{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,4,13]],"date-time":"2026-04-13T14:47:19Z","timestamp":1776091639204,"version":"3.50.1"},"reference-count":57,"publisher":"Springer Science and Business Media LLC","issue":"1","license":[{"start":{"date-parts":[[2018,1,10]],"date-time":"2018-01-10T00:00:00Z","timestamp":1515542400000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0"},{"start":{"date-parts":[[2018,1,10]],"date-time":"2018-01-10T00:00:00Z","timestamp":1515542400000},"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>Spheroid culture has gained increasing popularity, arising as a promising tool for regenerative medicine applications. Importantly, spheroids may present advantages over single-cell suspensions in cell-based therapies (CT). Unfortunately, most growth media used for spheroid culture contain animal origin-components, such as fetal bovine serum (FBS). The presence of FBS compromises the safety of CT and presents economic and ethical constraints. SCC (supplement for cell culture) is a novel xeno-free (XF) industrial cell culture supplement, derived from well-controlled pooled human plasma and processed under good manufacturing practice rules. Here, we developed a XF SCC-based formulation for 2D-culture of outgrowth endothelial cells (OEC), and then used it for generating co-culture spheroids of OEC and mesenchymal stem cells (MSC). XF MSC-OEC spheroids were characterized in detail and compared to spheroids cultured in FBS-supplemented medium. XF spheroids presented comparable integrity, size and morphology as the reference culture. The use of both media resulted in spheroids with similar structure, abundant extracellular matrix deposition and specific patterns of OEC distribution and organization. Notably, XF spheroids presented significantly enhanced angiogenic potential, both <jats:italic>in vitro<\/jats:italic> (fibrin sprouting assay) and <jats:italic>in vivo<\/jats:italic> (CAM assay). These findings are particularly promising in the context of potential therapeutic applications.<\/jats:p>","DOI":"10.1038\/s41598-017-18431-6","type":"journal-article","created":{"date-parts":[[2018,1,4]],"date-time":"2018-01-04T09:53:33Z","timestamp":1515059613000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":37,"title":["Xeno-free pre-vascularized spheroids for therapeutic applications"],"prefix":"10.1038","volume":"8","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-4162-2813","authenticated-orcid":false,"given":"E.","family":"Bauman","sequence":"first","affiliation":[]},{"given":"T.","family":"Feij\u00e3o","sequence":"additional","affiliation":[]},{"given":"D. T. O.","family":"Carvalho","sequence":"additional","affiliation":[]},{"given":"P. L.","family":"Granja","sequence":"additional","affiliation":[]},{"given":"C. C.","family":"Barrias","sequence":"additional","affiliation":[]}],"member":"297","published-online":{"date-parts":[[2018,1,10]]},"reference":[{"key":"18431_CR1","doi-asserted-by":"publisher","first-page":"35","DOI":"10.1038\/nm.3028","volume":"19","author":"P Bianco","year":"2013","unstructured":"Bianco, P. et al. The meaning, the sense and the significance: translating the science of mesenchymal stem cells into medicine. Nat med. 19, 35\u201342 (2013).","journal-title":"Nat med."},{"key":"18431_CR2","doi-asserted-by":"publisher","first-page":"566","DOI":"10.1016\/j.stem.2008.03.003","volume":"2","author":"F Belema-Bedada","year":"2008","unstructured":"Belema-Bedada, F., Uchida, S., Martire, A., Kostin, S. & Braun, T. Efficient homing of multipotent adult mesenchymal stem cells depends on FROUNT-mediated clustering of CCR2. Cell stem cell. 2, 566\u2013575 (2008).","journal-title":"Cell stem cell."},{"key":"18431_CR3","doi-asserted-by":"crossref","first-page":"38008","DOI":"10.18632\/oncotarget.16682","volume":"23","author":"F Mao","year":"2017","unstructured":"Mao, F. et al. Mesenchymal stem cells and their therapeutic applications in inflammatory bowel disease. Oncotarget. 23, 38008\u201338021 (2017).","journal-title":"Oncotarget."},{"key":"18431_CR4","doi-asserted-by":"publisher","first-page":"133","DOI":"10.1016\/j.tibtech.2016.08.004","volume":"35","author":"MW Laschke","year":"2017","unstructured":"Laschke, M. W. & Menger, M. D. Life is 3D: boosting spheroid function for tissue engineering. Trends Biotechnol. 35, 133\u2013144 (2017).","journal-title":"Trends Biotechnol."},{"key":"18431_CR5","doi-asserted-by":"publisher","first-page":"733","DOI":"10.1016\/j.tibtech.2016.03.002","volume":"34","author":"J Rouwkema","year":"2016","unstructured":"Rouwkema, J. & Khademhosseini, A. Vascularization and angiogenesis in tissue engineering: beyond creating static networks. Trends Biotechnol. 34, 733\u2013745 (2016).","journal-title":"Trends Biotechnol."},{"key":"18431_CR6","doi-asserted-by":"publisher","first-page":"310","DOI":"10.1089\/ten.tea.2011.0193","volume":"18","author":"SH Bhang","year":"2012","unstructured":"Bhang, S. H. et al. Three-dimensional cell grafting enhances the angiogenic efficacy of human umbilical vein endothelial cells. Tissue Eng Part A. 18, 310\u2013319 (2012).","journal-title":"Tissue Eng Part A."},{"key":"18431_CR7","doi-asserted-by":"publisher","first-page":"46","DOI":"10.1016\/j.jbiotec.2010.03.002","volume":"148","author":"JM Kelm","year":"2010","unstructured":"Kelm, J. M. et al. A novel concept for scaffold-free vessel tissue engineering: self-assembly of microtissue building blocks. J Biotechnol. 148, 46\u201355 (2010).","journal-title":"J Biotechnol."},{"key":"18431_CR8","doi-asserted-by":"publisher","first-page":"13724","DOI":"10.1073\/pnas.1008117107","volume":"107","author":"TJ Bartosh","year":"2010","unstructured":"Bartosh, T. J. et al. Aggregation of human mesenchymal stromal cells (MSCs) into 3D spheroids enhances their antiinflammatory properties. Proc Natl Acad Sci USA 107, 13724\u201313729 (2010).","journal-title":"Proc Natl Acad Sci USA"},{"key":"18431_CR9","doi-asserted-by":"publisher","first-page":"584","DOI":"10.5966\/sctm.2013-0007","volume":"2","author":"NC Cheng","year":"2013","unstructured":"Cheng, N. C., Chen, S. Y., Li, J. R. & Young, T. H. Short-term spheroid formation enhances the regenerative capacity of adipose-derived stem cells by promoting stemness, angiogenesis, and chemotaxis. Stem Cells Transl Med. 2, 584\u2013594 (2013).","journal-title":"Stem Cells Transl Med."},{"key":"18431_CR10","doi-asserted-by":"publisher","DOI":"10.1038\/srep41160","volume":"7","author":"DP Ivanov","year":"2017","unstructured":"Ivanov, D. P. & Grabowska, A. M. Spheroid arrays for high-throughput single-cell analysis of spatial patterns and biomarker expression in 3D. Sci Rep. 7, 41160, https:\/\/doi.org\/10.1038\/srep41160 (2017).","journal-title":"Sci Rep."},{"key":"18431_CR11","doi-asserted-by":"publisher","first-page":"108","DOI":"10.1016\/j.tibtech.2012.12.003","volume":"31","author":"E Fennema","year":"2013","unstructured":"Fennema, E., Rivron, N., Rouwkema, J., van Blitterswijk, C. & de Boer, J. Spheroid culture as a tool for creating 3D complex tissues. Trends Biotechnol. 31, 108\u2013115 (2013).","journal-title":"Trends Biotechnol."},{"key":"18431_CR12","doi-asserted-by":"publisher","first-page":"2015","DOI":"10.1089\/ten.tea.2008.0318","volume":"15","author":"J Rouwkema","year":"2009","unstructured":"Rouwkema, J., Westerweel, P. E., de Boer, J., Verhaar, M. C. & van Blitterswijk, C. A. The use of endothelial progenitor cells for prevascularized bone tissue engineering. Tissue Eng Part A. 15, 2015\u20132027 (2009).","journal-title":"Tissue Eng Part A."},{"key":"18431_CR13","doi-asserted-by":"publisher","first-page":"11","DOI":"10.1016\/j.stem.2015.06.007","volume":"17","author":"A Trounson","year":"2015","unstructured":"Trounson, A. & McDonald, C. Stem cell therapies in clinical trials: progress and challenges. Cell stem cell. 17, 11\u201322 (2015).","journal-title":"Cell stem cell."},{"key":"18431_CR14","doi-asserted-by":"publisher","first-page":"141","DOI":"10.1016\/j.stem.2014.01.013","volume":"14","author":"M Mendicino","year":"2014","unstructured":"Mendicino, M., Bailey, A. M., Wonnacott, K., Puri, R. K. & Bauer, S. R. MSC-based product characterization for clinical trials: an FDA perspective. Cell stem cell. 14, 141\u2013145 (2014).","journal-title":"Cell stem cell."},{"key":"18431_CR15","doi-asserted-by":"publisher","first-page":"7","DOI":"10.2217\/rme.11.112","volume":"7","author":"DA Brindley","year":"2012","unstructured":"Brindley, D. A. et al. Peak serum: implications of serum supply for cell therapy manufacturing. Regen Med. 7, 7\u201313 (2012).","journal-title":"Regen Med."},{"key":"18431_CR16","doi-asserted-by":"publisher","first-page":"155","DOI":"10.1016\/j.jcyt.2016.11.011","volume":"19","author":"O Karnieli","year":"2017","unstructured":"Karnieli, O. et al. A consensus introduction to serum replacements and serum-free media for cellular therapies. Cytotherapy. 19, 155\u2013169 (2017).","journal-title":"Cytotherapy."},{"key":"18431_CR17","doi-asserted-by":"publisher","DOI":"10.1038\/srep16570","volume":"5","author":"A Oikonomopoulos","year":"2015","unstructured":"Oikonomopoulos, A. et al. Optimization of human mesenchymal stem cell manufacturing: the effects of animal\/xeno-free media. Sci Rep. 5, 16570 (2015).","journal-title":"Sci Rep."},{"key":"18431_CR18","doi-asserted-by":"publisher","unstructured":"Ylostalo, J. H., Bazhanov, N., Mohammadipoor, A. & Bartosh, T. J. Production and administration of therapeutic mesenchymal stem\/stromal cell (MSC) spheroids primed in 3-D cultures under xeno-free conditions. J Vis Exp. 121, https:\/\/doi.org\/10.3791\/55126 (2017).","DOI":"10.3791\/55126"},{"key":"18431_CR19","doi-asserted-by":"publisher","first-page":"e99145","DOI":"10.1371\/journal.pone.0099145","volume":"9","author":"ML Chou","year":"2014","unstructured":"Chou, M. L., Burnouf, T. & Wang, T. J. Ex vivo expansion of bovine corneal endothelial cells in xeno-free medium supplemented with platelet releasate. PloS One. 9, e99145, https:\/\/doi.org\/10.1371\/journal.pone.0099145 (2014).","journal-title":"PloS One."},{"key":"18431_CR20","doi-asserted-by":"publisher","first-page":"6716","DOI":"10.1182\/blood-2008-09-181362","volume":"113","author":"A Reinisch","year":"2009","unstructured":"Reinisch, A. et al. Humanized large-scale expanded endothelial colony-forming cells function in vitro and in vivo. Blood. 113, 6716\u20136725 (2009).","journal-title":"Blood."},{"key":"18431_CR21","doi-asserted-by":"publisher","first-page":"712","DOI":"10.3109\/14653249.2010.548380","volume":"13","author":"L Huang","year":"2011","unstructured":"Huang, L., Critser, P. J., Grimes, B. R. & Yoder, M. C. Human umbilical cord blood plasma can replace fetal bovine serum for in vitro expansion of functional human endothelial colony-forming cells. Cytotherapy. 13, 712\u2013721 (2011).","journal-title":"Cytotherapy."},{"key":"18431_CR22","doi-asserted-by":"publisher","first-page":"e75224","DOI":"10.1371\/journal.pone.0075224","volume":"8","author":"SH Moon","year":"2013","unstructured":"Moon, S. H. et al. Development of a xeno-free autologous culture system for endothelial progenitor cells derived from human umbilical cord blood. PloS One. 8, e75224, https:\/\/doi.org\/10.1371\/journal.pone.0075224 (2013).","journal-title":"PloS One."},{"key":"18431_CR23","doi-asserted-by":"publisher","first-page":"28","DOI":"10.1186\/s13287-015-0016-2","volume":"6","author":"JM Diez","year":"2015","unstructured":"Diez, J. M., Bauman, E., Gajardo, R. & Jorquera, J. I. Culture of human mesenchymal stem cells using a candidate pharmaceutical grade xeno-free cell culture supplement derived from industrial human plasma pools. Stem Cell Res Ther. 6, 28 (2015).","journal-title":"Stem Cell Res Ther."},{"key":"18431_CR24","doi-asserted-by":"crossref","first-page":"36","DOI":"10.1002\/stem.248","volume":"28","author":"I Rodriguez-Piza","year":"2010","unstructured":"Rodriguez-Piza, I. et al. Reprogramming of human fibroblasts to induced pluripotent stem cells under xeno-free conditions. Stem Cells. 28, 36\u201344 (2010).","journal-title":"Stem Cells."},{"key":"18431_CR25","doi-asserted-by":"publisher","first-page":"103","DOI":"10.1186\/s13287-017-0552-z","volume":"8","author":"A Blazquez-Prunera","year":"2017","unstructured":"Blazquez-Prunera, A., Diez, J. M., Gajardo, R. & Grancha, S. Human mesenchymal stem cells maintain their phenotype, multipotentiality, and genetic stability when cultured using a defined xeno-free human plasma fraction. Stem Cell Res Ther. 8, 103 (2017).","journal-title":"Stem Cell Res Ther."},{"key":"18431_CR26","doi-asserted-by":"crossref","unstructured":"Cimino, M. et al. The optimization of the use of a pharmaceutical grade xeno-free medium for in vitro expansion of human mesenchymal stem\/stromal cells. In press (2017).","DOI":"10.1155\/2017\/6597815"},{"key":"18431_CR27","doi-asserted-by":"publisher","first-page":"352","DOI":"10.1007\/s10439-013-0953-9","volume":"42","author":"MA Kinney","year":"2014","unstructured":"Kinney, M. A., Hookway, T. A., Wang, Y. & McDevitt, T. C. Engineering three-dimensional stem cell morphogenesis for the development of tissue models and scalable regenerative therapeutics. Ann Biomed Eng. 42, 352\u2013367 (2014).","journal-title":"Ann Biomed Eng."},{"key":"18431_CR28","first-page":"2730472","volume":"2017","author":"L Xie","year":"2017","unstructured":"Xie, L., Mao, M., Zhou, L., Zhang, L. & Jiang, B. Signal factors secreted by 2D and spheroid mesenchymal stem cells and by cocultures of mesenchymal stem cells derived microvesicles and retinal photoreceptor neurons. Stem Cells Int. 2017, 2730472 (2017).","journal-title":"Stem Cells Int."},{"key":"18431_CR29","doi-asserted-by":"publisher","first-page":"222","DOI":"10.22203\/eCM.v026a16","volume":"26","author":"R Walser","year":"2013","unstructured":"Walser, R. et al. Generation of co-culture spheroids as vascularisation units for bone tissue engineering. Eur Cell Mater. 26, 222\u2013233 (2013).","journal-title":"Eur Cell Mater."},{"key":"18431_CR30","doi-asserted-by":"publisher","first-page":"879","DOI":"10.1038\/nbt1109","volume":"23","author":"S Levenberg","year":"2005","unstructured":"Levenberg, S. et al. Engineering vascularized skeletal muscle tissue. Nat Biotechnol. 23, 879\u2013884 (2005).","journal-title":"Nat Biotechnol."},{"key":"18431_CR31","doi-asserted-by":"publisher","first-page":"1296","DOI":"10.1177\/0022034514550040","volume":"93","author":"WL Dissanayaka","year":"2014","unstructured":"Dissanayaka, W. L., Zhu, L., Hargreaves, K. M., Jin, L. & Zhang, C. Scaffold-free prevascularized microtissue spheroids for pulp regeneration. J Dent Res. 93, 1296\u20131303 (2014).","journal-title":"J Dent Res."},{"key":"18431_CR32","doi-asserted-by":"publisher","first-page":"35","DOI":"10.1186\/s13018-015-0173-0","volume":"10","author":"D Murata","year":"2015","unstructured":"Murata, D. et al. A preliminary study of osteochondral regeneration using a scaffold-free three-dimensional construct of porcine adipose tissue-derived mesenchymal stem cells. J Orthop Surg Res. 10, 35 (2015).","journal-title":"J Orthop Surg Res."},{"key":"18431_CR33","doi-asserted-by":"publisher","first-page":"254","DOI":"10.1007\/s10856-015-5591-3","volume":"26","author":"H Suenaga","year":"2015","unstructured":"Suenaga, H., Furukawa, K. S., Suzuki, Y., Takato, T. & Ushida, T. Bone regeneration in calvarial defects in a rat model by implantation of human bone marrow-derived mesenchymal stromal cell spheroids. J Mater Sci Mater Med. 26, 254 (2015).","journal-title":"J Mater Sci Mater Med."},{"key":"18431_CR34","doi-asserted-by":"publisher","first-page":"2752","DOI":"10.1182\/blood-2004-04-1396","volume":"104","author":"DA Ingram","year":"2004","unstructured":"Ingram, D. A. et al. Identification of a novel hierarchy of endothelial progenitor cells using human peripheral and umbilical cord blood. Blood. 104, 2752\u20132760 (2004).","journal-title":"Blood."},{"key":"18431_CR35","doi-asserted-by":"publisher","first-page":"671","DOI":"10.1111\/j.1365-2443.2010.01409.x","volume":"15","author":"SM Zeisberger","year":"2010","unstructured":"Zeisberger, S. M. et al. Optimization of the culturing conditions of human umbilical cord blood-derived endothelial colony-forming cells under xeno-free conditions applying a transcriptomic approach. Genes Cells. 15, 671\u2013687 (2010).","journal-title":"Genes Cells."},{"key":"18431_CR36","doi-asserted-by":"publisher","first-page":"125","DOI":"10.1290\/1071-2690(2000)036<0125:HMECOO>2.0.CO;2","volume":"36","author":"TT Terramani","year":"2000","unstructured":"Terramani, T. T. et al. Human macrovascular endothelial cells: optimization of culture conditions. In Vitro Cell Dev Biol Anim. 36, 125\u2013132 (2000).","journal-title":"In Vitro Cell Dev Biol Anim."},{"key":"18431_CR37","doi-asserted-by":"publisher","first-page":"937","DOI":"10.1038\/nature04479","volume":"438","author":"L Coultas","year":"2005","unstructured":"Coultas, L., Chawengsaksophak, K. & Rossant, J. Endothelial cells and VEGF in vascular development. Nature 438, 937\u2013945 (2005).","journal-title":"Nature"},{"key":"18431_CR38","doi-asserted-by":"publisher","first-page":"2330","DOI":"10.1089\/ten.tea.2012.0750","volume":"19","author":"ML Skiles","year":"2013","unstructured":"Skiles, M. L., Sahai, S., Rucker, L. & Blanchette, J. O. Use of culture geometry to control hypoxia-induced vascular endothelial growth factor secretion from adipose-derived stem cells: optimizing a cell-based approach to drive vascular growth. Tissue Eng Part A. 19, 2330\u20132338 (2013).","journal-title":"Tissue Eng Part A."},{"key":"18431_CR39","doi-asserted-by":"publisher","first-page":"1486","DOI":"10.1016\/j.jcyt.2014.07.010","volume":"16","author":"JH Ylostalo","year":"2014","unstructured":"Ylostalo, J. H., Bartosh, T. J., Tiblow, A. & Prockop, D. J. Unique characteristics of human mesenchymal stromal\/progenitor cells pre-activated in 3-dimensional cultures under different conditions. Cytotherapy. 16, 1486\u20131500 (2014).","journal-title":"Cytotherapy."},{"key":"18431_CR40","doi-asserted-by":"publisher","first-page":"1216","DOI":"10.1126\/science.1176009","volume":"326","author":"RO Hynes","year":"2009","unstructured":"Hynes, R. O. The extracellular matrix: not just pretty fibrils. Science. 326, 1216\u20131219 (2009).","journal-title":"Science."},{"issue":"Suppl 1","key":"18431_CR41","doi-asserted-by":"publisher","first-page":"32","DOI":"10.1111\/j.1538-7836.2007.02569.x","volume":"5","author":"RO Hynes","year":"2007","unstructured":"Hynes, R. O. Cell-matrix adhesion in vascular development. J Thromb Haemost. 5(Suppl 1), 32\u201340 (2007).","journal-title":"J Thromb Haemost."},{"key":"18431_CR42","doi-asserted-by":"publisher","first-page":"1619","DOI":"10.1242\/dev.01037","volume":"131","author":"E Poschl","year":"2004","unstructured":"Poschl, E. et al. Collagen IV is essential for basement membrane stability but dispensable for initiation of its assembly during early development. Development. 131, 1619\u20131628 (2004).","journal-title":"Development."},{"key":"18431_CR43","doi-asserted-by":"publisher","first-page":"365","DOI":"10.1089\/ten.teb.2013.0537","volume":"20","author":"S Sart","year":"2014","unstructured":"Sart, S., Tsai, A. C., Li, Y. & Ma, T. Three-dimensional aggregates of mesenchymal stem cells: cellular mechanisms, biological properties, and applications. Tissue Eng Part B Rev. 20, 365\u2013380 (2014).","journal-title":"Tissue Eng Part B Rev."},{"key":"18431_CR44","doi-asserted-by":"publisher","first-page":"380","DOI":"10.1002\/path.3000","volume":"226","author":"ML Taddei","year":"2012","unstructured":"Taddei, M. L., Giannoni, E., Fiaschi, T. & Chiarugi, P. Anoikis: an emerging hallmark in health and diseases. J Pathol. 226, 380\u2013393 (2012).","journal-title":"J Pathol."},{"key":"18431_CR45","doi-asserted-by":"crossref","unstructured":"Saleh, F. A., Whyte, M. & Genever, P. G. Effects of endothelial cells on human mesenchymal stem cell activity in a three-dimensional in vitro model. Eur Cells Mater. 22, 242\u2013257; discussion 257 (2011).","DOI":"10.22203\/eCM.v022a19"},{"key":"18431_CR46","doi-asserted-by":"publisher","first-page":"7295","DOI":"10.1016\/j.biomaterials.2014.05.033","volume":"35","author":"SH Hsu","year":"2014","unstructured":"Hsu, S. H. et al. Substrate-dependent modulation of 3D spheroid morphology self-assembled in mesenchymal stem cell-endothelial progenitor cell coculture. Biomaterials. 35, 7295\u20137307 (2014).","journal-title":"Biomaterials."},{"key":"18431_CR47","doi-asserted-by":"publisher","first-page":"1097","DOI":"10.1042\/CBI20100718","volume":"35","author":"CW Eckermann","year":"2011","unstructured":"Eckermann, C. W., Lehle, K., Schmid, S. A., Wheatley, D. N. & Kunz-Schughart, L. A. Characterization and modulation of fibroblast\/endothelial cell co-cultures for the in vitro preformation of three-dimensional tubular networks. Cell Biol Int. 35, 1097\u20131110 (2011).","journal-title":"Cell Biol Int."},{"key":"18431_CR48","doi-asserted-by":"publisher","first-page":"1761","DOI":"10.1634\/stemcells.2007-0022","volume":"25","author":"IA Potapova","year":"2007","unstructured":"Potapova, I. A. et al. Mesenchymal stem cells support migration, extracellular matrix invasion, proliferation, and survival of endothelial cells in vitro. Stem cells. 25, 1761\u20131768 (2007).","journal-title":"Stem cells."},{"key":"18431_CR49","doi-asserted-by":"publisher","first-page":"C1385","DOI":"10.1152\/ajpcell.00248.2005","volume":"290","author":"LA Kunz-Schughart","year":"2006","unstructured":"Kunz-Schughart, L. A. et al. Potential of fibroblasts to regulate the formation of three-dimensional vessel-like structures from endothelial cells in vitro. Am J Physiol Cell Physiol. 290, C1385\u20131398 (2006).","journal-title":"Am J Physiol Cell Physiol."},{"key":"18431_CR50","doi-asserted-by":"publisher","first-page":"2685","DOI":"10.1089\/ten.2006.12.2685","volume":"12","author":"J Rouwkema","year":"2006","unstructured":"Rouwkema, J., de Boer, J. & Van Blitterswijk, C. A. Endothelial cells assemble into a 3-dimensional prevascular network in a bone tissue engineering construct. Tissue Eng. 12, 2685\u20132693 (2006).","journal-title":"Tissue Eng."},{"key":"18431_CR51","doi-asserted-by":"publisher","first-page":"332","DOI":"10.1002\/biof.46","volume":"35","author":"BJ Nieves","year":"2009","unstructured":"Nieves, B. J., D\u2019Amore, P. A. & Bryan, B. A. The function of vascular endothelial growth factor. Biofactors. 35, 332\u2013337 (2009).","journal-title":"Biofactors."},{"key":"18431_CR52","doi-asserted-by":"publisher","first-page":"98","DOI":"10.1186\/s13018-014-0098-z","volume":"9","author":"K Ishihara","year":"2014","unstructured":"Ishihara, K., Nakayama, K., Akieda, S., Matsuda, S. & Iwamoto, Y. Simultaneous regeneration of full-thickness cartilage and subchondral bone defects in vivo using a three-dimensional scaffold-free autologous construct derived from high-density bone marrow-derived mesenchymal stem cells. J Orthop Surg Res. 9, 98 (2014).","journal-title":"J Orthop Surg Res."},{"key":"18431_CR53","doi-asserted-by":"publisher","first-page":"155","DOI":"10.1089\/ten.tec.2010.0355","volume":"17","author":"F Urciuolo","year":"2011","unstructured":"Urciuolo, F., Imparato, G., Palmiero, C., Trilli, A. & Netti, P. A. Effect of process conditions on the growth of three-dimensional dermal-equivalent tissue obtained by microtissue precursor assembly. Tissue Eng Part C Methods. 17, 155\u2013164 (2011).","journal-title":"Tissue Eng Part C Methods."},{"key":"18431_CR54","doi-asserted-by":"publisher","first-page":"213","DOI":"10.1016\/j.jbiosc.2015.12.019","volume":"122","author":"T Okudaira","year":"2016","unstructured":"Okudaira, T., Amimoto, N., Mizumoto, H. & Kajiwara, T. Formation of three-dimensional hepatic tissue by the bottom-up method using spheroids. J Biosci Bioeng. 122, 213\u2013218 (2016).","journal-title":"J Biosci Bioeng."},{"key":"18431_CR55","doi-asserted-by":"publisher","first-page":"165","DOI":"10.1186\/s13287-016-0426-9","volume":"7","author":"A Al Madhoun","year":"2016","unstructured":"Al Madhoun, A. et al. Defined three-dimensional culture conditions mediate efficient induction of definitive endoderm lineage from human umbilical cord Wharton\u2019s jelly mesenchymal stem cells. Stem Cell Res Ther. 7, 165 (2016).","journal-title":"Stem Cell Res Ther."},{"key":"18431_CR56","doi-asserted-by":"publisher","first-page":"e0123437","DOI":"10.1371\/journal.pone.0123437","volume":"10","author":"PA Williams","year":"2015","unstructured":"Williams, P. A. et al. Hypoxia augments outgrowth endothelial cell (OEC) sprouting and directed migration in response to sphingosine-1-phosphate (S1P). PloS One. 10, e0123437, https:\/\/doi.org\/10.1371\/journal.pone.0123437 (2015).","journal-title":"PloS One."},{"key":"18431_CR57","first-page":"473","volume":"3","author":"B Page","year":"1993","unstructured":"Page, B., Page, M. & Noel, C. A new fluorometric assay for cytotoxicity measurements in-vitro. Int J Oncol. 3, 473\u2013476 (1993).","journal-title":"Int J Oncol."}],"container-title":["Scientific Reports"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.nature.com\/articles\/s41598-017-18431-6.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/www.nature.com\/articles\/s41598-017-18431-6","content-type":"text\/html","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/www.nature.com\/articles\/s41598-017-18431-6.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2022,12,24]],"date-time":"2022-12-24T01:52:08Z","timestamp":1671846728000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.nature.com\/articles\/s41598-017-18431-6"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2018,1,10]]},"references-count":57,"journal-issue":{"issue":"1","published-online":{"date-parts":[[2018,12]]}},"alternative-id":["18431"],"URL":"https:\/\/doi.org\/10.1038\/s41598-017-18431-6","relation":{},"ISSN":["2045-2322"],"issn-type":[{"value":"2045-2322","type":"electronic"}],"subject":[],"published":{"date-parts":[[2018,1,10]]},"assertion":[{"value":"24 August 2017","order":1,"name":"received","label":"Received","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"12 December 2017","order":2,"name":"accepted","label":"Accepted","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"10 January 2018","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":"230"}}