Cryoprotectant kinetic analysis of a human articular cartilage vitrification protocol

AbstractWe recently published a protocol to vitrify human articular cartilage and a method of cryoprotectant removal in preparation for transplantation. The current study’s goal was to perform a cryoprotectant kinetic analysis and theoretically shorten the procedure used to vitrify human articular cartilage. First, the loading of the cryoprotectants was modeled using Fick’s law of diffusion, and this information was used to predict the kinetics of cryoprotectant efflux after the cartilage sample had been warmed. We hypothesized that diffusion coefficients obtained from the permeation of individual cryoprotectants into porcine articular cartilage could be used to provide a reasonable prediction of the cryoprotectant loading and of the combined cryoprotectant efflux from vitrified human articular cartilage. We tested this hypothesis with experimental efflux measurements. Osteochondral dowels from three patients were vitrified, and after warming, the articular cartilage was immersed in 3 mL X-VIVO at 4 °C in two consecutive solutions, each for 24 h, with the solution osmolality recorded at various times. Measured equilibrium values agreed with theoretical values within a maximum of 15% for all three samples. The results showed that diffusion coefficients for individual cryoprotectants determined from experiments with 2-mm thick porcine cartilage can be used to approximate the rate of efflux of the combined cryoprotectants from vitrified human articular cartilage of similar thickness. Finally, Fick’s law of diffusion was used in a computational optimization to shorten the protocol with the constraint of maintaining the theoretical minimum cryoprotectant concentration needed to achieve vitrification. The learning provided by this study will enable future improvements in tissue vitrification

Surface tension models for binary aqueous solutions: a review and intercomparison

The liquid–air surface tension of aqueous solutions is a fundamental quantity in multi-phase thermodynamics and fluid dynamics and thus relevant in many scientific and engineering fields. Various models have been proposed for its quantitative description. This Perspective gives an overview of the most popular models and their ability to reproduce experimental data of ten binary aqueous solutions of electrolytes and organic molecules chosen to be representative of different solute types. In addition{,} we propose a new model which reproduces sigmoidal curve shapes (Sigmoid model) to empirically fit experimental surface tension data. The surface tension of weakly surface-active substances is well reproduced by all models. In contrast{,} only few models successfully model the surface tension of aqueous solutions with strongly surface-active substances. For substances with a solubility limit{,} usually no experimental data is available for the surface tension of supersaturated solutions and the pure liquid solute. We discuss ways in which these can be estimated and emphasize the need for further research. The newly developed Sigmoid model best reproduces the surface tension of all tested solutions and can be recommended as a model for a broad range of binary mixtures and over the entire concentration range.ISSN:1463-9084ISSN:1463-907

Homogeneous freezing of water droplets for different volumes and cooling rates

To understand the crystallization of aqueous solutions in the atmosphere, biological specimens, or pharmaceutical formulations, the rate at which ice nucleates from pure liquid water must be quantified. There is still an orders-of-magnitude spread in the homogeneous nucleation rate of water measured using different instruments, with the most important source of uncertainty being that of the measured temperature. Microfluidic platforms can generate hundreds to thousands of monodisperse water-in-oil droplets, unachievable by most other techniques. However, most microfluidic devices previously used to quantify homogeneous ice nucleation rates have reported high temperature uncertainties, between +/- 0.3 and +/- 0.7 K. We use the recently developed Microfluidic Ice Nuclei Counter Zurich (MINCZ) to observe the freezing of spherical water droplets with two diameters (75 and 100 mu m) at two cooling rates (1 and 0.1 K min(-1)). By varying both droplet volume and cooling rate, we were able to probe a temperature range of 236.5-239.3 K with an accuracy of +/- 0.2 K, providing reliable data where previously determined nucleation rates suffered from large uncertainties and inconsistencies, especially at temperatures above 238 K. From these data and from Monte Carlo simulations, we demonstrate the importance of obtaining a sufficiently large dataset so that underlying nucleation rates are not overestimated at higher temperatures. Finally, we obtain new parameters for a previous parameterisation by fitting to our newly measured nucleation rates, enabling its use in applications where ice formation needs to be predicted.ISSN:1463-9084ISSN:1463-907

The Microfluidic Ice Nuclei Counter Zurich (MINCZ): a platform for homogeneous and heterogeneous ice nucleation

Ice nucleation in the atmosphere is the precursor to important processes that determine cloud properties and lifetime. Computational models that are used to predict weather and project future climate changes require parameterizations of both homogeneous nucleation (i.e. in pure water) and heterogeneous nucleation (i.e. catalysed by ice-nucleating particles, INPs). Microfluidic systems have gained momentum as a tool for obtaining such parameterizations and gaining insight into the stochastic and deterministic contributions to ice nucleation. To overcome the shortcomings of polydimethylsiloxane (PDMS) microfluidic devices with regard to temperature uncertainty and droplet instability due to continuous water adsorption by PDMS, we have developed a new instrument: the Microfluidic Ice Nuclei Counter Zurich (MINCZ). In MINCZ, droplets with a diameter of 75 mu m are generated using a PDMS chip, and hundreds of these droplets are then stored in fluoropolymer tubing that is relatively impermeable to water and solvents. Droplets within the tubing are cooled in an ethanol bath. We validate MINCZ by measuring the homogeneous freezing temperatures of water droplets and the heterogeneous freezing temperatures of aqueous suspensions containing microcline, a common and effective INP in the atmosphere. We obtain results with a high accuracy of 0.2 K in measured droplet temperature. Pure water droplets with a diameter of 75 mu m freeze at a median temperature of 237.3 K with a standard deviation of 0.1 K. Additionally, we perform several freeze-thaw cycles. In the future, MINCZ will be used to investigate the freezing behaviour of INPs, motivated by a need for better-constrained parameterizations of ice nucleation in weather and climate models, wherein the presence or absence of ice influences cloud optical properties and precipitation formation.ISSN:1867-1381ISSN:1867-854