Developing a High-Flow Acoustofluidic Loading Platform for Research Cell Stabilization in the Anhydrous State

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Project Summary Model cells for cancer research and drug discovery are currently stored using cryogenic methods that necessitate powerful freezers and liquid nitrogen. Frozen cells require regular upkeep, have high energy costs, take up a lot of space, and rapidly degrade if cooling devices fail. Efforts to develop a lyophilization protocol for model cell lines have been ongoing for decades, however, loading protective agents into nucleated cells has proven challenging. DesiCorp has developed a novel and innovative cell loading device, termed the high flow acoustofluidic loading (HFAL) that can deliver protective osmolytes, proteins, and nucleotides into eukaryotic cells. Unlike microfluidic loading techniques, HFAL uses large channel dimensions that allow substantially larger volumes of cell suspension to flow through it at once. The cells are mixed with cationic lipid-coated microbubbles that resonate with ultrasound waves, inducing cavitation. These cavitation-induced microjets physically inject compounds from the surrounding media into the cells in suspension, which then repair any membrane damage and seal membrane-impermeant compounds inside of the cell. This technology has previously been utilized to load human erythrocytes with protective compounds, allowing them to be lyophilized, stored at ambient temperatures, then rehydrated with over 90% cell recovery. A notable innovation of the HFAL platform is the ability to load cells at high rates, exceeding 500 billion erythrocytes per minute. However, the unique challenges of protecting organelles requires additional work to design and validate the HFAL platform for eukaryotic cell stabilization during lyophilization. The specific aims for this proposal are 1) to develop a complete, sterile acoustofluidic loading system that loads HepG2 and HEK 293 cells with over 60% cell recovery, which will be measured using trypan-blue exclusion; 2) to achieve compound loading into HepG2 and HEK293 that is sufficient for their stabilization in the desiccated state, which will be measured using enzymatic and colorimetric assays; and 3) to lyophilize HepG2 and HEK293 cells, store them at ambient temperature, and rehydrate them with high enough recovery for culturing and their organelle structure should resemble that of frozen and thawed model cells, which will be measured using fluorescence microscopy. The main criterion for success of this phase I proposal is the fabrication of a fully integrated acoustofluidic loading platform that successfully loads eukaryotic cells with protective agents that allow them to be lyophilized, stored, rehydrated, and cultured. Lyophilized cell stocks have several advantages over cryogenically preserved cells, such as the lack of ongoing maintenance and energy costs. Large storage devices, such as freezers or liquid nitrogen dewars, are unnecessary for lyophilized products because they are thermally stable. If this proposed work is successful, then cell stocks could be stored in drawers, binders, or even with the notebooks detailing their creation, modification, and data associated with grants or publications.