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In this work, synthetic gelation of the cytosol in living cells is used to induce reversible molecular stasis. Through the sequential lipofectamine\u2010mediated transfection of complementary poly(ethylene glycol) macromers into mammalian cells, intracellular crosslinking occurs through bio\u2010orthogonal strain\u2010promoted azide\u2013alkyne cycloaddition click reactions. This achieves efficient polymer uptake with minimal cell death (99% viable). Intracellular crosslinking decreases DNA replication and protein synthesis, and increases the quiescent population by 2.5\u2010fold. Real\u2010time tracking of single cells containing intracellular crosslinked polymers identifies increases in intermitotic time (15 h\u00a0vs 19 h) and decreases in motility (30 \u00b5m h<jats:sup>\u22121<\/jats:sup>\u00a0vs 15\u00a0\u00b5m h<jats:sup>\u22121<\/jats:sup>). The cytosol viscosity increases threefold after intracellular crosslinking and results in disordered cytoskeletal structure in addition to the disruption of cellular coordination in a scratch assay. By incorporating photodegradable nitrobenzyl moieties into the polymer backbone, the effects of intracellular crosslinking are reversed upon exposure to light, thereby restoring proliferation (80% phospho\u2010Rb+ cells), protein translation, and migration. Reversible intracellular crosslinking provides a novel method for dynamic manipulation of intracellular mechanics, altering essential processes that determine cellular function.<\/jats:p>","DOI":"10.1002\/adma.202202882","type":"journal-article","created":{"date-parts":[[2022,6,7]],"date-time":"2022-06-07T18:18:41Z","timestamp":1654625921000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":26,"title":["Intracellular Crowding by Bio\u2010Orthogonal Hydrogel Formation Induces Reversible Molecular Stasis"],"prefix":"10.1002","volume":"34","author":[{"given":"Laura J.","family":"Macdougall","sequence":"first","affiliation":[{"name":"Department of Chemical and Biological Engineering University of Colorado Boulder  Boulder CO 80303 USA"},{"name":"The BioFrontiers Institute University of Colorado Boulder  Boulder CO 80303 USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Timothy 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