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While in vitro patient-derived xenografts (PDX) lines are useful for studying GBM, they often exclude astrocytes and macrophages, which contribute significantly to tumor growth, invasion, and chemoradioresistance. Integrating these cells into tumor models is difficult due to their need for serum, which triggers GBM-PDX lines to lose their stem-like properties. The aim of this study was to develop a serum-free triculture model of GBM-PDX lines, normal human astrocytes (NHAs), and macrophages. Serum-free media alternatives were formulated for NHAs and identified for THP-1 macrophages, then combined with GBM PDX media to establish \u201cPSX,\u201d an experimental maintenance media. Cells were transitioned to serum-free media alternatives and functionally assessed through several parameters unique to each cell type. In addition to assessing GBM \u201cstemness,\u201d a custom 350-gene NanoString chip was used to assess differential gene expression in monocultured PDX cells versus PDX cells exposed to NHAs and macrophages. PSX maintained canonical function in astrocytes and macrophages while preserving the stem-like properties of GBM-PDX cells. Tri-culturing all three cells increased the expression of stemness-associated transcription factors and increased the expression of genes related to stemness and hypoxia in GBM cells. GBM PDX cells exposed to NHAs and macrophages in direct triculture exhibit increases in markers of stemness and hypoxia. These findings suggest that the serum-free triculture model presented herein may better recapitulate the tumoral heterogeneity of GBM in vitro, providing a novel model to utilize in current research.<\/jats:p>","DOI":"10.3390\/ijms26199335","type":"journal-article","created":{"date-parts":[[2025,9,25]],"date-time":"2025-09-25T08:56:44Z","timestamp":1758790604000},"page":"9335","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["A Novel Serum-Free Triculture Model of Glioblastoma, Astrocytes, and Macrophages"],"prefix":"10.3390","volume":"26","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-7240-8748","authenticated-orcid":false,"given":"Hasan","family":"Alrefai","sequence":"first","affiliation":[{"name":"Department of Radiation Oncology, Heersink School of Medicine, The University of Alabama at Birmingham, Birmingham, AL 35233, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4096-9716","authenticated-orcid":false,"given":"Lauren C.","family":"Nassour-Caswell","sequence":"additional","affiliation":[{"name":"Department of Radiation Oncology, Heersink School of Medicine, The University of Alabama at Birmingham, Birmingham, AL 35233, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7270-7898","authenticated-orcid":false,"given":"Manoj","family":"Kumar","sequence":"additional","affiliation":[{"name":"Department of Radiation Oncology, Heersink School of Medicine, The University of Alabama at Birmingham, Birmingham, AL 35233, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9361-5917","authenticated-orcid":false,"given":"Benjamin","family":"Lin","sequence":"additional","affiliation":[{"name":"Division of Neuropathology, Department of Pathology, Heersink School of Medicine, The University of Alabama at Birmingham, Birmingham, AL 35233, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Taylor L.","family":"Schanel","sequence":"additional","affiliation":[{"name":"Department of Radiation Oncology, Heersink School of Medicine, The University of Alabama at Birmingham, Birmingham, AL 35233, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nicholas J.","family":"Eustace","sequence":"additional","affiliation":[{"name":"Department of Radiation Oncology, City of Hope National Medical Center, Duarte, CA 91010, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5246-3371","authenticated-orcid":false,"given":"Jianqing","family":"Zhang","sequence":"additional","affiliation":[{"name":"Department of Radiation Oncology, Heersink School of Medicine, The University of Alabama at Birmingham, Birmingham, AL 35233, 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