-specific tolerance through the transfusion of appropriately modified leukocytes from the donor. Vitrified cadaver allografts of the future may well be banked in conjunction with cryopreserved blood cells from the donor. The necessity for immunological preparation of the recipient over an interval of two or more weeks will mandate cryopreservation of the allografts. Although conventional cryoprotectant freezing may be an acceptable method for some tissues where a degree of injury can be tolerated, the inevitable mechanical damage from ice formation will place a restrictive limit on the use of freezing, particularly of highly vasularized tissues in which the vascular endothelium is particularly vulnerable to freezing injury. Vitrification avoids he problems assotiated with ice formation, cell dehydration and the control of freezing rates and thus should have many applications. Much of the challenge assotiated with vitrification now concerns the toxicity with of vitrifiable perfusion solutions, but this is a relatively new topic, as yet poorly understood and with much opportunity for developments of practical importance. No persuasive evidence has as yet been presented to contradict thhe expectation that the cryopreservation of tissues and organs is indeed possible and that we are on the right track. =-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-= [4] Physical Problems with the Vitrification of Large Biological Systems (Gregory M. Fahy, Joseph Saur, Robert J. Williams), CRYOBIOLOGY 27, 429-510 (1990). ABSTRACT Vitrification is an attractive potential pathway to the successful cryopreservation of mature mammalian organs, but modern cryobiological research on vitrification to date has been devoted mostly to experiments with solutions and with biological systems ranging in diameter from about 6 through about 100 um. The present paper focuses on concerns which are particularly relevant to large biological systems, i.e. those systems ranging in size from approximately 10 ml to approximately 1.5 liters. New qualitative data are provided on the effect of sample size on the probability of nucleation and the ultimate size of the resulting ice crystals as well as on the probability of fracture at or below T_g. Nucleation, crystal growth, and fracture depend on cooling velocity and the magnitude of thermal gradients in the sample, which in turn depend on the sample size, geometry and cooling technique (environmental thermal history and thermal uniformity). Quantitative data on thermal gradients, cooling rates, and fracture temperatures are provided as a function of sample size. The main conclusions are as follows. First, cooling rate (from about 0.2 to about 2.5 deg C/min) has a profound influence on the temperature-dependant processes of nucleation and crystal growth in 47-50% (w/w) solutions of propylene glycol. Second, fracturing depends strongly on cooling rate and thermal uniformity and can be postponed to about 25 deg C below T_g for a 482-ml sample if cooling is slow and uniform. Third, the presence of a carrier solution reduces the concentration of cryoprotectant needed for vitrification (C_v). Hoever, the C_v of samples larger than about 10 ml is significantly higher than the C_v of smaller samples whether a carrier solution is present or not. CONCLUSION In this paper we have provided preliminary results on the problems of vitrifying large specimens. The primary problems considered here are fracturing and crystallization during cooling. It seems likely that fracturing can be prevented by careful sample handling even for samples as large as a human kidney. Crystallization is a more serious problem, and much more must be learned about the magnitude of this problem in different circumstances. It is likely that crystallization can be reduced by the removal of heterogenous nucleating agents and the use of specific crystal-growth inhibitors, but it is not clear whether these growth inhibitors can lower C_v back to values which have acceptable toxicity or whether they can themselves induce damage (1, 9). However, rabbit kidneys are small enough to be cooled at rates compatible with vitrification at concentrations studied in the past, so the study of cryopreservation of this organ by vitrification can continue as the more formidable problems of vitrifying much larger organs are addressed. =-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-= .. to be continued... -- Eugene =-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-= Rate This Message: http://www.cryonet.org/cgi-bin/rate.cgi?msg=4793