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Osteoclast research is limited by the heterogeneity of osteoclast populations generated in vitro, where the mixture of undifferentiated monocytes, binuclear pre-osteoclasts and multinucleated osteoclasts has by necessity been considered a single osteoclast population. This study describes the differentiation of primary human CD14+ monocyte-derived osteoclasts in 3D collagen gels. These osteoclasts remained small (&gt;95% with \u22645 nuclei) but were viable and active; when released from the gel with collagenase, they fused rapidly when reseeded onto solid substrates and resorbed dentine for 2\u20133 weeks. 3D-generated osteoclasts expressed cell surface markers of osteoclast differentiation (e.g., CD9, RANK, OSCAR, CD63, CD51\/61) which, with their small size, enabled live cell sorting of highly enriched viable subpopulations of human osteoclasts that retained full functional resorption capacity. Low-yield osteoclast preparations were strongly enriched to remove undifferentiated cells (e.g., 13.3% CD51\/61+ to 84.2% CD51\/61+), and subpopulations of CD9+CD51\/61\u2212 early osteoclasts and CD9+CD51\/61+ mature cells were distinguished. This novel approach allows the study of selected populations of differentiating osteoclasts in vitro and opens the door to in-depth transcriptomic and proteomic analysis of these cells, increasing our ability to study human osteoclast molecular mechanisms relevant to development, aging and disease.<\/jats:p>","DOI":"10.3390\/cells11243973","type":"journal-article","created":{"date-parts":[[2022,12,9]],"date-time":"2022-12-09T02:50:51Z","timestamp":1670554251000},"page":"3973","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":9,"title":["A New Method to Sort Differentiating Osteoclasts into Defined Homogeneous Subgroups"],"prefix":"10.3390","volume":"11","author":[{"given":"Philippa A.","family":"Hulley","sequence":"first","affiliation":[{"name":"Botnar Institute for Musculoskeletal Sciences, Nuffield Department of Orthopaedics Rheumatology & Musculoskeletal Sciences, University of Oxford, Oxford OX3 7LD, UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9575-6067","authenticated-orcid":false,"given":"Helen J.","family":"Knowles","sequence":"additional","affiliation":[{"name":"Botnar Institute for Musculoskeletal Sciences, Nuffield Department of Orthopaedics Rheumatology & Musculoskeletal Sciences, University of Oxford, Oxford OX3 7LD, UK"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2022,12,8]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"e10539","DOI":"10.1002\/jbm4.10539","article-title":"New Insights into Osteoclast Biology","volume":"5","author":"McDonald","year":"2021","journal-title":"JBMR Plus"},{"key":"ref_2","doi-asserted-by":"crossref","unstructured":"Gambari, L., Grassi, F., Roseti, L., Grigolo, B., and Desando, G. 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