Artificial cells with Xenopus leavis eggs cell-free extracts

The large (1mm diameter), easily accessible eggs of the frog Xenopus laevis (100-1000 eggs at once per frog) offer the opportunity to reconstitute cell cycle events in-vitro by generating cell-free extracts which retain all the biochemical components that regulate cell cycle progression, as well as all the organelles and cytoskeletal networks. In summary, ovulation is induced in frogs by subcutaneous injection of chorionic gonadotropin. After about 16 hours, eggs are collected, inspected for quality, washed, and processed using several centrifugation steps to obtain the cytoplasmic cell-free extract. Extracts can either be arrested in a cell-cycle phase, or being cycling (i.e., oscillating between interphase and mitosis). Cycling extracts are obtained by activating the eggs with calcium ionophore, which mimics fertilization and activates the biochemical processes of the cell cycle. To mimic cellular behavior, experiments require cell-sized compartments with realistic shapes and boundaries. This is achieved in two distinct ways: (i) by encapsulating extracts in surfactant-stabilized droplets, termed artificial cells, formed by vortexing frog egg extracts with surfactants and oil; (ii) by generating droplets using droplets-microfluidics. Cell cycle event are observed and recorded using timelapse fluorescence microscopy.