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Efforts are currently directed towards improving the quality of vegetables after freezing and thawing. One of the methods under investigation is isochoric freezing. In this study, we evaluated isochoric freezing for preserving the quality of baby‐leaf spinach. We compared the properties of thawed spinach frozen to −4°C in an isochoric system with those of fresh spinach, thawed spinach frozen to −4°C in an isobaric system and thawed spinach that were commercially frozen. Spinach leaves frozen under isobaric conditions lost mass and thickness, making them softer and translucent. They also lost much of their nutrient content. In comparison, isochoric freezing maintained cell integrity and turgidity. Thawed leaves remained crunchy with characteristics similar to fresh leaves. Isochoric freezing also preserved nutritional content better than isobaric freezing, although significant nutrient losses still occurred.

Due to the perishable nature of fruits and the importance of reducing food waste, an effective preservation technique is required to prolong the shelf life and maintain the physical and nutritional properties of seasonal fruits. In this study, we evaluated isochoric freezing for preserving the quality of sweet cherries. We examined the physical characteristics and nutritional values of thawed cherries frozen to −4 °C or −7 °C in an isochoric system and compared them with those of fresh cherries, thawed cherries that were individually quick frozen and thawed cherries frozen to −4 °C or −7 °C in an isobaric system. We found that isochoric freezing decreased the drip loss and better preserved the color, texture, structure, ascorbic acid, phenolic and antioxidant content of frozen cherries, thereby proving their potential in frozen fruit applications.

A new technique is introduced which addresses the need for faster additive manufacturing methods for tissue scaffolds and frozen foods in large-scale industrial applications, inspired by print lithography. It is particularly relevant to biological matter, which is composed mostly of water. Instead of point-by-point printing in three dimensions (3D) with 3D printers, multiple single 2D layers can be assembled or printed separately, in parallel, on areas coated with hydrophilic materials to bind water-based compounds and hydrophobic materials to reject water-based compounds and bind hydrophobic molecules. This technique keeps the layers attached to the surface, opposing gravity, and thereby facilitating the transport and the assembly of the 2D layers, regardless of the direction of the surface relative to gravity. The individual layers are deposited one on top of the other and linked by chemical cross-linking and freezing to generate a 3D structure. Examples show how complex and large hydrogel-based structures can be manufactured by multi-layer cryolithography from fusion and freezing of 2D layers. Applications involve tissue engineering and food engineering, with particular emphasis on the ability to assemble a biological object, while every volume is frozen under optimal conditions during the assembly. Scanning electron microscopy demonstrates the ability to control and produce uniform microstructures in the 3D objects produced by cryolithography.

We have recently shown that, a living organism, which succumbs to freezing to −4 °C in an isobaric thermodynamic system (constant atmospheric pressure), can survive freezing to −4 °C in an isochoric thermodynamic system (constant volume). It is known that the mechanism of cell damage in an isobaric system is the freezing caused increase in extracellular osmolality, and, the consequent cell dehydration. An explanation for the observed survival during isochoric freezing is the thermodynamic modeling supported hypothesis that, in the isochoric frozen solution the extracellular osmolality is comparable to the cell intracellular osmolality. Therefore, cells in the isochoric frozen organism do not dehydrate, and the tissue maintains its morphological integrity. Comparing the histology of: a) fresh fish white muscle, b) fresh muscle frozen to −5 °C in an isobaric system and c) fresh muscle frozen to −5 °C I in an isochoric system, we find convincing evidence of the mechanism of cell dehydration during isobaric freezing. In contrast, the muscle tissue frozen to −5 °C in an isochoric system appears morphologically identical to fresh tissue, with no evidence of dehydration. This is the first experimental evidence in support of the hypothesis that in isochoric freezing there is no cellular dehydration and therefore the morphology of the frozen tissue remains intact.

Horizontal directional solidification techniques have been broadly utilized for the freezing of biological matter under conditions in which the freezing rate during solidification must be controlled and known. Directional solidification is used for diverse applications such as fundamental research on freezing of biological materials, cryopreservation of biological matter, and tissue engineering. This study is motivated by our intent to use directional solidification as a simplified model for the study of three-dimensional (3D) cryoprinting. In evaluating directional solidification in the context of 3D cryoprinting, we realized that current mathematical models of directional solidification are not adequately representative for this purpose, because they are simplified and one-dimensional (1D). Here, we introduce an experimentally verified and more representative two-dimensional (2D) mathematical model of directional solidification that can aid in the fundamental study of freezing of biological matter, in particular during 3D cryoprinting. The mathematical model was used to develop correlations between the freezing rates that a layer of an aqueous solution experiences during directional solidification and the various design parameters such as thickness of the sample and temperature gradients in the substrate. Results show that the freezing rates can be higher than those suggested by the previously used simplified 1D mathematical models. The results can be used for developing simplified models of 3D cryoprinting. In addition, the results suggest that many experimental studies on directional solidification of aqueous solutions and biological matter may require readjustment of analysis, in view of these findings.

While three-dimensional (3D) printing of biological matter is of increasing interest, current linear 3D printing processes lack the efficiency at scale required to mass manufacture products made of biological matter. This paper introduces a device for a newly developed parallel additive manufacturing technology for production of 3D objects, which addresses the need for faster, industrial scale additive manufacturing methods. The technology uses multilayer cryolithography (MLCL) to make biological products faster and in larger quantities by simultaneously printing two-dimensional (2D) layers in parallel and assembling the layers into a 3D structure at an assembly site, instead of sequentially and linearly assembling a 3D object from individual elements as in conventional 3D printing. The technique uses freezing to bind the 2D layers together into a 3D object. This paper describes the basic principles of MLCL and demonstrates the technology with a new device used to manufacture a very simple product that could be used for tissue engineering, as an example. An evaluation of the interlayer bonding shows that a continuous and coherent structure can be made from the assembly of distinct layers using MLCL.

The stability of solid–liquid interfaces during solidification is a physical phenomenon of fundamental interest with a wide range of practical applications, including the freezing of biological matter for medical and agricultural purposes. Much of the classical research in this field treats solidification in isobaric (constant-pressure) systems in which the phase transition typically occurs under constant atmospheric pressure. Recent research has found, however, that freezing in isochoric (constant-volume) systems in which the solidifying material is confined within a high-strength constant-volume chamber held at subfreezing temperatures gives rise to many atypical physical phenomena, and understanding of the solid–liquid interface behavior under isochoric conditions is currently lacking. In this work, we study the stability and propagation of the solid–liquid interface during isochoric freezing of aqueous solutions. Using a mathematical model of heat and mass transfer during solidification coupled with multiple criteria for predicting the emergence of interfacial instabilities based on temperature and concentration gradients in the phase transition region, we find that isochoric freezing significantly stabilizes the solid–liquid interface relative to isobaric freezing, suggesting the potential for extended growth of planar, non-dendritic interfaces.

Three-dimensional (3D) bioprinting is one of the fastest advancing and most promising techniques for tissue engineering. However, despite numerous developments in 3D bioprinting, issues of size scalability remain. Two primary factors limiting the scale of printable objects are the structural properties of the bioinks and the unsustainable time it takes to bioprint large structures. Freezing has been proposed as a possible solution to both, improving the mechanical properties of the soft aqueous hydrogel bioinks and reducing the adverse effects of cell metabolism with the lowered temperature. In addition, it eases the longer-term cryopreservation of the larger tissue constructs as they can be frozen element by element at faster (more preferable) and uniform cooling rates during printing rather than whole after 3D bioprinting. However, for freezing to be a feasible 3D bioprinting technique, there is a need to better understand the effect of phase change during printing on biological matter. This can be done by rigorous experimentation or alternatively, using mathematical tools and models. Though, most techniques of 3D printing are inherently thermal processes, limited thermal analysis has been done on extrusion-based (fused deposition modeling) printing processes to characterize thermal effects. This paper introduces a model for analysis of the thermal phase change process during the 3D bioprinting of biological matter at low temperature. The model was developed and experimentally validated for an extrusion-based printing process of aqueous biological material. It was used to investigate the conditions under which a constant freezing rate could be achieved during multilayer printing and the size limitations in conduction dependent freezing from a cold surface during printing. Overall, this model could be a useful tool for the design and control of the 3D printing protocols.

Background: Food preservation is essential to the growing world population, food economy. Freezing is a commonly used method for food preservation. While extending the life of the product, freezing has detrimental effects. It is causing loss of food weight and is causing changes in food quality, e.g. enzymatic browning. Method: Freezing of food is usually done under constant atmospheric pressure (isobaric). We have developed a new technology in which biological materials are preserved at subfreezing temperatures in an isochoric (constant volume) system. Experiments were performed with a food product, potato, in a thermodynamic isochoric device designed by us, that is robust and has no moving parts. Results: We have shown that under similar storage conditions, freezing to -5°C, the isochoric preserved potato experienced no weight loss and limited enzymatic browning. In contrast the -5°C isobaric frozen potato experienced substantial weight loss and substantial enzymatic browning. Microscopic analysis, shows that the mechanism responsible for the different results is related to the integrity of the cell and the cell membrane, which are maintain during freezing in the isochoric system and lost during freezing in the isobaric system. Discussion: The main mechanism of cell damage during isobaric freezing are the increase in extracellular osmolality and the mechanical damage by ice crystals. In contrast, during isochoric freezing the cells in the preserved material are under conditions in which the intracellular osmolality is comparable to the extracellular osmolality and they are not affected by ice mechanical damage. The conditions during isochoric freezing result in improved quality of the preserved food products. Conclusion: We have shown that the quality of food products preserved by isochoric freezing is better than the quality of food preserved to the same temperature in isobaric conditions. This is only a preliminary study on isochoric preservation of food. However, it illustrates the potential of the technology.

Author(s): Ukpai, Gideon; Nastase, Gabriel; Șerban, Alexandru; Rubinsky, Boris | Abstract: ObjectivePreservation of biological materials at subzero Centigrade temperatures, cryopreservation, is important for the field of tissue engineering and organ transplantation. Our group is studying the use of isochoric (constant volume) systems of aqueous solution for cryopreservation. Previous studies measured the pressure-temperature relations in aqueous isochoric systems in the temperature range from 0°C to - 20°C. The goal of this study is to expand the pressure-temperature measurement beyond the range reported in previous publications.Materials and methodsTo expand the pressure-temperature measurements beyond the previous range, we have developed a new isochoric device capable of withstanding liquid nitrogen temperatures and pressures of up to 413 MPa. The device is instrumented with a pressure transducer than can monitor and record the pressures in the isochoric chamber in real time. Measurements were made in a temperature range from - 5°C to liquid nitrogen temperatures for various solutions of pure water and Me2SO (a chemical additive used for protection of biological materials in a frozen state and for vitrification (glass formation) of biological matter). Undissolved gaseous are is carefully removed from the system.ResultsTemperature-pressure data from - 5°C to liquid nitrogen temperature for pure water and other solutions are presented in this study. Following are examples of some, temperature-pressure values, that were measured in an isochoric system containing pure water: (- 20°C, 187 MPa); (-25°C, 216 MPa); (- 30°C, 242.3 MPa); (-180°C, 124 MPa). The data is consistent with the literature, which reports that the pressure and temperature at the triple point, between ice I, ice III and water is, - 21.993°C and 209.9 MPa, respectively. It was surprising to find that the pressure in the isochoric system increases at temperatures below the triple point and remains high to liquid nitrogen temperatures. Measurements of pressure-temperature relations in solutions of pure water and Me2SO in different concentrations show that, for concentrations in which vitrification is predicted, no increase in pressure was measured during rapid cooling to liquid nitrogen temperatures. However, ice formation either during cooling or warming to and from liquid nitrogen temperatures produced an increase in pressure.ConclusionsThe data obtained in this study can be used to aid in the design of isochoric cryopreservation protocols. The results suggest that the pressure measurement is important in the design of "constant volume" systems and can provide a simple means to gain information on the occurrence of vitrification and devitrification during cryopreservation processes of aqueous solutions in an isochoric system.