{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,10,17]],"date-time":"2025-10-17T00:19:16Z","timestamp":1760660356731,"version":"build-2065373602"},"reference-count":25,"publisher":"MDPI AG","issue":"5","license":[{"start":{"date-parts":[[2025,10,16]],"date-time":"2025-10-16T00:00:00Z","timestamp":1760572800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"NIH\u2014NIGMS","award":["R01GM143938"],"award-info":[{"award-number":["R01GM143938"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["MPs"],"abstract":"<jats:p>Background: Extensive skin injuries from severe burns or chronic non-healing ulcers overwhelm the body\u2019s natural repair mechanisms, while current therapeutic approaches relying on autologous skin grafting are limited by donor site availability. Three-dimensional epithelial spheroid cultures enhance stem cell regenerative potential, but standardized comparative methodologies are lacking. Methods: We established a comprehensive framework comparing scaffold-free and scaffold-based epithelial spheroid systems using HaCaT keratinocytes. High-throughput approaches utilized BioFloat and ELPLASIA 96-well platforms, while low-throughput 6-well ULA plates generated heterogeneous populations (holospheres, merospheres, paraspheres). Scaffold-based studies embedded spheroids in Matrigel to evaluate outgrowth capacity. ROCK1 inhibitor treatment was assessed for stemness enhancement. Results: High-throughput systems generated uniform spheroids with high reproducibility and consistent circularity. Low-throughput cultures produced heterogeneous populations with distinct size distributions (holospheres: 408.7 \u03bcm2, merospheres: 99 \u03bcm2, paraspheres: 14.1 \u03bcm2). In Matrigel scaffolds, merospheres and paraspheres migrated outward, forming epithelial sheets, while holospheres remained intact as BMI-1+ stem cell reservoirs. ROCK1 inhibition enhanced holosphere formation, preserved stemness markers, and reduced premature differentiation. Conclusions: This standardized toolbox demonstrates scaffold-free systems optimize scalability for screening while scaffold-based approaches enable physiologically relevant regenerative studies. Integration of both methodologies provides flexibility matching experimental design to scientific objectives, accelerating translation to clinical applications.<\/jats:p>","DOI":"10.3390\/mps8050123","type":"journal-article","created":{"date-parts":[[2025,10,16]],"date-time":"2025-10-16T12:17:02Z","timestamp":1760617022000},"page":"123","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Standardized Methodology of Scaffold-Free and Scaffold-Based 3D Epithelial Spheroid Culture for Skin Regenerative Research"],"prefix":"10.3390","volume":"8","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-0049-2140","authenticated-orcid":false,"given":"Mariana B.","family":"Ramos-Pinto","sequence":"first","affiliation":[{"name":"Laboratory of Epithelial Biology, Department of Periodontics and Oral Medicine, School of Dentistry, University of Michigan, Ann Arbor, MI 48104, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Maria Leticia","family":"de Almeida Lan\u00e7a","sequence":"additional","affiliation":[{"name":"Laboratory of Epithelial Biology, Department of Periodontics and Oral Medicine, School of Dentistry, University of Michigan, Ann Arbor, MI 48104, USA"},{"name":"Department of Bioscience and Oral Diagnosis, Institute of Science and Technology, S\u00e3o Paulo State University\u2014UNESP, S\u00e3o Jos\u00e9 dos Campos 12245-000, SP, Brazil"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6782-3429","authenticated-orcid":false,"given":"Cristiane H.","family":"Squarize","sequence":"additional","affiliation":[{"name":"Laboratory of Epithelial Biology, Department of Periodontics and Oral Medicine, School of Dentistry, University of Michigan, Ann Arbor, MI 48104, USA"},{"name":"Rogel Cancer Center, University of Michigan, Ann Arbor, MI 48109, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5358-612X","authenticated-orcid":false,"given":"Rogerio M.","family":"Castilho","sequence":"additional","affiliation":[{"name":"Laboratory of Epithelial Biology, Department of Periodontics and Oral Medicine, School of Dentistry, University of Michigan, Ann Arbor, MI 48104, USA"},{"name":"Rogel Cancer Center, University of Michigan, Ann Arbor, MI 48109, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2025,10,16]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Centonze, G., Centonze, S., Ponzone, L., and Calautti, E. 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