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.

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.

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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 cm 3 , 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.