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In this study, we present HepG2 and LX-2 cocultured spheroids as a three-dimensional in vitro model of liver fibrosis and highlight the potential of hyperspectral Raman imaging as a label-free analytical approach for assessing fibrosis-associated responses. HepG2 cells and LX-2 cells served as hepatocytes and HSCs, respectively, within the coculture spheroids. Hyperspectral Raman imaging allowed for the acquisition of fingerprint spectra from individual cells, revealing distinct cytochrome-related band patterns that facilitated cell-type segmentation without labeling. To induce fibrosis, spheroids were treated with fibrosis activation factors: free fatty acids (FFAs) and transforming growth factor-\u03b2 (TGF-\u03b2). FFA administration induced steatotic features in the coculture spheroids, as demonstrated by a direct correlation between the increasing lipid band intensity in the Raman spectra and FFA concentrations. Concurrently, a reduced cytochrome-related band intensity was observed in these spheroids. Furthermore, LX-2 spheroids but not HepG2 spheroids exhibited signs of fibrosis after TGF-\u03b2 activation. Raman analysis revealed that the amide I and III bands reflected extracellular matrix, especially collagen accumulation, applied as fibrosis markers. Notably, in the 2:1 coculture spheroids, we observed enhanced fibrosis-related bands in the LX-2-enriched regions. The consistency between Raman imaging results and immunofluorescent images underscores the potential of hyperspectral Raman imaging as a sensitive and noninvasive method for assessing liver fibrosis.<\/jats:p>","DOI":"10.1021\/cbmi.5c00243","type":"journal-article","created":{"date-parts":[[2026,3,15]],"date-time":"2026-03-15T13:52:16Z","timestamp":1773582736000},"page":"1695-1705","source":"Crossref","is-referenced-by-count":1,"title":["Label-Free Hyperspectral\nRaman Imaging and Analysis\nof a 3D Spheroid Model for Liver Fibrosis Study"],"prefix":"10.1021","volume":"4","author":[{"given":"Gunganist","family":"Kongklad","sequence":"first","affiliation":[{"name":"The University of Osaka , , 2-1 Yamadaoka , , ,","place":["Suita, Osaka, Japan, 565-0871"]},{"name":"Chulabhorn Royal Academy , 906, Kamphaeng Phet 6 Rd, Talat Bang Khen , , ,","place":["Lak Si, Bangkok, Thailand, 10200"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2223-1881","authenticated-orcid":true,"given":"Menglu","family":"Li","sequence":"additional","affiliation":[{"name":"The University of Osaka , , 2-1 Yamadaoka , , ,","place":["Suita, Osaka, Japan, 565-0871"]},{"name":"Shenzhen Medical Academy of Research and Translation , , , ,","place":["Guangming, Shenzhen, China, 518107"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tomoko","family":"Yamashita","sequence":"additional","affiliation":[{"name":"The University of Osaka , , 2-1 Yamadaoka , , ,","place":["Suita, Osaka, Japan, 565-0871"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tomoaki","family":"Okumura","sequence":"additional","affiliation":[{"name":"The University of Osaka , , 2-1 Yamadaoka , , ,","place":["Suita, Osaka, Japan, 565-0871"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1139-7002","authenticated-orcid":true,"given":"Satoshi","family":"Fujita","sequence":"additional","affiliation":[{"name":"National Institute of Advanced Industrial Science and Technology (AIST) , , 2-1 Yamadaoka , , ,","place":["Suita, Osaka, Japan, 565-0871"]},{"name":"National Institute of Advanced Industrial Science and Technology (AIST) , , Central fifth, 1-1-1 Higashi , , ,","place":["Tsukuba, Ibaraki, Japan, 305-8565"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2284-375X","authenticated-orcid":true,"given":"Katsumasa","family":"Fujita","sequence":"additional","affiliation":[{"name":"The University of Osaka , , 2-1 Yamadaoka , , ,","place":["Suita, Osaka, Japan, 565-0871"]},{"name":"National Institute of Advanced Industrial Science and Technology (AIST) , , 2-1 Yamadaoka , , ,","place":["Suita, Osaka, Japan, 565-0871"]},{"name":"The University of Osaka , , 2-1 Yamadaoka , , ,","place":["Suita, Osaka, Japan, 565-0871"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2026,3,15]]},"reference":[{"key":"2026083104100379600_cit1","doi-asserted-by":"publisher","first-page":"38","DOI":"10.1016\/S0168-8278(02)00429-4","article-title":"Liver fibrosis\n\u2013 from bench to bedside","volume":"38","author":"Friedman","year":"2003","journal-title":"J. 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