Abstract
The process of cryopreservation subjects cells to gross changes in the composition of the solution that surrounds them, changes that cause the cells first to shrink and then to swell by an osmotic mechanism. Empirical methods have been developed that permit many cells to survive freezing and thawing, but the cornea, which is crucially dependent upon the function of its endothelial monolayer, has proved quite refractory. In this paper we explore the osmotic response of the corneal endothelium of the rabbit to solutions ranging in osmolality from 0.25 to 8.6 × isotonic. Boyle van't Hoff behavior was observed between 0.43 and 8.6 × isotonic, and there was an apparent nonosmotic volume of 33.6%. However, ultrastructural damage was observed at the limits of this range, and it appeared that the tolerated range was 0.64–4.4 × isotonic. We show the extent to which dimethyl sulfoxide (Me2SO) would be expected to moderate changes in volume during freezing and suggest that its initial concentration should be at least 2M to prevent excessive shrinkage. We also show that cell swelling during removal of Me2SO is especially likely to be hazardous.
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References
Lovelock, J. E. (1953),Biochim. Biophys. Acta 10, 414.
Mazur, P. (1977), Slow-freezing injury in mammalian cells, inThe Freezing of Mammalian Embryos (Elliott, K., and Whelan, J., eds.), Elsevier, Amsterdam, pp. 19–42.
Lovelock, J. E. (1953),Biochim. Biophys. Acta,11, 28.
Mazur, P., Leibo, S. P., Farrant, J., Chu, E. H. Y., Hanna, M. G., Jr., and Smith, L. H. (1970), Interactions of cooling rate, warming rate, and protective additive on the survival of frozen mammalian cells, inThe Frozen Cell. Ciba Symposium (Wolstenholme, G. E. W., and O'Connor, M., eds.), J. and A. Churchill, London, pp. 69–85.
Meryman, H. T. (1970), The exceeding of a minimum tolerable cell volume in hypertonic suspension as a cause of freezing injury, inThe Frozen Cell (Wolstenholme, G. E. W., and O'Connor, M., eds.), J. and A. Churchill, London, pp. 51–64.
Steponkus, P. L. (1984),Ann. Rev. Plant. Physiol. 35, 543.
Fong, L. P., Hunt, C. J., Taylor, M. J., and Pegg, D. E. (1986),Br. J. Ophthalmol. 70, 751.
Taylor, M. J. (1986),Cryobiology 23, 323.
Maurice, D. M. (1984), The cornea and sclera, inThe Eye. Vegetative Physiology and Biochemistry (Davson, H., ed.), Academic, New York, NY, Third Ed., vol. 1b.
Taylor, M. J., Hunt, C. J., and Sherrard, E. S. (1981), Assessment of corneal endothelial integrity by specular microscopy after cryopreservation, inThe Cornea in Health and Disease, Academic, and RSM, London, pp. 437–444.
Madden, P. W. and Easty, D. L. (1982),Cryobiology,19, 666.
Taylor, M. J., and Hunt, C. J. (1985),Curr. Eye Res. 4, 963.
Mazur, P., and Schneider, U. (1986),Cell Biophys.
Dick, D. A. T. (1966),Cell Water, Butterworths, Oxford.
Hazelwood, C. F. (1979), A view of the significance and understanding of the physical properties of cell-associated water, inCell-Associated Water, (Drost-Hansen, W., and Clegg, J., eds.), Academic, New York, NY, pp. 165–259.
Ling, G. N., and Ochsenfeld, M. M. (1973),Ann. NY Acad. Sci. 204, 325.
Garlid, K. D. (1979), Aqueous phase structure in cells and organelles, inCell Associated Water (Drost-Hansen, W., and Clegg, J., eds.), Academic, New York, NY, pp. 293–361.
Dick, D. A. T. (1959),Int. Rev. Cytol. 8, 387.
Armitage, W. J. (1986),J. Physiol. 374, 375.
McConaghey, P. D., and Maizels, M. (1961),J. Physiol. 155, 28.
Fong, L. P., Hunt, C. J., and Pegg, D. E. (1987),Current Eye Research Vol. 6, pp. 569.
Hayes, A. R. (1978),J. Clin. Pathol. 31, 696.
Luft, J. H. (1971),Anat. Rec. 171, 369.
Lee, R. M. K. W. (1984), A critical appraisal of the effects of fixation, dehydration, and embedding on cell volume, inScience of Biological Specimen Preparation (Revel, J. P., Barnard, T., and Haggis, G. H., eds.), SEM Inc., AMF O'Hare, IL, pp. 61–70.
Bone, Q., and Denton, E. J., (1971),J. Cell. Biol. 49, 571.
Pentilla, A., Kalimo, H., and Trump, B. F. (1974),J. Cell Biol. 63, 197.
Arborgh, B., Bell, P., Brunk, V., and Collins, V. P. (1976),J. Ultrastruct. Res. 56, 339.
Barnard, T. (1976),J. Ultrastruc. Res. 54, 478.
Lee, R. M. K. W., McKenzie, R., Kobayashi K., Garfield, R. E., Forrest, J. B., and Daniel, E. E. (1982),J. Microsc. 125, 77.
Mathieu, O., Claassen, H., and Weibel, E. R. (1978),J. Ultrastruc. Res. 63, 20.
Hillman, H., and Deutsch, K. (1978),J. Microsc. 114, 77.
Jensen, O. A., Prause, J. U., and Laursen, H. (1981),Albrecht Graefes. Arch. Klin. Ophthalmol. 215, 233.
Prickaerts, J. P., Wilson, G. J., Bayliss, C. E., and Baffour, R. (1984),J. Microsc. 135, 169.
Carstensen, E. L., Aldridge, W. G., Child, S. Z., Sullivan, P., and Brown, H. H. (1971),J. Cell Biol. 50, 529.
Wangensteen, D., Bachofen, H., and Weibel, E. R. (1981),J. Microsc. 124, 189.
Weibel, E. R., and Knight, B. W. (1964),J. Cell Biol. 21, 367.
Gusnard, D., and Kirschner, R. H. (1977),J. Microsc. 110, 51.
Boyde, A., and Boyde, S. (1980), Further studies of specimen volume changes during processing for SEM: Including some plant tissue, inScanning Electron Microscopy II, SEM, AMF O'Hare, IL, pp. 117–124.
Eins, S., and Wilhelms, E. (1976),Microscopie 24, 29.
Tooze, J. (1964),J. Cell Biol. 22, 551.
Elbers, P. F. (1966),Biochim. Biophys. Acta 112, 318.
Edelhauser, H. F., Hanneken, A. M., Pederson, H. J., and Van Horn, D. L. (1981),Arch. Ophthalmol. 99, 1281.
Armitage, W. J. (1986),Cryobiology 23, 566.
Pegg, D. E. (1984),Cryobiology 21, 234.
Pegg, D. E. (1983),Cryo Lett. 4, 259.
Pegg, D. E. (1986),Cryo Lett. 7, 387.
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Pegg, D.E., Hunt, C.J. & Fong, L.P. Osmotic properties of the rabbit corneal endothelium and their relevance to cryopreservation. Cell Biophysics 10, 169–189 (1987). https://doi.org/10.1007/BF02797398
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DOI: https://doi.org/10.1007/BF02797398