Journal: Advanced Science

Article Title: Understanding Glioblastoma Dynamics Using 3D Organoids and Engineered Extracellular Matrix

doi: 10.1002/advs.202522926

Figure Lengend Snippet: Establishing stemness, proliferation, and hypoxia profiles in GSC organoids co‐cultured with endothelial cells. (A) Schematic of experimental design. Glioma stem‐like cells (pGSC: proneural, mGSC: mesenchymal) were cultured for either 3 or 14 days under three conditions: spheroid (pGSCs/mGSCs), Matrigel spheroid (pGSCms/mGSCms), and Matrigel spheroid co‐cultured with endothelial cells (EC) via Transwell inserts (pGSCms/EC, mGSCms/EC). Schematic was designed in Adobe Illustator using icons sourced from Biorender.com. (B) Representative immunofluorescence images and quantification of percent area of fluorescent signal (normalized to DAPI area) for markers of stemness (CD133), proliferation (Ki67), and hypoxia (CAIX) at Days 3 and 14. Marker signal is shown in red; nuclei are counterstained with DAPI (blue). Scale bars = 500 µm. Data are presented as mean ± SD ( N = 3) with the following exceptions due to sample loss: mGSCmsec‐d03 (N = 2). Statistical analysis was performed using two‐way ANOVA with Tukey's post hoc test (* p ≤ 0.05, ** p ≤ 0.005, *** p ≤ 0.0005, **** p ≤ 0.00005).

Article Snippet: Co‐culture medium was prepared using Endothelial Cell Basal Medium (PromoCell, Cat. No. C‐22210) supplemented with the following components: 1× GlutaMAX (from 100× stock; Thermo Fisher Scientific, Cat. No. 35050061), 1× antibiotic‐antimycotic (from 100× stock; Thermo Fisher Scientific, Cat. No. 15240096), 2× B‐27 Supplement minus vitamin A (from 50× stock; Thermo Fisher Scientific, Cat. No. 12587010), 20 ng/mL human EGF recombinant protein (Cat. No. PHG0311L), 20 ng/mL human FGF‐basic recombinant protein (Cat. No. PHG0261), 0.2 μg/mL hydrocortisone (from 50 μg/mL stock; PromoCell, Cat. No. C‐39211), 0.4% endothelial cell growth supplement (PromoCell, Cat. No. C‐39210), and 0.5 ng/mL VEGF 165 (from 10 μg/mL stock; PromoCell, Cat. No. C‐39211).

Techniques: Cell Culture, Immunofluorescence, Marker

Journal: Advanced Science

Article Title: Understanding Glioblastoma Dynamics Using 3D Organoids and Engineered Extracellular Matrix

doi: 10.1002/advs.202522926

Figure Lengend Snippet: Endothelial co‐culture enhances lineage differentiation of GSC Matrigel spheroids. GSC (pGSC: proneural; mGSC: mesenchymal) were cultured for either 3 or 14 days as spheroids (pGSCs/mGSCs), Matrigel spheroids (pGSCms/mGSCms), or Matrigel spheroids co‐cultured with EC via Transwell inserts (pGSCms/EC, mGSCms/EC). Samples were fixed and immunostained for astrocytic (GFAP), oligodendrocytic (MOG), pericyte (CD248), and endothelial (CD31) markers. Representative immunofluorescence images show red signal for the respective markers and blue for DAPI nuclear staining. Quantification of the percent area of fluorescent signal (normalized to DAPI area) demonstrated a significant increase in marker expression over time and in the presence of EC, indicating enhanced lineage differentiation within the Matrigel co‐culture system. Scale bars = 500 µm. Data are presented as mean ± SD ( N = 3) with the following exceptions due to sample loss: mGSCmsec‐d03 (N = 2), pGSCmsec‐d03 (N = 2), and mGSCms‐d14 (N = 2). Statistical analysis was performed using two‐way ANOVA with Tukey's post hoc test (* p ≤ 0.05, ** p ≤ 0.005, *** p ≤ 0.0005, **** p ≤ 0.00005).

Article Snippet: Co‐culture medium was prepared using Endothelial Cell Basal Medium (PromoCell, Cat. No. C‐22210) supplemented with the following components: 1× GlutaMAX (from 100× stock; Thermo Fisher Scientific, Cat. No. 35050061), 1× antibiotic‐antimycotic (from 100× stock; Thermo Fisher Scientific, Cat. No. 15240096), 2× B‐27 Supplement minus vitamin A (from 50× stock; Thermo Fisher Scientific, Cat. No. 12587010), 20 ng/mL human EGF recombinant protein (Cat. No. PHG0311L), 20 ng/mL human FGF‐basic recombinant protein (Cat. No. PHG0261), 0.2 μg/mL hydrocortisone (from 50 μg/mL stock; PromoCell, Cat. No. C‐39211), 0.4% endothelial cell growth supplement (PromoCell, Cat. No. C‐39210), and 0.5 ng/mL VEGF 165 (from 10 μg/mL stock; PromoCell, Cat. No. C‐39211).

Techniques: Co-Culture Assay, Cell Culture, Immunofluorescence, Staining, Marker, Expressing

Journal: Advanced Science

Article Title: Understanding Glioblastoma Dynamics Using 3D Organoids and Engineered Extracellular Matrix

doi: 10.1002/advs.202522926

Figure Lengend Snippet: Establishing the engineered extracellular matrix (eECM) encapsulation and endothelial co‐culture model (A) Schematic representation of the experimental workflow. Glioblastoma stem‐like cells (GSC) were cultured as Matrigel spheroids (pGSCms, mGSCms) for 14 days, then encapsulated in engineered extracellular matrix (eECM) and maintained alone (pGSCems, mGSCems) or co‐cultured with HUVEC (pGSCems/EC, mGSCems/EC) for an additional 3 or 14 days. Schematic were created using Adobe Illustator with icons borrowed from Biorender.com. (B) Phase contrast images of GSC grown in all 3D culture conditions (GSC, GSCms, GSCms/EC, GSCems, and GSCems/EC) used at days 3 and 14. (C) Representative images of encapsulated pGSC and mGSC Matrigel spheroids that were co‐cultured with HUVEC for 14 days before being stained for GFAP (red). Images were enhanced by overlaying the GFAP fluorescent Matrigel spheroid over the same sample's phase contrast image showing the encapsulation.

Article Snippet: Co‐culture medium was prepared using Endothelial Cell Basal Medium (PromoCell, Cat. No. C‐22210) supplemented with the following components: 1× GlutaMAX (from 100× stock; Thermo Fisher Scientific, Cat. No. 35050061), 1× antibiotic‐antimycotic (from 100× stock; Thermo Fisher Scientific, Cat. No. 15240096), 2× B‐27 Supplement minus vitamin A (from 50× stock; Thermo Fisher Scientific, Cat. No. 12587010), 20 ng/mL human EGF recombinant protein (Cat. No. PHG0311L), 20 ng/mL human FGF‐basic recombinant protein (Cat. No. PHG0261), 0.2 μg/mL hydrocortisone (from 50 μg/mL stock; PromoCell, Cat. No. C‐39211), 0.4% endothelial cell growth supplement (PromoCell, Cat. No. C‐39210), and 0.5 ng/mL VEGF 165 (from 10 μg/mL stock; PromoCell, Cat. No. C‐39211).

Techniques: Encapsulation, Co-Culture Assay, Cell Culture, Staining

Journal: Advanced Science

Article Title: Understanding Glioblastoma Dynamics Using 3D Organoids and Engineered Extracellular Matrix

doi: 10.1002/advs.202522926

Figure Lengend Snippet: Engineered extracellular matrix (eECM) encapsulation and endothelial co‐culture promote GSC differentiation and stemness loss. The top row displays IF staining and quantification of stemness (CD133), proliferation (Ki67), and hypoxia (CAIX) markers in pGSC and mGSC spheroids under encapsulated and co‐culture conditions. Bottom row shows IF staining and quantification of astrocytic (GFAP), pericytic (CD248), and endothelial (CD31) differentiation markers, highlighting enhanced lineage specification with EC co‐culture. Data are presented as mean ± SD ( N = 3) with the following exceptions due to sample loss: pGSCemsec‐d03 (N = 2) and mGSCems‐d03 (N = 2). Statistical analysis was performed using two‐way ANOVA with Tukey's post hoc test (* p ≤ 0.05, ** p ≤ 0.005, *** p ≤ 0.0005, **** p ≤ 0.00005).

Article Snippet: Co‐culture medium was prepared using Endothelial Cell Basal Medium (PromoCell, Cat. No. C‐22210) supplemented with the following components: 1× GlutaMAX (from 100× stock; Thermo Fisher Scientific, Cat. No. 35050061), 1× antibiotic‐antimycotic (from 100× stock; Thermo Fisher Scientific, Cat. No. 15240096), 2× B‐27 Supplement minus vitamin A (from 50× stock; Thermo Fisher Scientific, Cat. No. 12587010), 20 ng/mL human EGF recombinant protein (Cat. No. PHG0311L), 20 ng/mL human FGF‐basic recombinant protein (Cat. No. PHG0261), 0.2 μg/mL hydrocortisone (from 50 μg/mL stock; PromoCell, Cat. No. C‐39211), 0.4% endothelial cell growth supplement (PromoCell, Cat. No. C‐39210), and 0.5 ng/mL VEGF 165 (from 10 μg/mL stock; PromoCell, Cat. No. C‐39211).

Techniques: Encapsulation, Co-Culture Assay, Staining