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Raman spectroscopy has been gaining in popularity for noninvasive analysis of single cells. https://www.selleckchem.com/ and images deliver meaningful information regarding the biochemical, biophysical, and structural properties of cells in various states. Low-temperature Raman spectroscopy has been applied to verify the presence of ice inside a frozen cell and to illustrate the distribution of both penetrating and non-penetrating cryoprotectants. This chapter delineates Raman cryomicroscopic imaging of single cells as well as sample handling for spectroscopic measurements at subzero temperature. The experimental setup is depicted with a special emphasis on a custom-built temperature-controlled cooling stage. The use of Raman cryomicroscopic imaging is demonstrated using Jurkat cells cryopreserved in a sucrose solution. Moreover, strategies for determining intracellular ice formation (IIF) and analysis of sucrose partitioning across the cell membrane are presented.In this chapter, we describe how Fourier transform infrared spectroscopy (FTIR) can be applied in cryobiological research to study structure and thermal properties of biomolecules in cells and tissues, physical properties of cryopreservation and freeze-drying formulations, and permeation of molecules into cells and tissues. An infrared spectrum gives information about characteristic molecular vibrations of specific groups in molecules, whereas the temperature dependence of specific infrared bands may reveal information about conformational and phase changes. Infrared spectroscopy is minimally invasive and does not require labeling, whereas spectra can be recorded in any physical state of a sample. Data acquisition and spectral processing procedures are described to study phase state changes of protective formulations, cell membrane phase behavior during freezing and drying, protein denaturation during heating, and permeation of protective molecules into tissues. The latter can be used to estimate incubation times needed to load tissues with sufficient amounts of protective agents for cryopreservation or freeze-drying.Cryoprotectants are essential to prevent ice formation during tissue cryopreservation procedures. However, the control of their concentration and spatial distribution in the tissue is necessary to avoid toxicity and other damages associated with the cryopreservation procedures, especially for bulky samples such as tissues and organs. X-ray computed tomography measures the attenuation of an X-ray beam when it passes through a substance, depending on the material properties of the samples. The high electronic density of the sulfur atom of the dimethyl sulfoxide makes it an excellent cryoprotectant to be assessed by X-ray CT, and its concentration is proportional to the X-ray attenuation either at room or cryogenic temperatures. In addition, this imaging technique also allows to detect the formation of ice and eventual fractures within tissues during the cooling and warming processes. Therefore, X-ray CT technology is an excellent tool to assess and develop new cryopreservation procedures for tissues and organs.Quantification of the amount of cryoprotective agent (CPA) in a tissue is an essential step in the design of successful cryopreservation protocols. This chapter details two inexpensive methods to measure cryoprotective agent permeation into tissues as functions of time. One of the methods to measure the CPA permeation is to permeate a series of tissue samples from a surrounding solution at a specified concentration of CPA, each sample for a different amount of time, and then to quantitate the amount of CPA that was taken up in the tissue during that time period. The quantification is performed by equilibrating the permeated tissue with a surrounding solution and then measuring the osmolality of the solution to determine the amounts of CPAs that have come out of each tissue sample corresponding to each permeation time. An alternative method to measuring the CPA permeation as a function of time, which requires fewer tissue samples, is to measure the CPA efflux as a function of time. In the efflux method, a CPA-permeated tissue sample is placed in a surrounding solution, and solution samples are taken at different time points throughout the efflux to quantitate how much CPA has left the tissue by each time point.The development of freezing and freeze-drying processes for biological samples requires knowledge of the thermophysical properties of the biomaterial and protectant solutions involved. This chapter provides an introduction on the use of differential scanning calorimetry (DSC) to study thermophysical properties of biomaterials in protective solutions. It covers specific methods to study thermal events related to freezing and drying processes including crystallization, eutectic formation, glass transition, devitrification, recrystallization, melting, molecular relaxation, and phase separation.Ice recrystallization inhibition assays are used to screen for compounds that possess the ability to inhibit ice recrystallization. The most common of these assays are the splat cooling assay (SCA) and sucrose sandwich assay (SSA). These two assays possess similarities; however, they vary in their sample size, cooling rate, and the solution used to dissolve the analyte. In this chapter, both assay methods are described in detail, and we perform a direct comparison of the assays by evaluating the IRI activity of an antifreeze protein (AFP I). IRI activity is quantified by using ImageJ software to analyze ice crystals, and a quantitative value describing the efficiency of the inhibitor is generated. This analysis emphasizes the importance of choosing the right assay to measure IRI activity.Quantitative information about the kinetics and cumulative probability of intracellular ice formation is necessary to develop minimally damaging freezing procedures for the cryopreservation of cells and tissues. Conventional cryomicroscopic assays, which rely on indirect evidence of intracellular freezing (e.g., opacity changes in the cell cytoplasm), can yield significant errors in the estimated kinetics. In contrast, the formation and growth of intracellular ice crystals can be accurately detected using temporally resolved imaging methods (i.e., video recording at sub-millisecond resolution). Here, detailed methods for the setup and operation of a high-speed video cryomicroscope system are described, including protocols for imaging of intracellular ice crystallization events and stochastic analysis of the ice formation kinetics in a cell population. Recommendations are provided for temperature profile design, sample preparation, and configuration of the video acquisition parameters. Throughout this chapter, the protocols incorporate best practices that have been drawn from two decades of experience with high-speed video cryomicroscopy in our laboratory.Dry preservation has become an attractive approach for the long-term storage of biologics. #link# By removing water from the matrix to solidify the sample, refrigeration needs are reduced, and thus storage costs are minimized and shipping logistics greatly simplified. This chapter describes two energy deposition technologies, namely, microwave and laser systems, that have recently been used to enhance the rate and nature of solution densification for the purpose of anhydrous preservation of feline oocytes, sperm, and egg white lysozyme in trehalose glass. Several physical screening methodologies used to determine the suitability of an amorphous matrix for biopreservation are also introduced in this chapter.From early dry-ice-based freezers and passive coolers, cryopreservation devices have come a long way. With increasing interest in the field of cryobiology from new scientific applications, the importance of reliable, traceable, and reproducible cold chain devices is sure to increase, ensuring more precise cryopreservation and enabling better post-thaw outcomes, both for the user and for biological samples. As with any cryopreservation process, it is important to optimize each part of the cold chain for each lab's biological samples, cryocontainers used, and logistical restraints. In this chapter we describe how freezing technology can be used for cryopreservation of cells.Mass transfer of protectant chemicals is a fundamental aspect of cryopreservation and freeze-drying protocols. As such, mass transfer modeling is useful for design of preservation methods. Cell membrane transport modeling has been successfully used to guide design of preservation methods for isolated cells. For tissues, though, there are several mass transfer modeling challenges that arise from phenomena associated with cells being embedded in a tissue matrix. Both cells and the tissue matrix form a barrier to the free diffusion of water and protective chemicals. Notably, the extracellular space becomes important to model. link2 The response of cells embedded in the tissue is dependent on the state of the extracellular space which varies both spatially and temporally. Transport in the extracellular space can also lead to changes in tissue size. In this chapter, we describe various mass transfer models that can be used to describe transport phenomena occurring during loading of tissues with protective molecules for cryopreservation applications. Assumptions and simplifications that limit the applicability of each of these models are discussed.Cryobiology is a multiscale and interdisciplinary field. The scope and scale of interactions limit the gains that can be made by one theory or experiment alone. Because of this, modeling has played a critical role in both explaining cryobiological phenomena and predicting improved protocols. Modeling facilitates understanding of the biophysical and some of the biochemical mechanisms of damage during all phases of cryopreservation including CPA equilibration and cooling and warming. Moreover, as a tool for optimization of cryopreservation protocols, modeling has yielded many successes. Modern cryobiological modeling includes very detailed descriptions of the physical phenomena that occur during freezing, including ice growth kinetics and spatial gradients that define heat and mass transport models. Here we reduce the complexity and approach only a small but classic subset of these problems. Namely, here we describe the process of building and using a mathematical model of a cell in suspension where spatial homogeneity is assumed for all quantities. We define the models that describe the critical cell quantities used to describe optimal and suboptimal protocols and then give an overview of classical methods of how to determine optimal protocols using these models. We include practical considerations of modeling in cryobiology, including fitting transport models to cell volume data, performing optimization with cell volume constraints, and a look at expanding cost functions to cooling regimes.Freeze-drying is a complex process despite the relatively small number of steps involved, since the freezing, sublimation, desorption, and reconstitution processes all play a part in determining the success or otherwise of the final product qualities, and each stage can impose different stresses on a product. This is particularly the case with many fragile biological samples, which require great care in the selection of formulation additives such as protective agents and other stabilizers. link3 Despite this, the process is widely used, not least because once any such processing stresses can be overcome, the result is typically a significantly more stable product than was the case with the starting material. Indeed, lyophilization may be considered a gentler method than conventional air-drying methods, which tend to apply heat to the product rather than starting by removing heat as is the case here. Additionally, due to the high surface area to volume ratio, freeze-dried materials tend to be drier than their conventionally dried counterparts and also rehydrate more rapidly.