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Use of Freeze Substitution With Ultrarapid and Controlled-Rate Freezing to Determine Quality of Cryopreservation

Published online by Cambridge University Press:  02 July 2020

Cindy Hastings ,
Fred Lightfoot ,
Michael Taylor  and
Kelvin Brockbank
Cindy Hastings
Affiliation:
Central Arkansas Veterans Healthcare System, Little Rock, AR72205
Fred Lightfoot
Affiliation:
Organ Recovery Systems, Inc. 701 E. Bay Street, Charleston, SC29403
Michael Taylor
Affiliation:
Organ Recovery Systems, Inc. 701 E. Bay Street, Charleston, SC29403
Kelvin Brockbank
Affiliation:
Organ Recovery Systems, Inc. 701 E. Bay Street, Charleston, SC29403
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Extract

The field of cryobiology is expanding and incorporating conventional methods used in electron microscopy into protocols heretofore unexplored; two of those methods is that of ultrarapid freezing and freeze substitution. When tissues such as heart valves and/or vascular tissue is frozen for clinical applications it is imperative to know the quality of the freezing process.

Ultrarapid Freezing: Various methods of ultrarapid freezing have been employed routinely by those utilizing electron microscopy. In particular, questions involving immunocytochemistry and x-ray microanalysis may indeed require the use of one or more of these methods.. Metal mirror fixation (slamming) is the preferred method of cryopreservation for most subsequent protocols. It is also the preferred, and probably the only, method to check the accuracy of freeze substitution protocols. The metal mirror method utilizes a copper block, which is brought into contact with the tissue, at a predetermined speed, via a pneumatically operated system. The chilled metal block, devoid of scratches or imperfections, ensures maximal preservation of tissue to a depth of 15μm, which can serve as a control, for both fixation and the substitution process.

Type
Cryotechniques, Immunocytochemistry, and Electron Microscopy
Information
Microscopy and Microanalysis , Volume 5 , Issue S2: Proceedings: Microscopy & Microanalysis '99, Microscopy Society of America 57th Annual Meeting, Microbeam Analysis Society 33rd Annual Meeting, Portland, Oregon August 1-5, 1999 , August 1999 , pp. 448 - 449
Copyright
Copyright © Microscopy Society of America

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References

1.Feder, N. and Sidman, R., J. Biophysic and Biochem. Cytol., 1958, Vol.4, #5.CrossRefGoogle Scholar