{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,20]],"date-time":"2026-08-20T13:28:33Z","timestamp":1787232513629,"version":"build-2736575974"},"reference-count":91,"publisher":"MDPI AG","issue":"8","license":[{"start":{"date-parts":[[2026,8,20]],"date-time":"2026-08-20T00:00:00Z","timestamp":1787184000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Pharmaceutics"],"abstract":"<jats:p>Background: The clinical translation of cell-based therapies for knee articular cartilage repair is fundamentally restricted by the severe biological unpredictability of autologous cell sources, inherent manufacturing bottlenecks, and the rapid phenotypic dedifferentiation of cells expanded in conventional 2D monolayers. To overcome these translational hurdles, this study engineered a scaffold-free, 3D formulation of highly characterized allogeneic FE002-Cart chondroprogenitor spheroids. Methods: We systematically investigated the specific microenvironmental cues and Good Manufacturing Practice (GMP) formulation parameters required to direct functional chondrogenesis. The structural and biochemical performance of this allogeneic formulation was benchmarked against multiple primary adult autologous chondrocyte types. Finally, we evaluated the phenotypic resilience of the microtissues in simulated osteoarthritic (OA) environments and investigated both short-term liquid storage and advanced terminal preservation strategies to establish off-the-shelf logistical viability. Results: Precise microenvironmental regulation proved to be a critical biological prerequisite. The synergistic combination of physiological hypoxia (2% O2) and stringent glucocorticoid limitation (10 nM dexamethasone) induced robust glycosaminoglycan (GAG) deposition and a &gt; 200-fold upregulation of ACAN and COL2, while suppressing the terminal hypertrophic drift observed in adult chondrocytes. Benchmarking revealed that the allogeneic FE002-Cart formulation substantially mitigates the profound morphological and biochemical unpredictability inherent to adult autologous cell sources. Furthermore, the scaffold-free spheroid geometry yielded a 10-fold increase in GAG production per cell compared to traditional matrix-seeded (MACI) platforms. Transitioning to a GMP-compatible manufacturing process revealed extreme cellular sensitivities; excipients within standard pharmaceutical-grade dexamethasone severely aborted chondrogenic differentiation, emphasizing the necessity of rigorous raw-material qualification. Functionally, the 3D architecture acted as a protective physical shield, sustaining high cellular viability when subjected to severe inflammatory stress and 100% OA patient synovial fluid. Logistically, the viable spheroids maintained matrix integrity and inter-spheroid fusion potential for up to 7 days at ambient temperature in transport medium. Finally, advanced spheroid preservation via lyophilization and high-dose gamma irradiation eliminated biological viability but successfully transitioned the microtissues into highly organized, terminally irradiated matrices capable of heterologous in vitro structural merging. Conclusions: These findings define the critical biological thresholds for manufacturing, demonstrate the enhanced in vitro biosynthetic efficiency of 3D allogeneic microtissues compared to specific autologous and matrix-dependent baselines, and establish a highly practical, off-the-shelf logistical framework for the regenerative treatment of large knee chondral defects.<\/jats:p>","DOI":"10.3390\/pharmaceutics18081032","type":"journal-article","created":{"date-parts":[[2026,8,20]],"date-time":"2026-08-20T12:57:53Z","timestamp":1787230673000},"page":"1032","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Engineering Allogeneic FE002-Cart Chondroprogenitor Spheroids for Large Knee Chondral Defects: Investigating Microenvironmental Cues for Functional Control, GMP Formulation, and Logistical Viability"],"prefix":"10.3390","volume":"18","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-7346-9921","authenticated-orcid":false,"given":"Lee Ann","family":"Applegate","sequence":"first","affiliation":[{"name":"Regenerative Therapy Unit, Lausanne University Hospital, University of Lausanne, CH-1066 Epalinges, Switzerland"},{"name":"Center for Applied Biotechnology and Molecular Medicine, University of Zurich, CH-8057 Zurich, Switzerland"},{"name":"Oxford OSCAR Suzhou Center, Oxford University, Suzhou 215123, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Farid","family":"Hadjab","sequence":"additional","affiliation":[{"name":"Development Department, Albomed GmbH, D-90592 Schwarzenbruck, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sandra","family":"Jaccoud","sequence":"additional","affiliation":[{"name":"Regenerative Therapy Unit, Lausanne University Hospital, University of Lausanne, CH-1066 Epalinges, Switzerland"},{"name":"Laboratory of Biomechanical Orthopedics, Federal Polytechnic School of Lausanne, CH-1015 Lausanne, Switzerland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1603-2704","authenticated-orcid":false,"given":"Alexandre","family":"Porcello","sequence":"additional","affiliation":[{"name":"Development Department, LOUNA REGENERATIVE SA, CH-1228 Plan-les-Ouates, Switzerland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3544-0383","authenticated-orcid":false,"given":"Virginie","family":"Philippe","sequence":"additional","affiliation":[{"name":"Regenerative Therapy Unit, Lausanne University Hospital, University of Lausanne, CH-1066 Epalinges, Switzerland"},{"name":"Orthopedic Surgery Service, Fribourg Hospital, CH-1752 Villars-sur-Gl\u00e2ne, Switzerland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1220-0213","authenticated-orcid":false,"given":"Nathalie","family":"Hirt-Burri","sequence":"additional","affiliation":[{"name":"Regenerative Therapy Unit, Lausanne University Hospital, University of Lausanne, CH-1066 Epalinges, Switzerland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7682-9160","authenticated-orcid":false,"given":"Corinne","family":"Scaletta","sequence":"additional","affiliation":[{"name":"Regenerative Therapy Unit, Lausanne University Hospital, University of Lausanne, CH-1066 Epalinges, Switzerland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Brigitte M.","family":"Jolles","sequence":"additional","affiliation":[{"name":"Department of Musculoskeletal Medicine, Swiss BioMotion Lab, Lausanne University Hospital and University of Lausanne (CHUV-UNIL), CH-1011 Lausanne, Switzerland"},{"name":"Institute of Electrical and Micro Engineering, Ecole Polytechnique F\u00e9d\u00e9rale de Lausanne (EPFL), CH-1015 Lausanne, Switzerland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5535-5296","authenticated-orcid":false,"given":"Dominique P.","family":"Pioletti","sequence":"additional","affiliation":[{"name":"Laboratory of Biomechanical Orthopedics, Federal Polytechnic School of Lausanne, CH-1015 Lausanne, Switzerland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Robin","family":"Martin","sequence":"additional","affiliation":[{"name":"Orthopedic Surgery Service, Fribourg Hospital, CH-1752 Villars-sur-Gl\u00e2ne, Switzerland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5738-8702","authenticated-orcid":false,"given":"Alexis E.","family":"Laurent","sequence":"additional","affiliation":[{"name":"Manufacturing Department, LAM Biotechnologies SA, CH-1066 Epalinges, Switzerland"},{"name":"Manufacturing Department, TEC-PHARMA SA, CH-1038 Bercher, Switzerland"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,8,20]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Roseti, L., Cavallo, C., Desando, G., D\u2019Alessandro, M., and Grigolo, B. 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