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Cryomacroscopy

Cryomicroscopy versus cryomacroscopy: At the cellular level, visualization of physical events such as crystallization, devitrification, recrystallization, and fracture formation is commonly done with the cryomicroscope. Consistent with efforts to develop cryopreservation techniques for large-size specimens and organs over the past decade, an urgent need to visualize physical events in large samples has arisen. For this purpose, cryomacroscopy technology was invented at the BTTL and kept evolving over the past decade. The uses of the cryomicroscope and the cryomacroscope are deemed complementary while their applications for the benefit of cryopreservation are conceptually different. In cryomicroscopy, representative micro-slices are exposed to conditions similar to those that would prevail in a large-scale specimen at selected points, such that a complete picture of the process can be piecewise-assembled. In cryomacroscopy, the large-scale specimen is analyzed as a whole in situ. Below is a summary of key cryomacroscopy prototypes, while selected publications are listed at the bottom of the page.

Cryomacroscope I has been developed to study vitrification processes in a sample contained in a 15 mL vial. In this prototype, a passive cooling mechanism was used by applying liquid nitrogen-immersed thermal-resistance sleeves of variable thicknesses. The history of events during the cryogenic protocol was recorded with a HyperHAD monochrome camera on VHS tapes with visualization capabilities aimed at the scale range of 50 μm to 2 cm. The thermal history of the same protocol was recorded with a thermocouple, and analysis of experiments necessitated simultaneous analysis of video tape and thermal history recordings. Results of Cryomacroscope I studies demonstrated for the first time that micro fractures in the glassy state may serve as nucleation sites for devitrification. Cryomacroscope I was demonstrated as a critical tool for the observations of rewarming-phase fracturing and rewarming-phase crystallization (RPC). Results of this study were further used to investigate fracture formation induced by the contraction of the container wall.

Advanced studies with Cryomacroscope I explored the reasonable boundaries of cryopreservation via vitrification on a rabbit carotid artery model, with the applications of the CPA cocktail VS55. These studies were focused on the correlation between crystallization, fracture formation, and functional recovery of blood vessel specimens. Results of this investigation demonstrated that the vessel’s mechanical function was preserved at marginal cooling rates to facilitate vitrification, with a high contractile response of about 80% relative to the fresh specimen. These results further indicated localized events of ice crystallization around the temperature sensor (thermocouple) and at the cannulated ends of the blood vessel at marginal cooling rates, which correlated well with post-thawing contractility results.

Representative Cryomacroscopy I images of blood vessel segments immersed in 1 ml VS55 at the bottom of a 15 ml vial (top view), where the blood-vessel centerline is represented by a dash line: (left) before cooling, (center) crystallized blood vessel in a vitrified domain due to subcritical cooling rates, (right) vitrified blood vessel in a vitrified domain as a result of supercritical cooling rates. More information, and also here

Cryomacroscope II has been developed to study solid-mechanics effects in thin films. In particular, Cryomacroscope II was designed to measure the strain to fracture (the relative elongation at the onset of fracturing), the repeatability of fracturing events, patterns of fracture formation, and the effects of tissue specimens on stress concentration in a large vitrified domain. The main differences between Cryomacroscopes I and II are in the specimen setup and cooling mechanism; while Cryomacroscope I was designed to mimic a common cryopreservation protocol in a vial, Cryomacroscope II was designed to investigate specific conditions relevant to solid mechanics modeling. The thin-film model was chosen as it simplifies the corresponding solid mechanics analyses, while taking advantage of measurable substrate-induced forces.

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Cryomacroscopy II snapshots of fracture formation in a thin film (1.5 mm thick and 20 mm in diameter) of (a) 7.05M DMSO containing six bovine muscle segments on a copper base, (b) 8.4M DMSO containing five bovine muscle segments on a copper base, (c) VS55 on a copper base, and (d) VS55 on a on a 0.15 mm glass; note the different fracturing patterns. More information

Cryomacroscope III has been designed to investigate physical events associated with vitrification in the presence of synthetic ice modulators (SIMs). The main improvement in Cryomacroscope III over Cryomacroscope I is the cooling mechanism: while Cryomacroscope I used a tailor-made passive cooling mechanism, Cryomacroscope III was designed to replace the lid of a commercially available top-loading controlled-rate cooler. Cryomacroscope III further benefits from an improved high-speed digital camera and illumination via fiber optics. Cryomacroscopy III results indicate improved suppression of crystallization with the application of SIMs and unexpected precipitation of solutes during rewarming. See also movies on blood vessels vitrification using SIMs.

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Cryomacroscopy III snapshots of a crystallization process during cooling in a 15 ml vial (top view): (a) a 1 ml of DP6/H2O, and (b-d) a 10 ml of DP6/UHK-CV, where solute precipitation is evident. More information

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Cryomacroscopy III demonstration of various fracturing patterns: (a) isolated fractures in a 1 ml DP6+1,4CHD sample containing a blood vessel segment at a storage temperature of -129°C (10°C below Tg of -119°C); (b) circumferential fractures in a 10 ml 1.25×DP6 sample at a storage temperature of -148.0°C; (c) fracturing in a 10 ml 1.25×DP6 sample during cooling when the center of the sample is about 9°C above Tg while the surface is below Tg; and (d) radial fractures in a 10 ml 1.25×DP6 sample at -113.9°C, during rewarming from a storage temperature of -146°C. Tf is the temperature at the center of the sample. More information

Cryomacroscope IV has been design for viewing specimens larger than the field of view of the camera and is also known as the scanning cryomacroscope. Both Cryomacroscope I and Cryomacroscope III were designed to visualize physical events with a stationary camera in a similar arrangement to the cryomicroscope setup, which created an unmet need for the study of larger size samples—larger than the field of view of the optical system. Similar to Cryomacroscope III, the new prototype is also design to be an add-on device to a commercially available controlled-rate cooler. The development of Cryomacroscope IV includes proprietary software to control its scanning operation and for post-processing of a single digital movie for the entire experimental investigation, with all relevant data overlaid. Demonstrated effects in this study included glass formation, various regimes of crystallization, thermal contraction, and fracture formation.

Polarized light has been further integrated into the scanning cryomacroscope, to reveal additional effects otherwise not observed with regular light illumination. The following effects have been demonstrated with the polarized light setup: display of contaminants otherwise unobservable with diffuse light, observation of ice nuclei, improved contrast in fractured sites, and visualization of mechanically strained vitrified material, where the strained material is coded with the visible light spectrum (i.e., photoelastic effects).

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Demonstration of polarized light effects otherwise not observed with regular light illumination, in a cuvette on an experimental stage of Crymoacroscope IV (side view): (a) contamination, (b) crystallization, (c) stress concentration (bright light), and (d) fractures.

In a recently study, the scanning cryomacroscope has been demonstrated instrumental in identifying crystallization events in the investigation of thermal conductivity of CPAs. Below are selected images from that study, where visualization is critical for the interpretation of thermal conductivity measurements.

Scanning cryomacroscope visualization during hotwire thermal conductivity measurements in a cuvette (side view): (a) a vitrified 7.05M DMSO sample at a temperature of -147°C; (b) a partially crystallized 6M DMSO sample at a temperature of -58°C; (c) a 2M DMSO sample undergoing crystallization in the form of dendrites at a temperature of -10°, and (d) a completely crystallized 6M DMSO solution at a temperature of -65°C. Source

Selected publications:

• Solanki, P.K., Rabin, Y. (2018): Analysis of polarized-light effects in glass-promoting solutions with applications to cryopreservation and organ banking, 13(6): e0199155, PubMed, HHS Public Access, Plos ONE

• Ehrlich, L.E., Malen, J.A., Rabin, Y. (2016): Thermal conductivity of the cryoprotective cocktail DP6 in cryogenic temperatures, in the presence and absence of synthetic ice modulators, Cryobiology, 73(2):196-202 PubMed, HHS Public Access, ScienceDirect

• Feig, J.S.G., Solanki, P.K., Eisenberg, D.P., Rabin, Y. (2016): Polarized light scanning cryomacroscopy, Part II: thermal modeling and analysis of experimental observations, Cryobiology, 73(2):272-281 PubMed, HHS Public Access, ScienceDirect

• Feig, J.S.G., Eisenberg, D.P., Rabin, Y. (2016): Polarized light scanning cryomacroscopy, Part I: Experimental apparatus and observations of vitrification, Crystallization, and Photoelasticity Effects, Cryobiology, 73(2):261-71 PubMed, HHS Public Access, ScienceDirect

• Feig, J.S.G., Rabin, Y. (2014): The scanning cryomacroscope with applications to cryopreservation – a device prototype, Cryogenics, 62:118–128 HHS Public Access, ScienceDirect

• Feig, J.S.G., Rabin, Y. (2013): Integration of polarized light into scanning cryomacroscopy. CRYO2013-the 50^th Annual Meeting of the Society for Cryobiology, N. Bethesda, DC (July 28-31), Cryobiology, 67(3):399-400 ScienceDirect

• Rabin, Y., Taylor, M.J., Feig, J.S.G., Baicu, S., Chen, Z. (2013): A new cryomacroscope device (Type III) for visualization of physical events in cryopreservation with applications to vitrification and synthetic ice modulators, Cryobiology 67(3):264-73 PubMed, HHS Public Access, ScienceDirect

• Rabin, Y., Feig, J.S.G., Williams, A.C., Lin, C.C., Thaokar, C. (2012): Cryomacroscopy in 3D: a device prototype for the study of cryopreservation. ASME 2012 Summer Bioengineering Conference - SBC 2012, Fajardo, Puerto Rico, USA (June 20-23) ASME Digital Collection, BTTL Depository

• Baicu, S., Taylor, M.J., Chen, Z., Rabin, Y. (2008): Cryopreservation of carotid artery segments via vitrification subject to marginal thermal conditions: Correlation of freezing visualization with functional recovery. Cryobiology, 57(1):1-8 PubMed, HHS Public Access, ScienceDirect, BTTL Depository

• Steif, P.S., Palastro, M.C, Rabin, Y. (2008): Continuum mechanics analysis of fracture progression in the vitrified cryoprotective agent DP6. ASME Biomechanical Engineering, 130(2):021006 PubMed, HHS Public Access, ASME Digital Collection

• Rabin, Y., Steif, P.S., Hess, K.C., Jimenez-Rios, J.L., Palastro, M.C. (2006): Fracture formation in vitrified thin films of cryoprotectants. Cryobiology, 53:75-95 PubMed, HHS Public Access, ScienceDirect, BTTL Depository

• Baicu, S., Taylor, M.J., Chen, Z., Rabin, Y. (2006): Vitrification of carotid artery segments: An integrated study of thermophysical events and functional recovery towards scale-up for clinical applications. Cell Preservation Technology, 4(4):236-244 PubMed, HHS Public Access, BTTL Depository

• Rabin, Y., Steif, P.S. (2006): Solid mechanics aspect of cryobiology, In: Advances in Biopreservation (Baust, J.G., and Baust J.M., Eds.), CRC Taylor & Francis, Chap. 13, pp. 359-382

• Rabin, Y., Taylor, M.J., Walsh, J.R., Baicu, S., Steif, P.S. (2005): Cryomacroscopy of vitrification, Part I: A prototype and experimental observations on the cocktails VS55 and DP6. Cell Preservation Technology, 3(3):169-183 PubMed, HHS Public Access, BTTL Depository

• Steif, P.S., Palastro, M., Wen, C.R., Baicu, S., Taylor, M.J., Rabin, Y. (2005): Cryomacroscopy of vitrification, Part II: Experimental observations and analysis of fracture formation in vitrified VS55 and DP6. Cell Preservation Technology, 3(3):184-200 PubMed, HHS Public Access, BTTL Depository

Acknowledgements:

This research has been supported, in part, by:

• National Heart Lung and Blood Institute (NHLBI) Grant R01HL127618

• National Institute of Biomedical Imaging and Bioengineering (NIBIB) Grant R21EB011751

• National Center for Research Resources (NCRR) Grant R21RR026210

• National Institute of General Medical Sciences (NIGMS) Grant R21GM103407

• National Heart Lung and Blood Institute (NHLBI) Grant R01HL127618

• US Army – Defense Health Program Contract H151-013-0162

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