Magnetic Resonance Microscopy and Spectroscopy Reveal Kinetics of Cryoprotectant Permeation in a Multicompartmental Biological System

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 93; no. 15; pp. 7454 - 7459
• Main Authors: Hagedorn, Mary, Hsu, Edward W., Pilatus, Ulrich, Wildt, David E., Rall, William F., Blackband, Stephen J.
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences of the United States of America 23-07-1996; National Acad Sciences; National Academy of Sciences
• Subjects: Animals; Biological sciences; Biology; Blastoderm; Blastoderm - cytology; Blastoderm - physiology; Cell Membrane - physiology; Cell Membrane - ultrastructure; Cell Membrane Permeability; Cryopreservation; Cryoprotective Agents; Danio rerio; Diffusion coefficient; Dimethyl Sulfoxide; Embryo, Nonmammalian - cytology; Embryo, Nonmammalian - physiology; Embryos; Fish; Freshwater; Imaging; Kinetics; Magnetic Resonance Spectroscopy - methods; Microscopy; NMR; Nuclear magnetic resonance; Propylene Glycol; Propylene Glycols; Spectroscopy; Term weighting; Time Factors; Yolk Sac - cytology; Yolk Sac - physiology; Zebrafish
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.93.15.7454

Successful cryopreservation of most multicompartmental biological systems has not been achieved. One prerequisite for success is quantitative information on cryoprotectant permeation into and amongst the compartments. This report describes direct measurements of cryoprotectant permeation into a multicompartmental system using chemical shift selective magnetic resonance (MR) microscopy and MR spectroscopy. We used the developing zebrafish embryo as a model for studying these complex systems because these embryos are composed of two membrane-limited compartments: (i) a large yolk (surrounded by the yolk syncytial layer) and (ii) differentiating blastoderm cells (each surrounded by a plasma membrane). MR images of the spatial distribution of three cryoprotectants (dimethyl sulfoxide, propylene glycol, and methanol) demonstrated that methanol permeated the entire embryo within 15 min. In contrast, the other cryoprotectants exhibited little or no permeation over 2.5 h. MR spectroscopy and microinjections of cryoprotectants into the yolk inferred that the yolk syncytial layer plays a critical role in limiting the permeation of some cryoprotectants throughout the embryo. This study demonstrates the power of MR technology combined with micromanipulation for elucidating key physiological factors in cryobiology.