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However, naturally sourced polysaccharides often show batch-to-batch variability, impacting reproducibility. Biomanufactured polysaccharides overcome this drawback by providing consistent control over cultivation and production conditions. Here, we produced and characterized exopolysaccharides (EPS) from Rhodotorula toruloides and incorporated it into hydrogels for proof-of-concept use in 3DCC. Shake flask cultivation on glucose, mannose, and xylose yielded 1.68, 1.44, and 0.48 g L\u20131 EPS, respectively, consisting of similar monosaccharide subunits, suggesting a shared biosynthetic pathway. Structural characterization revealed a branched architecture with multiple glycosidic linkage types, and high polydispersity, showing three molecular-weight fractions of 1.8, 30.0, and 1000.0 kDa. This polydisperse EPS was combined with polyethylene glycol diacrylate (PEGDA) to engineer hydrogels with a semi-interpenetrating polymer network (semi-IPN) for embedding cancer spheroids. Different EPS\/PEGDA formulations were evaluated for rheological properties, compressive modulus, swelling, and stability. Increasing EPS and PEGDA concentrations increased precursor viscosity. While PEGDA concentration governed swelling, EPS modulated mechanical properties. Among the formulations tested, 4%EPS\u20136%PEGDA exhibited the most suitable compressive modulus for 3DCC, with a complex shear modulus of 115.8 \u00b1 5.9 Pa after crosslinking and a compressive modulus of 3.1 \u00b1 0.6 kPa, resembling the biomechanical attributes of breast tissue. The selected hydrogel maintained single-cell viability comparable to the PEGDA-only control and enabled the embedding and three-day fluorescence imaging of multicellular spheroids. To our knowledge, this is the first report combining a bioprocess analysis of EPS-producing R. toruloides, comprehensive characterization of its EPS, and a proof-of-concept demonstration of this EPS in hydrogel engineering to encapsulate cancer spheroids. Our study suggests R. toruloides as a promising platform for biomanufactured polysaccharides and demonstrates the potential of its EPS in advanced biomaterials development, which could be valuable for tissue engineering and in vitro disease modeling applications in the future.<\/jats:p>","DOI":"10.1021\/acsami.6c02843","type":"journal-article","created":{"date-parts":[[2026,9,17]],"date-time":"2026-09-17T23:01:13Z","timestamp":1789686073000},"page":"51972-51991","source":"Crossref","is-referenced-by-count":0,"title":["Engineering Hydrogels with Polydisperse Yeast Exopolysaccharides and PEGDA for Embedding Cancer Spheroids"],"prefix":"10.1021","volume":"18","author":[{"given":"Henrique Sep\u00falveda Del Rio","family":"Hamacek","sequence":"first","affiliation":[{"name":"Tallinn University of Technology (TalTech) , , ,","place":["Tallinn, Estonia, 12618"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tobias","family":"Butelmann","sequence":"additional","affiliation":[{"name":"University of Freiburg , , ,","place":["Freiburg, Germany, 79104"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Katharina","family":"Ostertag","sequence":"additional","affiliation":[{"name":"Tallinn University of Technology (TalTech) , , ,","place":["Tallinn, Estonia, 12618"]},{"name":"BOKU University , , ,","place":["Vienna, Austria, 1190"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kerit-Lii","family":"Joasoon","sequence":"additional","affiliation":[{"name":"Tallinn University of Technology (TalTech) , , ,","place":["Tallinn, Estonia, 12618"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Oksana","family":"Tingajeva","sequence":"additional","affiliation":[{"name":"Tallinn University of Technology (TalTech) , , ,","place":["Tallinn, Estonia, 12618"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Piia","family":"J\u00f5ul","sequence":"additional","affiliation":[{"name":"Tallinn University of Technology (TalTech) , , ,","place":["Tallinn, Estonia, 12618"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Petri-Jaan","family":"Lahtvee","sequence":"additional","affiliation":[{"name":"Tallinn University of Technology (TalTech) , , ,","place":["Tallinn, Estonia, 12618"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5125-9678","authenticated-orcid":true,"given":"V. 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