A Programmable Perfusion Platform with Temperature Monitoring Achieves Multiscale Cryopreservation.

The addition and removal of cryoprotective agents (CPAs) are critical steps in the cryopreservation of biological samples, and in most research settings, they are performed manually. During this process, the CPA formulation, the addition procedure, and the ambient temperature all have a significant impact on the sample. However, there is currently no standardized platform that can simultaneously control these variables and be applied to the cryopreservation of multiscale samples. To address these issues, we developed a portable and programmable CPA perfusion platform for the standardized processing of multiscale biological samples before and after cryopreservation. By combining cell encapsulation with a nylon mesh trapping method, the system enables integrated handling of samples with different sizes, ranging from cell suspensions (NK-92 cells, ∼10 μm) to single cells (mouse oocytes, ∼80 μm) and skin tissues (∼2 mm). In terms of the solution environment, program-controlled linear-gradient CPA perfusion precisely regulates cryoprotectant concentration changes, thereby reducing osmotic damage. In terms of the temperature environment, the device can be placed in a 4 °C refrigerator during processing, which helps reduce CPA-related cytotoxic damage. Experimental results demonstrate that this programmed perfusion significantly improves post-thaw recovery: the survival rates of mouse oocytes and NK-92 cells increased from 65.6% and 69.7% to 77.4% and 83.2%, respectively. Furthermore, histological analysis reveals that skin tissues processed by this platform exhibit superior structural integrity and reduced cryoinjury. In summary, this study provides a highly efficient and cost-effective solution for the standardized cryopreservation of multiscale biological samples.