{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,6,11]],"date-time":"2026-06-11T12:14:29Z","timestamp":1781180069214,"version":"3.54.1"},"reference-count":41,"publisher":"Springer Science and Business Media LLC","issue":"1","license":[{"start":{"date-parts":[[2017,5,8]],"date-time":"2017-05-08T00:00:00Z","timestamp":1494201600000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0"},{"start":{"date-parts":[[2017,5,8]],"date-time":"2017-05-08T00:00:00Z","timestamp":1494201600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0"}],"content-domain":{"domain":["link.springer.com"],"crossmark-restriction":false},"short-container-title":["Sci Rep"],"abstract":"<jats:title>Abstract<\/jats:title><jats:p>For chronic wounds, the delivery of stem cells in spheroidal structures can enhance graft survival and stem cell potency. We describe an easy method for the 3D culture of adipose-derived stem\/stromal cells (ASCs) to prepare a ready-to-use injectable. We transferred suspensions of monolayer-cultured ASCs to a syringe containing hyaluronic acid (HA) gel, and then incubated the syringe as a 3D culture vessel. Spheroids of cells formed after 12\u2009h. We found that 6\u2009\u00d7\u200910<jats:sup>6<\/jats:sup> ASCs\/ml in 3% HA gel achieved the highest spheroid density with appropriate spheroid sizes (20\u2013100\u2009\u00b5m). Immunocytology revealed that the stem cell markers, NANOG, OCT3\/4, SOX-2, and SSEA-3 were up-regulated in the ASC spheroids compared with those in nonadherent-dish spheroids or in monolayer cultured ASCs. In delayed wound healing mice models, diabetic ulcers treated with ASC spheroids demonstrated faster wound epithelialization with thicker dermis than those treated with vehicle alone or monolayer cultured ASCs. In irradiated skin ulcers in immunodeficient mice, ASC spheroids exhibited faster healing and outstanding angiogenic potential partly by direct differentiation into \u03b1-SMA+ pericytes. Our method of 3D in-syringe HA gel culture produced clinically relevant amounts of ready-to-inject human ASC microspheroids that exhibited superior stemness <jats:italic>in vitro<\/jats:italic> and therapeutic efficacy in pathological wound repair <jats:italic>in vivo<\/jats:italic>.<\/jats:p>","DOI":"10.1038\/s41598-017-01528-3","type":"journal-article","created":{"date-parts":[[2017,5,2]],"date-time":"2017-05-02T11:04:46Z","timestamp":1493723086000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":49,"title":["An injectable non-cross-linked hyaluronic-acid gel containing therapeutic spheroids of human adipose-derived stem cells"],"prefix":"10.1038","volume":"7","author":[{"given":"Jingwei","family":"Feng","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kazuhide","family":"Mineda","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Szu-Hsien","family":"Wu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Takanobu","family":"Mashiko","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kentaro","family":"Doi","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shinichiro","family":"Kuno","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kahori","family":"Kinoshita","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Koji","family":"Kanayama","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rintaro","family":"Asahi","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ataru","family":"Sunaga","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7152-5475","authenticated-orcid":false,"given":"Kotaro","family":"Yoshimura","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2017,5,8]]},"reference":[{"key":"1528_CR1","first-page":"145","volume":"30","author":"G Crovetti","year":"2004","unstructured":"Crovetti, G. et al. Platelet gel for healing cutaneous chronic wounds. Transfusion and Apheresis. Science. \n                           30, 145\u2013151 (2004).","journal-title":"Science."},{"key":"1528_CR2","doi-asserted-by":"publisher","first-page":"857","DOI":"10.1038\/nm.3653","volume":"20","author":"SJ Forbes","year":"2014","unstructured":"Forbes, S. J. & Rosenthal, N. Preparing the ground for tissue regeneration: from mechanism to therapy. Nat. Med. \n                           20, 857\u2013869 (2014).","journal-title":"Nat. Med."},{"key":"1528_CR3","doi-asserted-by":"publisher","first-page":"211","DOI":"10.1089\/107632701300062859","volume":"7","author":"PA Zuk","year":"2007","unstructured":"Zuk, P. A. et al. Multilineage cells from human adipose tissue: implications for cell-based therapies. Tissue Eng. \n                           7, 211\u2013228 (2007).","journal-title":"Tissue Eng."},{"key":"1528_CR4","doi-asserted-by":"publisher","first-page":"632","DOI":"10.5966\/sctm.2014-0235","volume":"4","author":"AL Strong","year":"2015","unstructured":"Strong, A. L. et al. Adipose stromal cells repair pressure ulcers in both young and elderly mice: potential role of adipogenesis in skin repair. Stem Cells Transl. Med. \n                           4, 632\u2013642 (2015).","journal-title":"Stem Cells Transl. Med."},{"key":"1528_CR5","doi-asserted-by":"publisher","first-page":"245","DOI":"10.1016\/j.jcyt.2013.11.011","volume":"16","author":"A Bura","year":"2014","unstructured":"Bura, A. et al. Phase I trial: the use of autologous cultured adipose-derived stroma\/stem cells to treat patients with non-revascularizable critical limb ischemia. Cytotherapy \n                           16, 245\u2013257 (2014).","journal-title":"Cytotherapy"},{"key":"1528_CR6","doi-asserted-by":"publisher","first-page":"36","DOI":"10.1016\/j.jss.2013.05.024","volume":"185","author":"G Marino","year":"2013","unstructured":"Marino, G. et al. Therapy with autologous adipose-derived regenerative cells for the care of chronic ulcer of lower limbs in patients with peripheral arterial disease. J. Surg. Res. \n                           185, 36\u201344 (2013).","journal-title":"J. Surg. Res."},{"key":"1528_CR7","doi-asserted-by":"publisher","first-page":"25","DOI":"10.1016\/j.stem.2015.12.005","volume":"18","author":"X Yin","year":"2016","unstructured":"Yin, X. et al. Engineering stem cell organoids. Cell Stem Cell \n                           18, 25\u201338 (2016).","journal-title":"Cell Stem Cell"},{"key":"1528_CR8","doi-asserted-by":"publisher","first-page":"1868","DOI":"10.1097\/TP.0b013e3181775136","volume":"85","author":"K Yoshimura","year":"2008","unstructured":"Yoshimura, K. et al. Ectopic fibrogenesis induced by transplantation of adipose-derived progenitor cell suspension immediately after lipoinjection. Transplantation \n                           85, 1868\u20131869 (2008).","journal-title":"Transplantation"},{"key":"1528_CR9","doi-asserted-by":"publisher","first-page":"1748","DOI":"10.1016\/j.biomaterials.2011.11.049","volume":"33","author":"NC Cheng","year":"2012","unstructured":"Cheng, N. C. et al. The influence of spheroid formation of human adipose-derived stem cells on chitosan films on stemness and differentiation capabilities. Biomaterials \n                           33, 1748\u20131758 (2012).","journal-title":"Biomaterials"},{"key":"1528_CR10","doi-asserted-by":"publisher","first-page":"9280","DOI":"10.1016\/j.biomaterials.2014.07.061","volume":"35","author":"IS Park","year":"2014","unstructured":"Park, I. S. et al. Enhanced angiogenic effect of adipose-derived stromal cell spheroid with low-level light therapy in hind limb ischemia mice. Biomaterials \n                           35, 9280\u20139289 (2014).","journal-title":"Biomaterials"},{"key":"1528_CR11","doi-asserted-by":"publisher","first-page":"83","DOI":"10.1016\/j.jconrel.2014.12.016","volume":"205","author":"K Sakaguchi","year":"2015","unstructured":"Sakaguchi, K. et al. Construction of three-dimensional vascularized cardiac tissue with cell sheet engineering. J. Control Release \n                           205, 83\u201388 (2015).","journal-title":"J. Control Release"},{"key":"1528_CR12","first-page":"922","volume":"34","author":"A Hartmann","year":"2008","unstructured":"Hartmann, A. et al. Transplantation of autologous keratinocyte suspension in fibrin matrix to chronic venous leg ulcers: improved long-term healing after removal of the fibrin carrier. Dermatol. Surg. \n                           34, 922\u2013929 (2008).","journal-title":"Dermatol. Surg."},{"key":"1528_CR13","doi-asserted-by":"publisher","first-page":"2723","DOI":"10.2337\/db14-1133","volume":"64","author":"Y Kato","year":"2015","unstructured":"Kato, Y. et al. Allogeneic transplantation of an adipose-derived stem cell sheet combined with artificial skin accelerates wound healing in a rat wound model of type 2 diabetes and obesity. Diabetes \n                           64, 2723\u20132734 (2015).","journal-title":"Diabetes"},{"key":"1528_CR14","doi-asserted-by":"publisher","first-page":"53","DOI":"10.1016\/j.biomaterials.2015.04.029","volume":"59","author":"Y Zeng","year":"2015","unstructured":"Zeng, Y. et al. Injectable microcryogels reinforced alginate encapsulation of mesenchymal stromal cells for leak-proof delivery and alleviation of canine disc degeneration. Biomaterials \n                           59, 53\u201365 (2015).","journal-title":"Biomaterials"},{"key":"1528_CR15","doi-asserted-by":"publisher","first-page":"1511","DOI":"10.5966\/sctm.2015-0037","volume":"4","author":"K Mineda","year":"2015","unstructured":"Mineda, K. et al. Therapeutic potential of human adipose-derived stem\/stromal cell micro-spheroids prepared by 3D culture in non-cross-linked hyaluronic acid gel. Stem Cells Transl. Med. \n                           4, 1511\u20131522 (2015).","journal-title":"Stem Cells Transl. Med."},{"key":"1528_CR16","doi-asserted-by":"publisher","first-page":"6929","DOI":"10.1016\/j.biomaterials.2011.05.092","volume":"32","author":"GS Huang","year":"2011","unstructured":"Huang, G. S. et al. Spheroid formation of mesenchymal stem cells on chitosan and chitosan-hyaluronan membranes. Biomaterials \n                           32, 6929\u20136945 (2011).","journal-title":"Biomaterials"},{"key":"1528_CR17","doi-asserted-by":"publisher","first-page":"28","DOI":"10.1089\/biores.2012.0285","volume":"2","author":"BH Liu","year":"2013","unstructured":"Liu, B. H. et al. Spheroid formation and enhanced cardiomyogenic potential of adipose-derived stem cells grown on chitosan. Biores. Open Access \n                           2, 28\u201339 (2013).","journal-title":"Biores. Open Access"},{"key":"1528_CR18","doi-asserted-by":"publisher","first-page":"9875","DOI":"10.1073\/pnas.1100816108","volume":"108","author":"S Wakao","year":"2011","unstructured":"Wakao, S. et al. Multilineage-differentiating stress-enduring (Muse) cells are a primary source of induced pluripotent stem cells in human fibroblasts. Proc. Natl. Acad. Sci. USA \n                           108, 9875\u20139880 (2011).","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"1528_CR19","doi-asserted-by":"publisher","first-page":"105","DOI":"10.1186\/s12896-014-0105-9","volume":"14","author":"Y Yamaguchi","year":"2014","unstructured":"Yamaguchi, Y. et al. Mesenchymal stem cell spheroids exhibit enhanced in-vitro and in-vivo osteoregenerative potential. BMC Biotechnol. \n                           14, 105 (2014).","journal-title":"BMC Biotechnol."},{"key":"1528_CR20","doi-asserted-by":"publisher","first-page":"26","DOI":"10.1016\/j.biomaterials.2015.01.078","volume":"52","author":"Y Shao","year":"2015","unstructured":"Shao, Y. et al. On human pluripotent stem cell control: The rise of 3D bioengineering and mechanobiology. Biomaterials \n                           52, 26\u201343 (2015).","journal-title":"Biomaterials"},{"key":"1528_CR21","doi-asserted-by":"publisher","first-page":"852","DOI":"10.1089\/ten.tec.2014.0376","volume":"21","author":"P Beauchamp","year":"2015","unstructured":"Beauchamp, P. et al. Development and characterization of a scaffold-free 3D spheroid model of induced pluripotent stem cell-derived human cardiomyocytes. Tissue Eng. Part C \n                           21, 852\u2013861 (2015).","journal-title":"Tissue Eng. Part C"},{"key":"1528_CR22","doi-asserted-by":"publisher","first-page":"8430","DOI":"10.1016\/j.biomaterials.2012.08.040","volume":"33","author":"T Anada","year":"2012","unstructured":"Anada, T. et al. An oxygen-permeable spheroid culture system for the prevention of central hypoxia and necrosis of spheroids. Biomaterial \n                           33, 8430\u20138441 (2012).","journal-title":"Biomaterial"},{"key":"1528_CR23","doi-asserted-by":"publisher","first-page":"297","DOI":"10.1007\/s00441-014-2055-x","volume":"360","author":"Y Li","year":"2015","unstructured":"Li, Y. et al. Three-dimensional spheroid culture of human umbilical cord mesenchymal stem cells promotes cell yield and stemness maintenance. Cell Tissue Res. \n                           360, 297\u2013307 (2015).","journal-title":"Cell Tissue Res."},{"key":"1528_CR24","doi-asserted-by":"publisher","first-page":"7012","DOI":"10.1016\/j.biomaterials.2010.05.078","volume":"31","author":"J Yu","year":"2010","unstructured":"Yu, J. et al. The use of human mesenchymal stem cells encapsulated in RGD modified alginate microspheres in the repair of myocardial infarction in the rat. Biomaterials \n                           31, 7012\u20137020 (2010).","journal-title":"Biomaterials"},{"key":"1528_CR25","unstructured":"Rubenstein, D., Yin, W. & Frame, M. D. Biofluid Mechanics: An Introduction to Fluid Mechanics, Macrocirculation, and Microcirculation. 2nd ed. Waltham, MA: Academic Press, 2011."},{"key":"1528_CR26","unstructured":"Robert, A. F. Nanomedicine, Volume I: Basic Capabilities. 1st ed. Georgetown, TX: Landes Bioscience, 1999."},{"key":"1528_CR27","doi-asserted-by":"publisher","first-page":"641","DOI":"10.1016\/j.jcyt.2013.02.006","volume":"15","author":"P Bourin","year":"2013","unstructured":"Bourin, P. et al. Stromal cells from the adipose tissue-derived stromal vascular fraction and culture expanded adipose tissue-derived stromal\/stem cells: a joint statement of the International Federation for Adipose Therapeutics and Science (IFATS) and the International Society for Cellular Therapy (ISCT). Cytotherapy \n                           15, 641\u2013648 (2013).","journal-title":"Cytotherapy"},{"key":"1528_CR28","doi-asserted-by":"publisher","first-page":"3945","DOI":"10.1016\/j.biomaterials.2014.01.066","volume":"35","author":"D L\u00fc","year":"2014","unstructured":"L\u00fc, D. et al. Differential regulation of morphology and stemness of mouse embryonic stem cells by substrate stiffness and topography. Biomaterials \n                           35, 3945\u20133955 (2014).","journal-title":"Biomaterials"},{"key":"1528_CR29","doi-asserted-by":"publisher","first-page":"39","DOI":"10.1016\/j.tice.2014.10.008","volume":"47","author":"H Li","year":"2015","unstructured":"Li, H. et al. Spheroid cultures promote the stemness of corneal stromal cells. Tissue Cell \n                           47, 39\u201348 (2015).","journal-title":"Tissue Cell"},{"key":"1528_CR30","doi-asserted-by":"publisher","first-page":"75","DOI":"10.1111\/j.1365-2184.2010.00732.x","volume":"44","author":"M\u00c1 Ram\u00edrez","year":"2011","unstructured":"Ram\u00edrez, M. \u00c1. et al. Effect of long-term culture of mouse embryonic stem cells under low oxygen concentration as well as on glycosaminoglycan hyaluronan on cell proliferation and differentiation. Cell Prolif. \n                           44, 75\u201385 (2011).","journal-title":"Cell Prolif."},{"key":"1528_CR31","doi-asserted-by":"publisher","first-page":"123","DOI":"10.1007\/978-94-007-4285-7_11","volume":"7","author":"Ramos-Ibease","year":"2012","unstructured":"Ramos-Ibease et al. Maintenance of pluripotency in mouse stem cells: use of hyaluronan in the long-term culture. Stem Cells and Cancer Stem Cells \n                           7, 123\u2013133 (2012).","journal-title":"Stem Cells and Cancer Stem Cells"},{"key":"1528_CR32","doi-asserted-by":"publisher","first-page":"17635","DOI":"10.1074\/jbc.M800109200","volume":"283","author":"LYW Bourguignon","year":"2008","unstructured":"Bourguignon, L. Y. W. et al. Hyaluronan\u2013CD44 interaction activates stem cell marker Nanog, Stat-3-mediated MDR1 gene expression, and ankyrin-regulated multidrug efflux in breast and ovarian tumor cells. J. Biol. Chem. \n                           283, 17635\u201317651 (2008).","journal-title":"J. Biol. Chem."},{"key":"1528_CR33","doi-asserted-by":"publisher","first-page":"86","DOI":"10.1016\/j.scr.2013.09.011","volume":"12","author":"SY Ju","year":"2014","unstructured":"Ju, S. Y. et al. Maintenance of the stemness in CD44(+) HCT-15 and HCT-116 human colon cancer cells requires miR-203 suppression. Stem Cell Res \n                           12, 86\u2013100 (2014).","journal-title":"Stem Cell Res"},{"key":"1528_CR34","doi-asserted-by":"publisher","first-page":"85","DOI":"10.1530\/REP-09-0300","volume":"139","author":"CE Forristal","year":"2010","unstructured":"Forristal, C. E. et al. Hypoxia inducible factors regulate pluripotency and proliferation in human embryonic stem cells cultured at reduced oxygen tensions. Reproduction \n                           139, 85\u201397 (2010).","journal-title":"Reproduction"},{"key":"1528_CR35","doi-asserted-by":"publisher","first-page":"e108309","DOI":"10.1371\/journal.pone.0108309","volume":"9","author":"R Petruzzelli","year":"2014","unstructured":"Petruzzelli, R. et al. HIF-2\u03b1 regulates NANOG expression in human embryonic stem cells following hypoxia and reoxygenation through the interaction with an Oct-Sox Cis regulatory element. PLoS One \n                           9, e108309 (2014).","journal-title":"PLoS One"},{"key":"1528_CR36","doi-asserted-by":"publisher","first-page":"526","DOI":"10.1093\/molehr\/gau016","volume":"20","author":"YH Lin","year":"2014","unstructured":"Lin, Y. H. et al. Hypoxia inducible factor 2\u03b1\/insulin-like growth factor receptor signal loop supports the proliferation and Oct-4 maintenance of mouse germline stem cells. Mol. Hum. Reprod. \n                           20, 526\u2013537 (2014).","journal-title":"Mol. Hum. Reprod."},{"key":"1528_CR37","doi-asserted-by":"publisher","first-page":"172","DOI":"10.1007\/s12195-014-0333-z","volume":"7","author":"Y Lei","year":"2014","unstructured":"Lei, Y. et al. Developing defined and scalable 3D culture systems for culturing human pluripotent stem cells at high densities. Cell Mol Bioeng. \n                           7, 172\u2013183 (2014).","journal-title":"Cell Mol Bioeng."},{"key":"1528_CR38","doi-asserted-by":"publisher","first-page":"1045","DOI":"10.3390\/cells1041045","volume":"1","author":"S Wakao","year":"2012","unstructured":"Wakao, S. et al. Regenerative Effects of Mesenchymal Stem Cells: Contribution of Muse Cells, a Novel Pluripotent Stem Cell Type that Resides in Mesenchymal Cells. Cells \n                           1, 1045\u20131060 (2012).","journal-title":"Cells"},{"key":"1528_CR39","doi-asserted-by":"publisher","first-page":"1391","DOI":"10.1038\/nprot.2013.076","volume":"8","author":"Y Kuroda","year":"2013","unstructured":"Kuroda, Y. et al. Isolation, culture and evaluation of multilineage-differentiating stress-enduring (Muse) cells. Nat Protoc. \n                           8, 1391\u20131415 (2013).","journal-title":"Nat Protoc."},{"key":"1528_CR40","doi-asserted-by":"publisher","first-page":"146","DOI":"10.5966\/sctm.2014-0181","volume":"4","author":"K Kinoshita","year":"2015","unstructured":"Kinoshita, K. et al. Therapeutic potential of adipose-derived SSEA-3-positive muse cells for treating diabetic skin ulcers. Stem Cells Transl. Med. \n                           4, 146\u2013155 (2015).","journal-title":"Stem Cells Transl. Med."},{"key":"1528_CR41","doi-asserted-by":"publisher","first-page":"246","DOI":"10.1111\/j.1742-481X.2008.00436.x","volume":"5","author":"A Olascoaga","year":"2008","unstructured":"Olascoaga, A. et al. Wound healing in radiated skin: pathophysiology and treatment options. Int. Wound J. \n                           5, 246\u2013257 (2008).","journal-title":"Int. Wound J."}],"container-title":["Scientific Reports"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.nature.com\/articles\/s41598-017-01528-3.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/www.nature.com\/articles\/s41598-017-01528-3","content-type":"text\/html","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/www.nature.com\/articles\/s41598-017-01528-3.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2022,12,23]],"date-time":"2022-12-23T04:01:54Z","timestamp":1671768114000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.nature.com\/articles\/s41598-017-01528-3"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2017,5,8]]},"references-count":41,"journal-issue":{"issue":"1","published-online":{"date-parts":[[2017,12]]}},"alternative-id":["1528"],"URL":"https:\/\/doi.org\/10.1038\/s41598-017-01528-3","relation":{},"ISSN":["2045-2322"],"issn-type":[{"value":"2045-2322","type":"electronic"}],"subject":[],"published":{"date-parts":[[2017,5,8]]},"assertion":[{"value":"6 October 2016","order":1,"name":"received","label":"Received","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"30 March 2017","order":2,"name":"accepted","label":"Accepted","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"8 May 2017","order":3,"name":"first_online","label":"First Online","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"The authors declare that they have no competing interests.","order":1,"name":"Ethics","group":{"name":"EthicsHeading","label":"Competing Interests"}}],"article-number":"1548"}}