Measurement of Cryoprotective Solvent Penetration into Intact Organ Tissues Using High-Field NMR Microimaging

Isbell, Stephanie A.; Fyfe, C. A.; Ammons, Richard Lewis Martin; Pearson, Betty

doi:10.1006/cryo.1997.2037

Cited by 26 publications

(17 citation statements)

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“…There are a number of non-invasive techniques, which have been used to assess chemical loading as this is important for protecting cells and tissues from ice formation during freezing. The most promising techniques have focused on the use of non-invasive MR imaging 2,17 or NMR spectroscopy. 7,37 Of these techniques, the most recent work has focused on rapid acquisition times (30 s) in order to visualize the relatively fast permeation of 50% v/v Dimethyl Sulfoxide (DMSO) in tissue constructs.…”

Section: Introductionmentioning

confidence: 99%

Use of X-ray Tomography to Map Crystalline and Amorphous Phases in Frozen Biomaterials

et al. 2006

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The outcome of both cryopreservation and cryosurgical freezing applications is influenced by the concentration and type of the cryoprotective agent (CPA) or the cryodestructive agent (i.e., the chemical adjuvants referred to here as CDA) added prior to freezing. It also depends on the amount and type of crystalline, amorphous and/or eutectic phases formed during freezing which can differentially affect viability. This work describes the use of X-ray computer tomography (CT) for non-invasive, indirect determination of the phase, solute concentration and temperature within biomaterials (CPA, CDA loaded solutions and tissues) by X-ray attenuation before and after freezing. Specifically, this work focuses on establishing the feasibility of CT (100-420 kV acceleration voltage) to accurately measure the concentration of glycerol or salt as model CPA and CDAs in unfrozen solutions and tissues at 20 degrees C, or the phase in frozen solutions and tissue systems at -78.5 and -196 degrees C. The solutions are composed of water with physiological concentrations of NaCl (0.88% wt/wt) and DMEM (Dulbecco's Modified Eagle's Medium) with added glycerol (0-8 M). The tissue system is chosen as 3 mm thick porcine liver slices as well as 2 cm diameter cores which were either imaged fresh (3-4 h cold ischemia) or after loading with DMEM based glycerol solutions (0-8 M) for times ranging from hours to 7 days at 4 degrees C. The X-ray attenuation is reported in Hounsfield units (HU), a clinical measurement which normalizes X-ray attenuation values by the difference between those of water and air. NaCl solutions from 0 to 23.3% wt/wt (i.e. water to eutectic concentration) were found to linearly correspond to HU in a range from 0 to 155. At -196 degrees C the variation was from -80 to 95 HU while at -78.5 degrees C all readings were roughly 10 HU lower. At 20 degrees C NaCl and DMEM solutions with 0-8 M glycerol loading show a linear variation from 0 to 145 HU. After freezing to -78.5 degrees C the variation of the NaCl and DMEM solutions is more than twice as large between -90 and +190 HU and was distinctly non-linear above 6 M. After freezing to -196 degrees C the variation of the NaCl and DMEM solutions increased even further to -80 to +225 HU and was distinctly non-linear above 4 M, which after modeling the phase change and crystallization process is shown to correlate with an amorphous phase. In all tissue systems the HU readings were similar to solutions but higher by roughly 30 HU, as well as showing some deviations at 0 M after storage, probably due to tissue swelling. The standard deviations in all measurements were roughly 5 HU or below in all samples. In addition, two practical examples for CT use were demonstrated including: (1) glycerol loading and freezing of tissue cores and, (2) a mock cryosurgical procedure. In the loading experiment CT was able to measure the permeation of the glycerol into the sample at 20 degrees C, as well as the evolution of distinct amorphous vs. crystalline phases after freezing to -196 degrees C....

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Section: Introductionmentioning

confidence: 99%

Use of X-ray Tomography to Map Crystalline and Amorphous Phases in Frozen Biomaterials

et al. 2006

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“…NMR has been applied as a more quantitative tool to study tissue permeation by cryoprotectants. 3,4,5,6 Fuller et al 3 assessed the extent of equilibration of Me 2 SO in the tissue after 10–15 minutes of perfusion with Me 2 SO and again after subsequent washout with Me 2 SO‐free medium by NMR spectroscopy. Zieger et al 4 used NMR to measure the diffusion kinetics of glycerol in skin at different temperatures.…”

Section: Introductionmentioning

confidence: 99%

Response Of A Liver Tissue Slab To An Hyperosmotic Sucrose Boundary Condition: Microscale Cellular And Vascular Level Effects^a

Bhowmick

Khamis

Bischof

1998

Annals of the New York Academy of Sciences

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Transport of a non-permeating CPA in liver tissue was studied by experimental and theoretical techniques. The system consisted of a 20 mm x 15 mm x 500 microns (thick) slab of liver tissue which was exposed to culture media and hyperosmotic sucrose (0.3 or 0.6 M) at the boundary. The volumetric changes of cell and vascular spaces within the tissue slab at 125 microns from one of the symmetric boundaries was studied by slam freezing followed by freeze substitution microscopy. The experimental data was then theoretically investigated using two models; one based on an effective diffusion coefficient for sucrose, and another which incorporated the convective flux of water out of the cells (and the tissue) while sucrose diffuses in. We estimate the effective diffusion of sucrose as 16-33% of the actual diffusivity of sucrose in bulk water. The role of convection of water out of the tissue is against the flow of sucrose and appears to be important in reducing the effective diffusivity of the sucrose. The role of vascular compliance, porosity and tortuosity are also discussed with respect to our results.

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“…The uptake of CPAs in animal and human tissues is enhanced with increasing temperature; for example in rat kidney cortex and rat liver [60], medaka fish embryos [61] and human oocytes [62]. For DMSO, enhanced uptake at higher temperatures may be due to the progressive destabilization of the phospholipid bilayers [63], which increases the risk of toxicity [64], greater oxidation of sulfhydryl groups [65] and, in the presence of glycerol, the formation of formaldehyde by non-enzymatic reactions [65].…”

Section: Discussionmentioning

confidence: 99%

Biophysical Characteristics of Successful Oilseed Embryo Cryoprotection and Cryopreservation Using Vacuum Infiltration Vitrification: An Innovation in Plant Cell Preservation

Jayanthi

Pritchard

2014

PLoS ONE

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Heterogeneity in morphology, physiology and cellular chemistry of plant tissues can compromise successful cryoprotection and cryopreservation. Cryoprotection is a function of exposure time × temperature × permeability for the chosen protectant and diffusion pathway length, as determined by specimen geometry, to provide sufficient dehydration whilst avoiding excessive chemical toxicity. We have developed an innovative method of vacuum infiltration vitrification (VIV) at 381 mm (15 in) Hg (50 kPa) that ensures the rapid (5 min), uniform permeation of Plant Vitrification Solution 2 (PVS2) cryoprotectant into plant embryos and their successful cryopreservation, as judged by regrowth in vitro. This method was validated on zygotic embryos/embryonic axes of three species (Carica papaya, Passiflora edulis and Laurus nobilis) up to 1.6 mg dry mass and 5.6 mm in length, with varying physiology (desiccation tolerances) and 80°C variation in lipid thermal profiles, i.e., visco-elasticity properties, as determined by differential scanning calorimetry. Comparisons between the melting features of cryoprotected embryos and embryo regrowth indicated an optimal internal PVS2 concentration of about 60% of full strength. The physiological vigour of surviving embryos was directly related to the proportion of survivors. Compared with conventional vitrification, VIV-cryopreservation offered a ∼ 10-fold reduction in PVS2 exposure times, higher embryo viability and regrowth and greater effectiveness at two pre-treatment temperatures (0°C and 25°C). VIV-cryopreservation may form the basis of a generic, high throughput technology for the ex situ conservation of plant genetic resources, aiding food security and protection of species from diverse habitats and at risk of extinction.

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Measurement of Cryoprotective Solvent Penetration into Intact Organ Tissues Using High-Field NMR Microimaging

Cited by 26 publications

References 10 publications

Use of X-ray Tomography to Map Crystalline and Amorphous Phases in Frozen Biomaterials

Use of X-ray Tomography to Map Crystalline and Amorphous Phases in Frozen Biomaterials

Response Of A Liver Tissue Slab To An Hyperosmotic Sucrose Boundary Condition: Microscale Cellular And Vascular Level Effects^a

Biophysical Characteristics of Successful Oilseed Embryo Cryoprotection and Cryopreservation Using Vacuum Infiltration Vitrification: An Innovation in Plant Cell Preservation

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Measurement of Cryoprotective Solvent Penetration into Intact Organ Tissues Using High-Field NMR Microimaging

Cited by 26 publications

References 10 publications

Use of X-ray Tomography to Map Crystalline and Amorphous Phases in Frozen Biomaterials

Use of X-ray Tomography to Map Crystalline and Amorphous Phases in Frozen Biomaterials

Response Of A Liver Tissue Slab To An Hyperosmotic Sucrose Boundary Condition: Microscale Cellular And Vascular Level Effectsa

Biophysical Characteristics of Successful Oilseed Embryo Cryoprotection and Cryopreservation Using Vacuum Infiltration Vitrification: An Innovation in Plant Cell Preservation

Contact Info

Product

Resources

About

Response Of A Liver Tissue Slab To An Hyperosmotic Sucrose Boundary Condition: Microscale Cellular And Vascular Level Effects^a