An enzyme-free alcohol-based organoid harvesting solution

Three-dimensional (3D) cell culture is a more physiologically relevant model for drug development than two-dimensional (2D) cell culture. A common method to culture cells in 3D consists in embedding cells in synthetic or animal-based matrices that provide structural support for cell growth. They partially mimic <i>in vivo</i> conditions and enable scalable culture. Here, we introduce an alcohol-based <b>S</b>olution for <b>H</b>arvesting <b>O</b>rganoids <b>E</b>fficiently, denoted <b>SHOE</b>. We tested its harvesting potential on 2 cell lines grown as spheroids and 2 patient-derived organoids. It enables rapid, high-yield cell recovery, at room temperature (RT), and bypasses prolonged cold incubation of standard protocols. It preserves 3D structure and growth in subsequent passages. Three-dimensional cell culture offers a more realistic biological model for drug testing than 2D culture. However, harvesting cells from 3D matrices is challenging and requires long incubation time with harvesting solutions, at cold temperatures. Here, we introduce a solution that bypasses these limitations. We describe a method to recover organoids from matrices used in 3D cell culture using a novel alcohol-based harvesting solution. The solution is added onto matrix-embedded cells, matrix domes are disrupted by pipetting after 1-minute incubation. Cells are recovered by centrifugation. Discloses a novel harvesting solution that enables harvesting of 3D models from common matrices within minutes, at room temperature.The solution preserves 3D structures and achieves high cell recovery yields.Proof-of-concept is demonstrated on 2 commercial cell lines and 2 patient-derived organoids. Discloses a novel harvesting solution that enables harvesting of 3D models from common matrices within minutes, at room temperature. The solution preserves 3D structures and achieves high cell recovery yields. Proof-of-concept is demonstrated on 2 commercial cell lines and 2 patient-derived organoids.