Investigation of Rapid Rewarming Chips for Cryopreservation by Joule Heating.

Rapid and uniform rewarming is critical to cryopreservation. Current rapid rewarming methods require complex physical field application devices (such as lasers or radio frequencies) and the addition of nanoparticles as heating media. These complex devices and nanoparticles limit the promotion of the rapid rewarming method and pose potential biosafety concerns. In this work, a joule heating-based rapid electric heating chip (EHC) was designed for cryopreservation. Uniform and rapid rewarming of biological samples in different volumes can be achieved through simple operations. EHC loaded with 0.28 mL of CPA solution can achieve a rewarming rate of 3.2 × 105 °C/min (2.8 mL with 2.3 × 103 °C/min), approximately 2 orders of magnitude greater than the rewarming rates observed with an equal capacity straw when combined with laser nanowarming or magnetic induction heating. In addition, the degree of supercooling can be significantly reduced without manual nucleation during the cooling of the EHC. Subsequently, the results of cryopreservation validation of cells and spheroids showed that the cell viability and spheroid structural integrity were significantly improved after cryopreservation. The viability of human lung adenocarcinoma (A549) cells postcryopreservation was 97.2%, which was significantly higher than 93% in the cryogenic vials (CV) group. Similar results were seen in human mesenchymal stem cells (MSCs), with 93.18% cell survival in the EHC group, significantly higher than 86.83% in the CV group, and cells in the EHC group were also significantly better than those in the CV group for further apoptosis and necrosis assays. This work provides an efficient rewarming protocol for the cryopreservation of biological samples, significantly improving the quantity and quality of cells and spheroids postcryopreservation.

A study on the relationship between the crystallization characteristics of quenched droplets and the effect of cell cryopreservation with Raman spectroscopy.

The cryopreservation method of microdroplets has steadily become widely employed in the cryopreservation of microscale biological samples such as various types of cells due to its fast cooling rate, significant reduction of the concentration of cryoprotectants, and practical liquid handling method. However, it is still necessary to consider the corresponding relationship between droplet size and concentration and the impact of crystallization during the cooling process on cell viability. The key may be a misunderstanding of the influencing factors of crystallization and vitrification behavior with concentration during cooling on the ultimate cell viability, which may be attributable to the inability to analyze the freezing state inside the microdroplets. Therefore, in this work, an in situ Raman observation system for droplet quenching was assembled to obtain Raman spectra in the frozen state, and the spectral characteristics of the crystallization and vitrification processes of microdroplets with varied concentrations and volumes were investigated. Furthermore, the degree of crystallization inside the droplets was quantitatively analyzed, and it was found that the ratio of the crystalline peak to hydrogen bond shoulder could clearly distinguish the degree of crystallization and the vitrified state, and the Raman crystallization characteristic parameters gradually increased with the decrease of concentrations. By obtaining the cooling curve and the overall cooling rate of quenching droplets, the vitrification state of the microdroplets was confirmed by theoretical analysis of the cooling characteristics of a DMSO solution system. In addition, the effect of cell cryopreservation was investigated using the microdroplet quenching device, and it was found that the key to cell survival during the quenching process of low-concentration microdroplets was dominated by the cooling rate and the internal crystallization degree, while the main influencing factor on high concentration was the toxic effect of a protective agent. In general, this work introduces a new nondestructive evaluation and analysis method for the cryopreservation of quenching microdroplets.

Structural design optimization and lipolytic effect prediction of vacuum suction cryolipolysis applicator: Simulation study.

BACKGROUND AND OBJECTIVES: Cryolipolysis is a popular noninvasive lipolytic method that uses low temperature to induce apoptosis or necrosis of adipocytes to reduce local fat in the human body. Vacuum suction applicator is a commonly used cryolipolysis equipment, which suction human skin and fat into a chamber for cooling. The structure of vacuum suction applicator is usually irregular, its cooling characteristic is also complex, and unreasonable suction structure will cause human discomfort. Biological experiments and clinical studies are often used to study the structural design of applicators, whereas these methods are impossible to obtain the three-dimensional cooling characteristic of applicator comprehensively and require a lot of costs. This study aims to optimize the structure of applicator for lowering discomfort, evaluate the cooling characteristic and lipolytic effect of applicators, which could provide guidance for clinical application of applicators and reduce costs. MATERIALS AND METHODS: Cryolipolysis applicators models with four vacuum suction angles were established, and COMSOL was used to compare the cooling performance parameters, cooling kinetics, and lipolytic effects of the applicators. Specific evaluation indicators also include: cooling capacity analysis, temperature field analysis, lipolytic percentage, lipolytic volume, lipolytic weight, lipolytic thickness, lipolytic waistline, and lipolysis temperature threshold analysis. RESULTS: The applicator with a small suction angle has a greater cooling capacity to cool deeper level of fat. When the cooling temperature is -10°C, the temperature of skin layer is about -10°C at 60 minutes, the temperature of fat layer is -7.36 to 3.01°C at 10 mm, -3.67 to 5.91°C at 20 mm and 2.01-10.81°C at 30 mm. The percentage of lipolytic declined with the increase of suction angle, the final lipolytic percentage (35.81%) of the 90° applicator is the highest, the percentage (28.72%) of 150° applicator (28.72%) is the lowest. The lipolytic volume, weight, and average thickness of applicator constantly increased with the increase of the suction angle, the final lipolytic volume range of the four suction angle applicators is 171.88-310.18 cm3 , the lipolytic weight range is 160.11-288.93 g, and the lipolytic average thickness range is 1.21-1.36 cm. Lower lipolysis temperature threshold will reduce the lipolysis effect, but it may also lead to another lipolysis mechanism-cell necrosis. CONCLUSION: Different suction angles significantly affect the cooling characteristics and lipolytic effects of cryolipolysis applicator. A reasonable suction angle is one of the critical factors to improve the efficiency and comfort of cryolipolysis.

New Cryoprotectant Loading Method for Cell Droplet Vitrification with Continuous Evaporation.

Droplet-based vitrification is considered to be a promising cryopreservation method, which achieves high cell viability through high cooling rates and low concentrations of cryoprotective agents (CPAs). However, the droplet vitrification cryopreservation process needs in-depth research, such as the balance of the CPA concentration and the cooling rate, the CPA loading process, and the droplet encapsulation method. Here, we developed a chip with a high cooling rate for vitrification droplet encapsulation and provided a new method for continuous loading of low-concentration CPA droplets by evaporation. The results showed that the CPA droplet volume decreased exponentially with the evaporation time, and the larger the initial droplet size, the longer the evaporation time to achieve the critical vitrification concentration. There was no significant difference in the viability of MSCs, NHEK, and A549 cells between the evaporation loading vitrification method and the traditional slow freezing method, but the former was easier to operate and can balance the cooling rate and concentration by controlling the evaporation time. Moreover, a theoretical model was proposed to predict the CPA concentration inside the microdroplets dependent on the evaporation time. The current work provides a potential method to load low-concentration CPAs for cell vitrification preservation, which is more beneficial for cell therapy and other regenerative medicine applications.

Quantitative assessment of intracellular/extracellular dimethyl sulfoxide concentrations during freezing with low-temperature confocal Raman micro-spectroscopy.

Although cryopreservation plays an indispensable role in the clinical application of cell therapy, the research on the osmotic behavior of cells during freezing is still at the level of theoretical models, and quantitative experimental data are still lacking. Therefore, the Raman spectra of dimethyl sulfoxide (DMSO) solutions with different standard concentrations (5%-80% v/v) were recorded experimentally to establish a quantitative evaluation method with the intensity ratio of different labeled peaks to the hydrogen bonding peak (as the internal standard) of water molecules in relation to different DMSO concentrations. By using this method, the characteristics of quantitative changes in intra- and extracellular concentrations under three different freezing methods were explored, including direct freezing, ice seeding freezing and vitrification. It was found that the intracellular concentration (@ -50 °C) after the ice seeding (@ -7 °C) freezing (1 °C min-1) method could reach 41.6%-49.2%, significantly higher than that of the direct freezing method (1 °C min-1 to -50 °C) of 32.4%-39.1%. Moreover, it is worth noting that the quantitative values of concentrations (@ -50 °C) of the ice seeding freezing are more consistent with the primary saturation curve of the DMSO solution. Thus, for the first time, it was revealed from the experimental data that the fundamental reason for the improvement of cell survival after ice seeding operation was pre-dehydration, higher concentration and smaller osmotic pressure difference between the inside and outside of the cell. These results also confirmed the validity of the famous two-factor hypothesis and more work will be carried out in depth.

Cryopreservation of organoids: Strategies, innovation, and future prospects.

Organoid technology has demonstrated unique advantages in multidisciplinary fields such as disease research, tumor drug sensitivity, clinical immunity, drug toxicology, and regenerative medicine. It will become the most promising research tool in translational research. However, the long preparation time of organoids and the lack of high-quality cryopreservation methods limit the further application of organoids. Although the high-quality cryopreservation of small-volume biological samples such as cells and embryos has been successfully achieved, the existing cryopreservation methods for organoids still face many bottlenecks. In recent years, with the development of materials science, cryobiology, and interdisciplinary research, many new materials and methods have been applied to cryopreservation. Several new cryopreservation methods have emerged, such as cryoprotectants (CPAs) of natural origin, ice-controlled biomaterials, and rapid rewarming methods. The introduction of these technologies has expanded the research scope of cryopreservation of organoids, provided new approaches and methods for cryopreservation of organoids, and is expected to break through the current technical bottleneck of cryopreservation of organoids. This paper reviews the progress of cryopreservation of organoids in recent years from three aspects: damage factors of cryopreservation of organoids, new protective agents and loading methods, and new technologies of cryopreservation and rewarming.

Droplet Generation, Vitrification, and Warming for Cell Cryopreservation: A Review.

Cryopreservation is currently a key step in translational medicine that could provide new ideas for clinical applications in reproductive medicine, regenerative medicine, and cell therapy. With the advantages of a low concentration of cryoprotectant, fast cooling rate, and easy operation, droplet-based printing for vitrification has received wide attention in the field of cryopreservation. This review summarizes the droplet generation, vitrification, and warming method. Droplet generation techniques such as inkjet printing, microvalve printing, and acoustic printing have been applied in the field of cryopreservation. Droplet vitrification includes direct contact with liquid nitrogen vitrification and droplet solid surface vitrification. The limitations of droplet vitrification (liquid nitrogen contamination, droplet evaporation, gas film inhibition of heat transfer, frosting) and solutions are discussed. Furthermore, a comparison of the external physical field warming method with the conventional water bath method revealed that better applications can be achieved in automated rapid warming of microdroplets. The combination of droplet vitrification technology and external physical field warming technology is expected to enable high-throughput and automated cryopreservation, which has a promising future in biomedicine and regenerative medicine.