Journal: Bioactive Materials

Article Title: Multimodal profiling of CAR T cells against glioblastoma using a microengineered 3D tumor-on-a-chip model

doi: 10.1016/j.bioactmat.2026.01.003

Figure Lengend Snippet: Generation of CAR T cell workflow, assessment of CAR transduction, and quantification of on-target antigens on U87. A) Pictographic representation of timeline for CAR T cell culturing and functional assessment. B) Flow cytometric gating strategy of representative donor to quantify CAR transduction applicable to both IL-13 and TV-13 CAR transduced cells. C) Comparative CAR expression distinguished between CD4 and CD8 from a representative donor of Control T Cells (UTD), TV-13, and IL-13 CARs. D) Flow cytometric verification of IL13Rα1 and IL13Rα2 expression on U87 cells.

Article Snippet: U87 Culture : The Uppsala 87 (U87) Malignant Glioma cell line (HTB-14, ATCC) performed as the target tumor for this study was cultured in complete media composed of Eagle's minimum essential medium (EMEM) with L-Glutamine, and supplemented with 10 % FBS, 1 % HEPES, and 1 % penicillin-streptomycin.

Techniques: Transduction, Cell Culture, Functional Assay, Expressing, Control

Journal: Bioactive Materials

Article Title: Multimodal profiling of CAR T cells against glioblastoma using a microengineered 3D tumor-on-a-chip model

doi: 10.1016/j.bioactmat.2026.01.003

Figure Lengend Snippet: 2D in vitro cytotoxic assessment of CARs polyfunctionality. A) Workflow for intracellular cytokine assay. Created with BioRender.com . B) Flow cytometric gating strategy of the representative donor to identify CAR + T cells from viable singlets. C) Comparative release of IL-2 and TNF-α by CAR + T cells from the representative donor between UTD, TV-13, and IL-13 CAR transduced cells. D ) Comparative release of IFN-γ from the representative between UTD, TV-13, and IL-13 CAR T cells. E ) Graphical display of perforin and granzyme B release ( n = 3 ). ∗ p < 0.05. F) Quantification of the amount of INF-γ released into bulk media across UTD, TV-13, and IL-13 ( n = 3 ). One-way ANOVA with Tukey's multiple comparisons test was utilized for statistical analysis. G) Lactate Dehydrogenase (LDH) based quantification rate of tumor lysis across different T cell treatment conditions ( n = 3 ). One-way ANOVA with Tukey's multiple comparisons test was utilized for statistical analysis. H) Simplified Presentation of Incredibly Complex Evaluations (SPICE) analysis showing the number of intracellular cytokines (TNF-α, IFN-γ, and IL-2) produced per T cell by TV-13 and IL-13 CAR T cells, in response to U87 target stimulation indicating their polyfunctionality. The purple quadrant denotes the percentage of T cells producing all three cytokines, green represents cells producing two cytokines, blue denotes cells producing one, and grey represents cells producing none. Comparable levels of polyfunctionality were observed between the TV-13 and IL-13 groups. Data collected from three biological replicates ( n = 3 ).

Article Snippet: U87 Culture : The Uppsala 87 (U87) Malignant Glioma cell line (HTB-14, ATCC) performed as the target tumor for this study was cultured in complete media composed of Eagle's minimum essential medium (EMEM) with L-Glutamine, and supplemented with 10 % FBS, 1 % HEPES, and 1 % penicillin-streptomycin.

Techniques: In Vitro, Cytokine Assay, Lysis, Produced

Journal: Bioactive Materials

Article Title: Multimodal profiling of CAR T cells against glioblastoma using a microengineered 3D tumor-on-a-chip model

doi: 10.1016/j.bioactmat.2026.01.003

Figure Lengend Snippet: Formation of 3D self-assembled microvascular network (μVN) and its influence on U87 cells. A) Establishment of the μVN. (i) Schematic representation detailing the formation of the self-assembled μVN, and (ii) Representative phase contrast tile image of the device showing the progression of μVN formation on day 0 (left) and day 7 (right). B) Characterization of the μVN. (i) 10X tile image of vascular region stained for endothelial marker CD31 (green), junctional protein CD144 (red), and counterstained for nuclei with DAPI (blue) (scale bar: 200 μm), (ii) Phase contrast region of interest (ROI) image highlighting the vascular bundle formed within the vascular region (left), alongside 20X immunofluorescent image showing the expression of CD31(middle), and wrapping of pericytes (α-SMA) around the vascular bundle (right). Scale bars: 100 μm. C) orthogonal sectioning of established μVN confirming the open lumen formation (white arrowhead indicates the open lumen in the orthogonal view). Scale bar: 50 μm. D) Representative immunofluorescent and phase contrast overlap image after injection of 70 kDa fluorescent dextran dye captured at 30s, 1,2, and 4min. Scale bars: 100 μm. E) Line graph image of co-localization of pericytes with endothelial cells based on the scan line (white line) from figure Bii (right). F) Representative immunofluorescent image captured after perfusion of 2 μm fluorescent bead (red) through the CD31 (green) stained vascular bundle. Scale bar: 100 μm. G) Characterization of the μVN in the presence of tumor cells. (i) 10X tile image showing the intact μVN in the vascular (V) region and the migration of the tumor cells (U87-green) from the tumor (T) to the stroma (S) region. Yellow dashed trapezoids and hexagons mark the microposts of the 3D GOC. Scale bar: 200 μm, and (ii) Orthogonal sectioning of the vascular region confirming the maintenance of lumens post U87 injection (white arrowhead indicates the open lumen with white dashed box showing a zoomed-in lumen). Scale bar: 50 μm. Actin acquired with Alexa 647 and CD31 stained with Alexa 555 were pseudo colored in gray and magenta, respectively, for visualization. T, S, V represent the tumor, stroma, and vascular regions of the GOC system.

Article Snippet: U87 Culture : The Uppsala 87 (U87) Malignant Glioma cell line (HTB-14, ATCC) performed as the target tumor for this study was cultured in complete media composed of Eagle's minimum essential medium (EMEM) with L-Glutamine, and supplemented with 10 % FBS, 1 % HEPES, and 1 % penicillin-streptomycin.

Techniques: Staining, Marker, Expressing, Injection, Migration

Journal: Bioactive Materials

Article Title: Multimodal profiling of CAR T cells against glioblastoma using a microengineered 3D tumor-on-a-chip model

doi: 10.1016/j.bioactmat.2026.01.003

Figure Lengend Snippet: Evaluation of cytotoxic abilities of T cells against GBM cells within the GOC model. A) Microfluidic 3D invasion assay. (i) Schematic representation depicting the culture of tumor cells with T cells on day 0 (top) and day 3 (bottom), (ii) Representative phase contrast tile image overlapped with GFP (tumor cells) channel captured on day 0 to show the distribution of tumor and T cells across the experimental conditions (Scale bars: 200 μm), and (iii) Representative phase contrast tile image overlapped with GFP channel showing the migration of the U87 cells (green) from the tumor region to the stroma region across three different T cell populations. The densities of U87 are kept consistent across all conditions, and the density of T cells varies from 4 × 10 6 to 15 × 10 6 cells/mL. Images were captured 72 h after the interaction of cells within the GOC model (Scale bars: 200 μm). T-tumor, S-stroma, and V-vascular regions of GOC. B) Assessment of tumor cell migration in the presence of different T cells. (i) Quantification of migration distance from the 3D microfluidic model showing dose-dependent inhibition of U87 migration by the CAR T cells. Data were measured on Day 3 from three biological replicates ( n = 3 ) and represented as mean ± SD, T cell donors: DN26, DN28, and DN31, ∗ p < 0.05, ∗∗∗ ∗p < 0.0001. Two-way ANOVA with Tukey's multiple comparisons test was utilized for statistical analysis, and (ii) Comparison of migration distance of the U87 cells in the presence of different concentrations of the T cell population. Analysis performed on samples captured on Day 3 of migration ( n = 3 ) and represented as mean ± SD, T cell donors: DN26, DN28, and DN31, ∗ p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗ ∗p < 0.0001. Two-way ANOVA with Tukey's multiple comparisons test was utilized for statistical analysis. C) xCELLigence-based real-time evaluation of T cell cytolytic capacity. (i) Time-course of the average cell index ( n = 3 donors ) for UTD, TV-13, and IL-13 CAR T cell groups under a 10:1 E: T condition over a 7-day co-culture, measured using the xCELLigence platform, (ii) Bar plot of xCELLigence data comparing averaged cell index values of tumor cells at Day 0 and Day 7 across UTD, TV-13, and IL-13 CAR T cell groups. Data represent mean ± SEM ( n = 3 donors ), ∗ p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗ ∗p < 0.0001, Two-way ANOVA with Tukey's multiple comparisons test was utilized for statistical analysis, (iii) xCELLigence data from a representative donor (Donor 31) showing dose-dependent killing of U87 cells achieved by five doses of TV-13 CAR T cells, and (iv) IL-13 CAR T cells during a 7-day co-culture period.

Article Snippet: U87 Culture : The Uppsala 87 (U87) Malignant Glioma cell line (HTB-14, ATCC) performed as the target tumor for this study was cultured in complete media composed of Eagle's minimum essential medium (EMEM) with L-Glutamine, and supplemented with 10 % FBS, 1 % HEPES, and 1 % penicillin-streptomycin.

Techniques: Invasion Assay, Migration, Inhibition, Comparison, Co-Culture Assay

Journal: Bioactive Materials

Article Title: Multimodal profiling of CAR T cells against glioblastoma using a microengineered 3D tumor-on-a-chip model

doi: 10.1016/j.bioactmat.2026.01.003

Figure Lengend Snippet: Assessment of migratory behavior and proliferative potential of GBM tumor cells in the presence of engineered T cells. A) Evaluation of changes in migratory behavior of tumor cells across UTD, TV-13, and IL-13 T cells based on cytoskeletal organization. (i) Representative tile image of the 3D GOC model stained for actin cytoskeleton (red) showing the tumor-stroma-vascular interface (left), zoomed-in view highlighting the chain migration of the tumor cells from the tumor to the stroma region (middle), 20X region of interest (ROI) showing the disruption in the migratory pattern of the tumor cells and the formation of immune synapse (IS) (right). The white dashed box represents the ROIs alongside an inset image (ROI1) that highlights the formation of multiple IS between the tumor (green) and T cell within the stroma interface. The white arrow shows the IS formation, and the white dashed arrow represents the line scan utilized for intensity profiling to confirm the reorganization of actin cytoskeleton at the tumor-T cell interface . Red- Actin, Green- U87 cells, and DAPI – Blue . Scale bars: 200 μm (left and middle), 50 μm (right). (ii) Quantification of the number of cells migrating in a chain from near and far regions across three different T cell conditions . Data are represented as mean ± SD measured from three biological replicates ( n = 3 ), T cell donors: DN26, DN28, and DN31, ∗∗∗ p < 0.001 , ∗∗∗ ∗p < 0.0001. Two-way ANOVA with Tukey's multiple comparisons test was utilized for statistical analysis, (iii) Quantification of the number of cells within a field of view (FOV) from two distinct areas, namely near and far regions, Data are represented as mean ± SD measured from three biological replicated ( n = 3 ), T cell donors: DN26, DN28, and DN31, ∗∗∗ ∗p < 0.0001. Two-way ANOVA with Tukey's multiple comparisons test was utilized for statistical analysis. B) Immunofluorescent images of the devices stained for proliferation marker Ki-67. (i) Representative 20X ROI image showing the Ki-67 (red) expression on U87 cells (green) and (ii) Quantification of the number of Ki-67 positive cells across each condition through the proliferative index (Ki-67/Nuclei Ratio), Data are represented as mean ± SD measured from three biological replicates ( n = 3 ), T cell donors: DN26, DN28, and DN31, ∗∗ p < 0.01. One-way ANOVA with Tukey's multiple comparisons test was utilized for statistical analysis.

Article Snippet: U87 Culture : The Uppsala 87 (U87) Malignant Glioma cell line (HTB-14, ATCC) performed as the target tumor for this study was cultured in complete media composed of Eagle's minimum essential medium (EMEM) with L-Glutamine, and supplemented with 10 % FBS, 1 % HEPES, and 1 % penicillin-streptomycin.

Techniques: Staining, Migration, Disruption, Marker, Expressing

Journal: Pharmaceutics

Article Title: Employing a Combination of Chemoattractants to Trap Glioblastoma Cells in a Macroporous Hydrogel

doi: 10.3390/pharmaceutics18020229

Figure Lengend Snippet: Determination of the optimal CXCL12 concentration for CXCR4 receptor activation in GBM cells. ( A ) The effect of CXCL12 at various concentrations (2, 4, 6, 8, and 10 nM) on activation of the CXCR4+ receptor measured by using the Fluo-4 NW Calcium Assay kit in U87, U87 CXCR4+, F98 and U118 glioma cell lines. ( B ) Inhibition of CXCR4 receptor activation by the allosteric antagonist AMD11070 .

Article Snippet: The murine cell line F98 and human cell lines U87 and U118 were obtained from the American Type Culture Collection (Manassas, VA, USA), while the U87 CXCR4+ cells were provided by the NIH Reagent Program, catalog # 4036 (Germantown, MD, USA).

Techniques: Concentration Assay, Activation Assay, Calcium Assay, Inhibition

Journal: Pharmaceutics

Article Title: Employing a Combination of Chemoattractants to Trap Glioblastoma Cells in a Macroporous Hydrogel

doi: 10.3390/pharmaceutics18020229

Figure Lengend Snippet: Relative expression of CXCR4 , normalized to housekeeping genes, assessed by semi-quantitative PCR in the cell lines U87, U87 CXCR4+, F98, and U118 after 24 h of incubation, with or without 10 ng/mL of IL-1β (* p < 0.05; ** p < 0.01).

Article Snippet: The murine cell line F98 and human cell lines U87 and U118 were obtained from the American Type Culture Collection (Manassas, VA, USA), while the U87 CXCR4+ cells were provided by the NIH Reagent Program, catalog # 4036 (Germantown, MD, USA).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Incubation

Journal: Pharmaceutics

Article Title: Employing a Combination of Chemoattractants to Trap Glioblastoma Cells in a Macroporous Hydrogel

doi: 10.3390/pharmaceutics18020229

Figure Lengend Snippet: Relative production levels of matrix metalloproteinases -2 and -9 (MMP-2 and -9) were measured in U87, U87 CXCR4+, F98, and U118 cell lines by gel zymography. The GBM cells were incubated with either with IL-1β (10 ng/mL), IL-6 (20 ng/mL), EGF (20 ng/mL), or CXCL12 (100 ng/mL), both individually and in combination. The first well of each zymography gel was loaded with MMP-2 (0.07 ng/well) or MMP-9 (0.3 ng/well) to confirm their location and to calculate their relative quantities in the other wells (* p < 0.05; ** p < 0.01; *** p < 0.001).

Article Snippet: The murine cell line F98 and human cell lines U87 and U118 were obtained from the American Type Culture Collection (Manassas, VA, USA), while the U87 CXCR4+ cells were provided by the NIH Reagent Program, catalog # 4036 (Germantown, MD, USA).

Techniques: Zymography, Incubation

Journal: Pharmaceutics

Article Title: Employing a Combination of Chemoattractants to Trap Glioblastoma Cells in a Macroporous Hydrogel

doi: 10.3390/pharmaceutics18020229

Figure Lengend Snippet: Induction of epithelial–mesenchymal transition in GBM cells in response to EGF. ( A ) Representative microscopic observations ( n = 3) of morphological changes in U87 cells in presence of 20 ng/mL EGF after 48 and 72 h. ( B ) Relative expression of E-cadherin and N-cadherin , normalized to housekeeping genes, in U87 cells at 48 and 72 h in presence of 20 ng/mL EGF (* p < 0.05; ** p < 0.01).

Article Snippet: The murine cell line F98 and human cell lines U87 and U118 were obtained from the American Type Culture Collection (Manassas, VA, USA), while the U87 CXCR4+ cells were provided by the NIH Reagent Program, catalog # 4036 (Germantown, MD, USA).

Techniques: Expressing

Journal: Pharmaceutics

Article Title: Employing a Combination of Chemoattractants to Trap Glioblastoma Cells in a Macroporous Hydrogel

doi: 10.3390/pharmaceutics18020229

Figure Lengend Snippet: Migration of U87, U87 CXCR4+, and U118 cells in the absence and presence of different combinations of cytokines at 48 h (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001). T = 0 (light gray bars) corresponds to the initial distribution of GBM cells in the upper Matrigel layer.

Article Snippet: The murine cell line F98 and human cell lines U87 and U118 were obtained from the American Type Culture Collection (Manassas, VA, USA), while the U87 CXCR4+ cells were provided by the NIH Reagent Program, catalog # 4036 (Germantown, MD, USA).

Techniques: Migration

Journal: Pharmaceutics

Article Title: Employing a Combination of Chemoattractants to Trap Glioblastoma Cells in a Macroporous Hydrogel

doi: 10.3390/pharmaceutics18020229

Figure Lengend Snippet: Wound healing assay to confirm the enhancement in GBM cell migration by cytokines. ( A ) Representative observation of the results obtained for the scratch assay for U87 cells in the absence and presence of the combination of IL-1β, CXCL12, and EGF at 0, 24, and 48 h (×25 magnification, n = 3). ( B ) Percentage scratch closure at different time intervals in the U87, U87-CXCR4+, F98, and U118 cell lines (* p < 0.05; ** p < 0.01; *** p < 0.001).

Article Snippet: The murine cell line F98 and human cell lines U87 and U118 were obtained from the American Type Culture Collection (Manassas, VA, USA), while the U87 CXCR4+ cells were provided by the NIH Reagent Program, catalog # 4036 (Germantown, MD, USA).

Techniques: Wound Healing Assay, Migration

Journal: Pharmaceutics

Article Title: Employing a Combination of Chemoattractants to Trap Glioblastoma Cells in a Macroporous Hydrogel

doi: 10.3390/pharmaceutics18020229

Figure Lengend Snippet: ( A ) A schematic illustration depicting GBM cells embedded in a layer of Matrigel, which is placed on the top surface of a hydrogel. The hydrogel is positioned on a porous membrane inside a migration chamber insert. A combination of cytokines was introduced into the lower compartment of the migration chamber. ( B ) Representative images ( n = 3) captured with the EVOS™ FL Auto Imaging System epifluorescence microscope of U87 cells accumulated at the L3 layer of the hydrogel composed of 1% alginate, 0.75% chitosan, and 0.05% genipin, and grafted with the adhesion peptide RGD. A magnified view of U87 cells in the hydrogel after 72 h of incubation with the cytokine combination IL-1β + CXCL12 + EGF.

Article Snippet: The murine cell line F98 and human cell lines U87 and U118 were obtained from the American Type Culture Collection (Manassas, VA, USA), while the U87 CXCR4+ cells were provided by the NIH Reagent Program, catalog # 4036 (Germantown, MD, USA).

Techniques: Membrane, Migration, Imaging, Microscopy, Incubation

Journal: Pharmaceutics

Article Title: Employing a Combination of Chemoattractants to Trap Glioblastoma Cells in a Macroporous Hydrogel

doi: 10.3390/pharmaceutics18020229

Figure Lengend Snippet: Migration and accumulation of U87 and U118 cells in the 1% alginate, 0.75% chitosan, and 0.05% genipin hydrogels after 72 h of incubation, in the absence or presence of the cytokine combination of IL-1β + CXCL12 + EGF (* p < 0.05; ** p < 0.01; **** p < 0.0001).

Article Snippet: The murine cell line F98 and human cell lines U87 and U118 were obtained from the American Type Culture Collection (Manassas, VA, USA), while the U87 CXCR4+ cells were provided by the NIH Reagent Program, catalog # 4036 (Germantown, MD, USA).

Techniques: Migration, Incubation