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However, effective biocontainment, defined as the ability to retain cells within 3D-printed matrices during the application period, remains a key limitation for ELMs requiring physical confinement. While synthetic biology provides cell-specific biocontainment strategies, materials-based approaches can enable generic cell retention capabilities. Therefore, we applied a materials-based strategy that combines pre-printing formulation development and post-printing processing to increase the retention of yeast Saccharomyces cerevisiae in Pluronic F127-bisurethane methacrylate (F127-BUM) hydrogels. F127-BUM precursor solutions were formulated with the comonomers methacrylic acid (MAA) and\/or 2-(dimethylamino)ethyl methacrylate (DMAEMA), extrusion-printed, UV-cross-linked, and immersed in 70 vol % ethanol for 60 or 300 s before cultivation. Comonomer addition preserved dimensional fidelity, thermoresponsiveness, and shear-thinning behavior for 3D printing. After immobilization and cross-linking, DMAEMA and higher concentrations of MAA considerably reduced yeast viability, whereas a 70\u201380% viability was observed for 0.03 and 0.06 mol L\u20131 MAA. With 0.06 mol L\u20131 MAA and 300 s ethanol immersion, the yeast retention time reached 98 \u00b1 12 h, which was approximately 48% longer than that of the correspondingly treated F127-BUM-only control and represented about a 465% increase relative to the untreated F127-BUM-only control. After ethanol immersion, a peripheral growth-free zone developed around a metabolically active core, enhancing cell retention and contributing to delayed cell escape. The selected MAA-containing ELMs remained metabolically active and showed nearly 38% higher apparent glucose-to-ethanol conversion than treated F127-BUM-only controls. The suspension cultures fermented faster and achieved higher yield. Our method achieved enhanced biocontainment, with an operational window of about 4\u20135 days. In conclusion, we demonstrate that combining pre-printing and post-printing processes can enhance biocontainment for different ELM applications. We suggest the integration of this method with synthetic-biology strategies to further extend biocontainment duration for metabolically active living materials.<\/jats:p>","DOI":"10.1021\/acsabm.6c01394","type":"journal-article","created":{"date-parts":[[2026,10,20]],"date-time":"2026-10-20T05:01:17Z","timestamp":1792472477000},"source":"Crossref","is-referenced-by-count":0,"title":["Additive Manufacturing of Metabolically Active Living Materials with Enhanced Biocontainment"],"prefix":"10.1021","author":[{"given":"Henrique Sep\u00falveda Del Rio","family":"Hamacek","sequence":"first","affiliation":[{"name":"Tallinn University of Technology , , ,","place":["Tallinn, Estonia, 19086"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shannon","family":"Daily","sequence":"additional","affiliation":[{"name":"University of Washington , , , ,","place":["Seattle, Washington, United States, 98195-1700"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"LeAnn","family":"Le","sequence":"additional","affiliation":[{"name":"University of Washington , , , ,","place":["Seattle, Washington, United States, 98195-1700"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4274-9936","authenticated-orcid":true,"given":"Sadia","family":"Khalid","sequence":"additional","affiliation":[{"name":"Tallinn University of Technology , , ,","place":["Tallinn, Estonia, 19086"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Pirjo","family":"Spuul","sequence":"additional","affiliation":[{"name":"Tallinn University of Technology , , ,","place":["Tallinn, Estonia, 19086"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Petri-Jaan","family":"Lahtvee","sequence":"additional","affiliation":[{"name":"Tallinn University of Technology , , ,","place":["Tallinn, Estonia, 19086"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8060-8611","authenticated-orcid":true,"given":"Alshakim","family":"Nelson","sequence":"additional","affiliation":[{"name":"University of Washington , , , ,","place":["Seattle, Washington, United States, 98195-1700"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2277-9831","authenticated-orcid":true,"given":"Rahul","family":"Kumar","sequence":"additional","affiliation":[{"name":"Tallinn University of Technology , , ,","place":["Tallinn, Estonia, 19086"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2026,10,19]]},"reference":[{"issue":"5","key":"2026102001010964000_cit1","doi-asserted-by":"publisher","first-page":"920","DOI":"10.1038\/nprot.2016.041","article-title":"3D Printing of Versatile Reactionware for Chemical Synthesis","volume":"11","author":"Kitson","year":"2016","journal-title":"Nat. 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