Autophagy is a lysosomal degradation system that involves de novo autophagosome formation. A lot of factors are involved in autophagosome formation, including dozens of Atg proteins that form supramolecular complexes, membrane structures including vesicles and organelles, and even membraneless organelles. Because these diverse higher-order structural components cooperate to mediate de novo formation of autophagosomes, it is too complicated to be elaborated only by cell biological approaches. Recent trials to regenerate each step of this phenomenon in vitro have started to elaborate on the molecular mechanisms of such a complicated process by simplification. In this review article, we outline the in vitro reconstitution trials in autophagosome formation, mainly focusing on the reports in the past few years and discussing the molecular mechanisms of autophagosome formation by comparing in vitro and in vivo observations.

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