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However, major challenges remain for developing a standardized and robust good manufacturing practice (GMP)-compliant cell product which is severely hampered by low frequency of Tregs in circulation and laborious ex vivo expansion.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Methods<\/jats:title>\n                    <jats:p>Paediatric thymuses routinely obtained during heart surgery have been shown by us and others to be a valuable source of large numbers of pure Tregs (Thy-Tregs). Here we show results from our process development approach including systematic laboratory-scale testing of activation reagents, restimulation timing, and cryopreservation to translate our expansion protocol of Thy-Tregs into a clinical grade cell product.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>\n                      Thy-Tregs obtained through CD8\n                      <jats:sup>+<\/jats:sup>\n                      cell depletion and subsequent CD25\n                      <jats:sup>+<\/jats:sup>\n                      enrichment were expanded with \u03b1CD3\/\u03b1CD28 beads in the presence of Rapamycin and IL-2 for 10\u201323 days using G-Rex bioreactors. We successfully embedded bead removal and final formulation of a cryopreserved cell product ready to be used at bedside transfusion.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Conclusion<\/jats:title>\n                    <jats:p>This process has proved the capability of efficiently producing high number of functional Thy-Tregs, which will be administered as cell therapy in children undergoing heart transplantation (ATT-Heart, ISRCTN15374803), and enhancing the potential of using expanded Thy-Tregs for broad-ranging therapeutic applications.<\/jats:p>\n                  <\/jats:sec>","DOI":"10.1186\/s12967-025-06561-9","type":"journal-article","created":{"date-parts":[[2025,6,3]],"date-time":"2025-06-03T04:31:29Z","timestamp":1748925089000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":16,"title":["Clinical grade expansion protocol for the manufacture of thymus-derived Treg cells for clinical application"],"prefix":"10.1186","volume":"23","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-2003-529X","authenticated-orcid":false,"given":"Giorgia","family":"Fanelli","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Philippa","family":"Marks","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Apoorva","family":"Aiyengar","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Marco","family":"Romano","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sakina","family":"Gooljar","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sandeep","family":"Kumar","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Michael","family":"Burch","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Giovanna","family":"Lombardi","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2025,6,3]]},"reference":[{"key":"6561_CR1","doi-asserted-by":"publisher","first-page":"43","DOI":"10.3389\/fimmu.2019.00043","volume":"10","author":"M Romano","year":"2019","unstructured":"Romano M, Fanelli G, Albany CJ, Giganti G, Lombardi G, Past. 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