Controlled evaporation for microdroplet-based cryopreservation of cells and 3D cell aggregates

Microdroplet-based cryopreservation methods are effective for long-term biological storage but suffer from challenges such as inconsistent droplet sizes and tedious loading processes, leading to variable cooling rates, vitrified states and preservation outcomes due to the varying sizes and concentration of microdroplets. We hypothesize that a new controlled pressure evaporation-driven concentrated method can rapidly load the cryoprotectants solute through the interface of bio-samples from microdroplets aqueous environment, achieving more efficient migration and permeation behavior of the cryoprotectant and outcomes of the microdroplet-based cryopreservation.

This study presents an innovative loading method for microdroplet-based cryopreservation using controlled negative pressure evaporation on the film chips at low temperature range. By adjusting evaporation temperature, environmental pressure, and initial cryoprotectant concentration, we could control the liquid evaporation behavior and the concentration of microdroplets on the film chips, allowing for a gentle loading of cryoprotectant solute into cells and 3D cell aggregates and reducing osmotic stress and cellular toxicity. The evaporation time and concentration were precisely matched with the critical cooling rate required for quenching microdroplets inside the film chips for a direct quenching vitrification operation. This method was compared with traditional programmed cryopreservation techniques to assess its impact on post-thawing survival rates, cellular functionality, and 3D aggregate integrity.

Our results demonstrate that the loading method driven by controlled-pressure evaporation (@4 °C and -0.09 MPa for 4-6 min) significantly enhances the evaporation rate at the droplet interface, improves the efficiency of water molecule migration from the droplet interface and the penetration of cryoprotectants into the cell membrane, and reduces osmotic stress and cytotoxic damage during the loading process. The optimizing evaporation times and operation protocol of microdroplet-based cryopreservation have mitigated the risk of ice crystal formation and growth within the biological samples during the microdroplet quenching cooling and rewarming processes, leading to higher post-thawing survival rates and improved cellular functionality, and better 3D aggregate integrity compared to traditional programmed freezing methods. This innovative approach of synergistically regulating the evaporation at the droplet interface and the permeation at the cell membrane interface not only offers a scalable solution for the preservation of diverse cell and cell aggregate types, but also paves the way for microdroplet-based cryopreservation in the advancement of biobanking and cell therapy.