{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,1,20]],"date-time":"2026-01-20T22:59:36Z","timestamp":1768949976312,"version":"3.49.0"},"posted":{"date-parts":[[2025,9,19]]},"group-title":"Neuroscience","reference-count":54,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2025,9,19]],"date-time":"2025-09-19T00:00:00Z","timestamp":1758240000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/www.biorxiv.org\/about\/FAQ#license"}],"funder":[{"name":"NIH","award":["UH3-NS115064"],"award-info":[{"award-number":["UH3-NS115064"]}]},{"name":"NIH","award":["F32AG072813"],"award-info":[{"award-number":["F32AG072813"]}]},{"name":"NIH","award":["K01AG083734"],"award-info":[{"award-number":["K01AG083734"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2025,9,25]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>\n                  Development and delivery of treatments for neurological diseases are limited by the tight and selective human blood\u2013brain barrier (BBB). Although animal models have been important research and preclinical tools, the rodent BBB exhibits species differences and fails to capture the complexity of human genetics. Microphysiological systems incorporating human-derived cells hold great potential for modeling disease and therapeutic development, with advantages in screening throughput, real-time monitoring, and tunable genetic backgrounds when combined with induced pluripotent stem cell (iPSC) technology. Existing 3D BBB-on-chip systems have incorporated iPSC-derived endothelial cells but not the other major brain cell types from iPSCs, each of which contributes to brain physiology and disease. Here we developed a 3D Brain-Chip system incorporating endothelial cells, pericytes, astrocytes, neurons, microglia, and oligodendroglia from iPSCs. To enable this multicellular 3D co-culture in-chip, we designed a GelChip microfluidic platform using a 3D printing-based approach and dextran-based engineered hydrogel. Leveraging this platform, we co-cultured and characterized iPSC-derived brain-on-chips and modeled the brain microvasculature of\n                  <jats:italic>APOE4<\/jats:italic>\n                  , the strongest known genetic risk factor for sporadic Alzheimer\u2019s disease. These 3D brain-on-chips provide a versatile system to assess BBB vascular morphology and function, investigate downstream neurological effects in disease, and screen therapeutics to optimize delivery to the brain.\n                <\/jats:p>\n                <jats:sec>\n                  <jats:title>Significance Statement<\/jats:title>\n                  <jats:p>\n                    The blood\u2013brain barrier (BBB) is both a contributing factor to neurological disease and a major obstacle to its treatment, yet human-relevant models remain limited. Most existing brain-on-chip systems incorporate only subsets of BBB cell types and cannot capture the full cellular complexity of the human neurovascular unit. Here, we establish a vascular-perfusable 3D Brain-Chip using human induced pluripotent stem cell-derived brain cells including endothelial cells, pericytes, astrocytes, neurons, microglia, and oligodendroglia. This system enables systematic analysis of human genetic risk factors, such as\n                    <jats:italic>APOE4<\/jats:italic>\n                    in Alzheimer\u2019s disease, and provides a powerful platform to investigate BBB function and dysfunction and accelerate the development of more effective neurological therapies.\n                  <\/jats:p>\n                <\/jats:sec>","DOI":"10.1101\/2025.09.18.676925","type":"posted-content","created":{"date-parts":[[2025,9,20]],"date-time":"2025-09-20T01:15:16Z","timestamp":1758330916000},"source":"Crossref","is-referenced-by-count":2,"title":["Vascular-Perfusable Human 3D Brain-on-Chip"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-8981-0629","authenticated-orcid":false,"given":"Alice E.","family":"Stanton","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5369-2317","authenticated-orcid":false,"given":"Rebecca L.","family":"Pinals","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Aaron","family":"Choi","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Nhat","family":"Truong","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Eulim","family":"Kang","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Alan","family":"Jiang","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Claudia F.","family":"Lozano Cruz","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Sydney","family":"Hawkins","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Ryana","family":"Sarcar","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Alexandra","family":"Volkova","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Ois\u00edn","family":"King","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Emre","family":"Agbas","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Masayuki","family":"Nakano","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Ching-Chi","family":"Chiu","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Adele","family":"Bubnys","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Shannon","family":"Wright","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Colin","family":"Staab","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Rami","family":"Bikdash","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Emily","family":"Forden","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4255-0492","authenticated-orcid":false,"given":"Robert","family":"Langer","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1262-0592","authenticated-orcid":false,"given":"Li-Huei","family":"Tsai","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"54368","reference":[{"key":"2025092801050642000_2025.09.18.676925v2.1","doi-asserted-by":"publisher","DOI":"10.1001\/jama.2024.5157"},{"key":"2025092801050642000_2025.09.18.676925v2.2","first-page":"11","article-title":"The Need for New Approaches in CNS Drug Discovery: Why Drugs Have Failed, and What Can Be Done to Improve Outcomes","volume":"120","year":"2016","journal-title":"Neuropharmacology"},{"key":"2025092801050642000_2025.09.18.676925v2.3","doi-asserted-by":"publisher","DOI":"10.1602\/neurorx.2.1.3"},{"key":"2025092801050642000_2025.09.18.676925v2.4","doi-asserted-by":"publisher","DOI":"10.1038\/s41598-021-81132-8"},{"key":"2025092801050642000_2025.09.18.676925v2.5","first-page":"e230010","article-title":"Endothelial structure contributes to heterogeneity in brain capillary diameter","volume":"5","year":"2023","journal-title":"Vasc. Biol"},{"key":"2025092801050642000_2025.09.18.676925v2.6","first-page":"78","article-title":"Basement membrane and blood\u2013brain barrier","volume":"4","year":"2018","journal-title":"Stroke Vasc. Neurol"},{"key":"2025092801050642000_2025.09.18.676925v2.7","doi-asserted-by":"publisher","DOI":"10.1212\/WNL.0000000000207379"},{"key":"2025092801050642000_2025.09.18.676925v2.8","first-page":"150","article-title":"Involvement of Astrocytes in the Formation, Maintenance, and Function of the Blood\u2013Brain Barrier","volume":"13","year":"2024","journal-title":"Cells 2024 Vol 13 Page 150"},{"key":"2025092801050642000_2025.09.18.676925v2.9","doi-asserted-by":"publisher","DOI":"10.1146\/annurev-neuro-071714-033835"},{"key":"2025092801050642000_2025.09.18.676925v2.10","doi-asserted-by":"crossref","unstructured":"Bisht, K. et al. Capillary-associated microglia regulate vascular structure and function through PANX1-P2RY12 coupling in mice. Nat. Commun. 12, (2021).","DOI":"10.1038\/s41467-021-25590-8"},{"key":"2025092801050642000_2025.09.18.676925v2.11","doi-asserted-by":"crossref","first-page":"24","DOI":"10.1186\/s12987-023-00425-4","article-title":"Direct association with the vascular basement membrane is a frequent feature of myelinating oligodendrocytes in the neocortex","volume":"20","year":"2023","journal-title":"Fluids Barriers CNS"},{"key":"2025092801050642000_2025.09.18.676925v2.12","doi-asserted-by":"publisher","DOI":"10.1038\/s41598-020-59121-0"},{"key":"2025092801050642000_2025.09.18.676925v2.13","doi-asserted-by":"publisher","DOI":"10.36255\/EXONPUBLICATIONS.ALZHEIMERSDISEASE.2020.CH7"},{"key":"2025092801050642000_2025.09.18.676925v2.14","doi-asserted-by":"publisher","DOI":"10.1523\/JNEUROSCI.4707-08.2009"},{"key":"2025092801050642000_2025.09.18.676925v2.15","doi-asserted-by":"publisher","DOI":"10.1002\/glia.23138"},{"key":"2025092801050642000_2025.09.18.676925v2.16","first-page":"1","article-title":"Induced pluripotent stem cells (iPSCs): molecular mechanisms of induction and applications","volume":"9","year":"2024","journal-title":"Signal Transduct. Target. Ther. 2024 91"},{"key":"2025092801050642000_2025.09.18.676925v2.17","first-page":"455689","article-title":"A novel transwell blood brain barrier model using primary human cells","volume":"13","year":"2019","journal-title":"Front. Cell. Neurosci"},{"key":"2025092801050642000_2025.09.18.676925v2.18","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-019-09078-0"},{"key":"2025092801050642000_2025.09.18.676925v2.19","doi-asserted-by":"publisher","DOI":"10.1186\/s12987-021-00303-x"},{"key":"2025092801050642000_2025.09.18.676925v2.20","doi-asserted-by":"crossref","unstructured":"Lu, T. M. et al. Pluripotent stem cell-derived epithelium misidentified as brain microvascular endothelium requires ETS factors to acquire vascular fate. Proc. Natl. Acad. Sci. 118, (2021).","DOI":"10.1073\/pnas.2016950118"},{"key":"2025092801050642000_2025.09.18.676925v2.21","doi-asserted-by":"publisher","DOI":"10.1038\/s41598-018-23276-8"},{"key":"2025092801050642000_2025.09.18.676925v2.22","doi-asserted-by":"crossref","first-page":"205","DOI":"10.1007\/978-1-0716-1174-6_14","article-title":"3D Self-Organized Human Blood\u2013Brain Barrier in a Microfluidic Chip","volume":"2258","year":"2021","journal-title":"Methods Mol. Biol"},{"key":"2025092801050642000_2025.09.18.676925v2.23","doi-asserted-by":"crossref","first-page":"1251195","DOI":"10.3389\/fbioe.2023.1251195","article-title":"Accelerating the in vitro emulation of Alzheimer\u2019s disease-associated phenotypes using a novel 3D blood-brain barrier neurosphere co-culture model","volume":"11","year":"2023","journal-title":"Front. Bioeng. Biotechnol"},{"key":"2025092801050642000_2025.09.18.676925v2.24","doi-asserted-by":"crossref","first-page":"122864","DOI":"10.1016\/j.biomaterials.2024.122864","article-title":"Engineered 3D human neurovascular model of Alzheimer\u2019s disease to study vascular dysfunction","volume":"314","year":"2025","journal-title":"Biomaterials"},{"key":"2025092801050642000_2025.09.18.676925v2.25","doi-asserted-by":"crossref","first-page":"1422707","DOI":"10.3389\/fneur.2025.1422707","article-title":"Decoding the genetic blueprints of neurological disorders: disease mechanisms and breakthrough gene therapies","volume":"16","year":"2025","journal-title":"Front. Neurol"},{"key":"2025092801050642000_2025.09.18.676925v2.26","doi-asserted-by":"publisher","DOI":"10.1038\/s41588-024-01685-y"},{"key":"2025092801050642000_2025.09.18.676925v2.27","first-page":"472","article-title":"Roles of glial ion transporters in brain diseases","volume":"68","year":"2019","journal-title":"Glia"},{"key":"2025092801050642000_2025.09.18.676925v2.28","first-page":"1","article-title":"Role of neuroinflammation in neurodegeneration development","volume":"8","year":"2023","journal-title":"Signal Transduct. Target. Ther. 2023 81"},{"key":"2025092801050642000_2025.09.18.676925v2.29","doi-asserted-by":"publisher","DOI":"10.1001\/archneur.59.10.1524"},{"key":"2025092801050642000_2025.09.18.676925v2.30","doi-asserted-by":"publisher","DOI":"10.1155\/2022\/3201644"},{"key":"2025092801050642000_2025.09.18.676925v2.31","doi-asserted-by":"publisher","DOI":"10.1101\/2023.08.15.553453"},{"key":"2025092801050642000_2025.09.18.676925v2.32","first-page":"561429","article-title":"Impact of Culture Medium on Cellular Interactions in in vitro Coculture Systems","volume":"8","year":"2020","journal-title":"Front. Bioeng. Biotechnol"},{"key":"2025092801050642000_2025.09.18.676925v2.33","doi-asserted-by":"publisher","DOI":"10.1016\/j.biomaterials.2020.119921"},{"key":"2025092801050642000_2025.09.18.676925v2.34","doi-asserted-by":"publisher","DOI":"10.1038\/nmat3357"},{"key":"2025092801050642000_2025.09.18.676925v2.35","doi-asserted-by":"crossref","first-page":"1673","DOI":"10.1002\/bit.28075","article-title":"Bioprinting small-diameter vascular vessel with endothelium and smooth muscle by the approach of two-step crosslinking process","volume":"119","year":"2022","journal-title":"Biotechnol. Bioeng"},{"key":"2025092801050642000_2025.09.18.676925v2.36","doi-asserted-by":"publisher","DOI":"10.1016\/j.biomaterials.2018.10.023"},{"key":"2025092801050642000_2025.09.18.676925v2.37","first-page":"1","article-title":"A novel three-dimensional computer-assisted method for a quantitative study of microvascular networks of the human cerebral cortex","volume":"13","year":"2006","journal-title":"Microcirc. N. Y. N 1994"},{"key":"2025092801050642000_2025.09.18.676925v2.38","doi-asserted-by":"publisher","DOI":"10.1039\/C6LC00638H"},{"key":"2025092801050642000_2025.09.18.676925v2.39","doi-asserted-by":"publisher","DOI":"10.1016\/0095-8522(58)90041-2"},{"key":"2025092801050642000_2025.09.18.676925v2.40","doi-asserted-by":"publisher","DOI":"10.1016\/S0022-510X(02)00288-5"},{"key":"2025092801050642000_2025.09.18.676925v2.41","doi-asserted-by":"crossref","unstructured":"Montagne, A. et al. APOE4 leads to blood-brain barrier dysfunction predicting cognitive decline. Nature 581, (2020).","DOI":"10.1038\/s41586-020-2247-3"},{"key":"2025092801050642000_2025.09.18.676925v2.42","doi-asserted-by":"publisher","DOI":"10.1038\/jcbfm.2015.44"},{"key":"2025092801050642000_2025.09.18.676925v2.43","doi-asserted-by":"publisher","DOI":"10.1177\/0271678X20952012"},{"key":"2025092801050642000_2025.09.18.676925v2.44","doi-asserted-by":"crossref","unstructured":"Barisano, G. et al. A \u2018multi-omics\u2019 analysis of blood-brain barrier and synaptic dysfunction in APOE4 mice. J. Exp. Med. 219, (2022).","DOI":"10.1084\/jem.20221137"},{"key":"2025092801050642000_2025.09.18.676925v2.45","first-page":"512","article-title":"Apolipoprotein E controls cerebrovascular integrity via cyclophilin A","volume":"485","year":"2012","journal-title":"Nat. 2012 4857399"},{"key":"2025092801050642000_2025.09.18.676925v2.46","doi-asserted-by":"publisher","DOI":"10.1038\/jcbfm.2014.172"},{"key":"2025092801050642000_2025.09.18.676925v2.47","doi-asserted-by":"publisher","DOI":"10.1038\/nature11087"},{"key":"2025092801050642000_2025.09.18.676925v2.48","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-018-06301-2"},{"key":"2025092801050642000_2025.09.18.676925v2.49","doi-asserted-by":"publisher","DOI":"10.1016\/j.mvr.2008.08.004"},{"key":"2025092801050642000_2025.09.18.676925v2.50","doi-asserted-by":"publisher","DOI":"10.1115\/1.4025892"},{"key":"2025092801050642000_2025.09.18.676925v2.51","doi-asserted-by":"crossref","first-page":"8083","DOI":"10.1038\/s41598-017-07416-0","article-title":"A Low Permeability Microfluidic Blood-Brain Barrier Platform with Direct Contact between Perfusable Vascular Network and Astrocytes","volume":"7","year":"2017","journal-title":"Sci. Rep"},{"key":"2025092801050642000_2025.09.18.676925v2.52","doi-asserted-by":"publisher","DOI":"10.1016\/j.neuron.2018.05.008"},{"key":"2025092801050642000_2025.09.18.676925v2.53","doi-asserted-by":"publisher","DOI":"10.1038\/s41596-021-00635-w"},{"key":"2025092801050642000_2025.09.18.676925v2.54","doi-asserted-by":"crossref","first-page":"e2200143","DOI":"10.1002\/smtd.202200143","article-title":"A Robust Method for Perfusable Microvascular Network Formation In Vitro","volume":"6","year":"2022","journal-title":"Small Methods"}],"container-title":[],"original-title":[],"link":[{"URL":"https:\/\/syndication.highwire.org\/content\/doi\/10.1101\/2025.09.18.676925","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,1,15]],"date-time":"2026-01-15T09:37:08Z","timestamp":1768469828000},"score":1,"resource":{"primary":{"URL":"http:\/\/biorxiv.org\/lookup\/doi\/10.1101\/2025.09.18.676925"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,9,19]]},"references-count":54,"URL":"https:\/\/doi.org\/10.1101\/2025.09.18.676925","relation":{},"subject":[],"published":{"date-parts":[[2025,9,19]]},"subtype":"preprint"}}