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Here, we developed a perfusable organ-on-a-chip platform for the in situ formation and culture of HepG2 spheroids and compared rectangle-, rhombus-, and round-chamber designs under matched key channel dimensions. Two-dimensional (2D) simulations revealed geometry-associated differences in intragel velocity and shear-rate distributions. Experimentally, chamber design was associated with differences in spheroid morphology, qualified spheroid proportion, and viability, with the round chamber showing the best overall performance under standardized conditions. Rho-associated coiled-coil-containing protein kinase (ROCK) inhibition impaired early spheroid compaction without a significant geometry-dependent treatment effect, whereas Matrigel supplementation improved spheroid morphology, hepatic functional readouts, and viability. Under nominal APAP and TBHP exposure conditions, chip and ultra-low-attachment plate cultures exhibited distinct concentration- and time-dependent injury responses. Together, these findings identify chamber architecture and extracellular matrix (ECM) composition as important design variables in perfused spheroid culture and provide a comparative platform for studying liver spheroid development and drug-induced injury under perfusion.<\/jats:p>","DOI":"10.1021\/acsbiomaterials.6c01462","type":"journal-article","created":{"date-parts":[[2026,10,7]],"date-time":"2026-10-07T12:01:38Z","timestamp":1791374498000},"page":"6447-6459","source":"Crossref","is-referenced-by-count":0,"title":["Chamber-Design-Associated\n                    <i>I<\/i>\n                    <i>n Situ<\/i>\n                    Formation of HepG2 Spheroids in a Perfusable Organ-on-a-Chip"],"prefix":"10.1021","volume":"12","author":[{"ORCID":"https:\/\/orcid.org\/0009-0006-1545-2677","authenticated-orcid":true,"given":"Juan","family":"Zhang","sequence":"first","affiliation":[{"name":"Shanghai University , , ,","place":["Shanghai, China, 200444"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hongze","family":"Yin","sequence":"additional","affiliation":[{"name":"Shanghai University , , ,","place":["Shanghai, China, 200444"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Huiying","family":"Yang","sequence":"additional","affiliation":[{"name":"Shanghai University , , ,","place":["Shanghai, China, 200444"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yue","family":"Wang","sequence":"additional","affiliation":[{"name":"Shanghai University , , ,","place":["Shanghai, China, 200444"]},{"name":"Zhejiang University , , ,","place":["Hangzhou, China, 310027"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yichun","family":"Xu","sequence":"additional","affiliation":[{"name":"Shanghai Biochip Limited Corporation , , ,","place":["Shanghai, China, 201203"]},{"name":"Tongji Hospital of Tongji University , , ,","place":["Shanghai, China, 200065"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8321-1898","authenticated-orcid":true,"given":"Tao","family":"Yue","sequence":"additional","affiliation":[{"name":"Shanghai University , , ,","place":["Shanghai, China, 200444"]},{"name":"Shanghai University , , ,","place":["Shanghai, China, 200444"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2026,10,7]]},"reference":[{"issue":"8","key":"2026110907082314900_cit1","doi-asserted-by":"publisher","first-page":"545","DOI":"10.1038\/s44222-023-00063-3","article-title":"Human disease models in drug development","volume":"1","author":"Loewa","year":"2023","journal-title":"Nat. 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