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

Journal: Advanced Materials (Deerfield Beach, Fla.)

Article Title: Multiphysics‐Driven Assembly of Biomimetic Vesicles

doi: 10.1002/adma.202518755

Figure Lengend Snippet: The representative (a) design of HB patterns (W HB : 10 µm, G HB : 10 µm with θ: SYM45°, SYM60°, and ST45°) and (b) schematic of assembled hink θ ‐µ‐chips connected to a peristaltic pump circulating the floating cells. (c) The U87 cell rupture efficiency in the µ‐chips (θ: SYM45°, SYM60°, and ST45°), cycling the floating U87 cells for 60 min (4 million cells, 2 mL). The U87‐AEVs produced in knife‐ and hink θ ‐µ‐chips, (d) AEV count per million cells (one‐way ANOVA with the Tukey correction with a normal distribution), (e) average diameter of AEVs, (f) representative size distribution, and (g) cryo‐electron microscopy (cryo‐EM) micrograph of U87‐AEVs. Raji‐AEVs produced in knife‐ and hink ST45 ‐µ‐chips, (h) yield per million cells, (i) protein content, (j) mean diameter, and (k) size distribution. (l) The representative analysis of PI uptake in U87 cells indicates membrane rupture during AEV formation in knife‐ and hink ST45 ‐µ‐chips, with greater disruption in ST45° channels. (m) The fluidic phenomena in the cell‐knife interaction and cell rupture, illustrating the estimated maximum net forces on particles (10 µm diameter, representing the properties of a floating cell) within µ‐chips. (n) The net force magnitudes on individual cells along the channel (normalized length) in hink θ ‐µ‐chips compared to knife‐µ‐chips without HB structures. (o) The calculated shear stress on the knife in different spots of hink θ ‐ and knife‐µ‐chips. All bar graphs plot data as means ± SD of ≥ 5 independent experiments.

Article Snippet: The human glioblastoma U87 cell line (ATCC, Manassas, VA, U.S.)—as well as a stable GFP‐expressing U87 cell line [ ] (Note ) used as a control—were cultured in supplemented Dulbecco's Modified Eagle Medium (DMEM, Sigma–Aldrich, Burlington, MA, U.S.) with glucose (4 g/L glucose, Sigma–Aldrich).

Techniques: Produced, Cryo-Electron Microscopy, Cryo-EM Sample Prep, Membrane, Disruption, Shear

Journal: Advanced Materials (Deerfield Beach, Fla.)

Article Title: Multiphysics‐Driven Assembly of Biomimetic Vesicles

doi: 10.1002/adma.202518755

Figure Lengend Snippet: (a) The representative schematic of forming DOX‐AEVs using a knife‐µ‐chip connected to a peristaltic pump, which circulates the floating U87 cells (1 million/mL, 60 min) with DOX (10 µM) for 60 min. (b) The DOX loading amount (µg) into AEVs using knife‐µ‐chip and hink ST45 ‐µ‐chip (Lognormal distribution analyzed by Mann‐Whitney test). (c) The measured encapsulation efficiency (the loaded amount of DOX into AEVs compared to the initial amount of DOX in the solution) shows a higher loading level for the formulated AEVs using the hink ST45 ‐µ‐chip (normal distribution analyzed by an unpaired two‐tailed t ‐test). (d) The size distribution (mean diameter) of DOX‐AEVs formulated using the knife‐µ‐chip and the hink ST45 ‐µ‐chip. (e) The representative FACS analysis of DOX‐AEVs compared to empty AEVs. (f) The schematics of forming DOX‐AEVs using acoustothermal‐integrated µ‐chips connected to a peristaltic pump, circulating the floating U87 cells (1 million/mL, 60 min circulation) with DOX (10 µM) for 60 min. (g) The total acoustic pressure (theoretical) was calculated by simulating the piezoelectric transducer at the actual frequency of 9.73 MHz (Movie ). (h) The representative simplified 2D simulation of traveling acoustic waves generated by the frequency of 9.73 MHz of the piezoelectric transducer at the applied 60 Vpp (assumptions: considering a uniform distribution of the acoustic pressure at the small section of the center of the transducer and channel, an acoustic wave propagation without the attenuation and reflection from the inner boundaries, and a perfect piezoelectric transducer materials and devices). (i) The simulated temperature profile of the surface of the piezoelectric transducer (9.73 MHz) with variable applicable Vpp at room temperature (RT). (j) The representative simulation of the temperature profile of acoustothermal‐integrated µ‐chips at the constant frequency of 9.73 MHz and 60 Vpp. (k) The effect of flow rate on the channel's temperature profile at the constant frequency of 9.73 MHz and 60 Vpp, and (l) calculated temperature profile of the channel's flow (2 µL/s). The temperature profile of the channel was calculated based on the concentrated heat flow generated within the multilayer structure, comprising the transducer, a glycerol coupling layer, and a silicon nitride wafer, rather than from direct acoustic heating of the fluid. The representative thermal images of (m) acoustothermal‐integrated µ‐chip (shown by a dashed rectangle), (n) its calculated temperature profile of the PDMS block (4 mm above the knife array), and (o) the sample reservoir at the end of the process (60 min). The entire formulation process was performed in a cold room refrigerator (5°C). (p) The DOX loading ratio into the formulated AEVs using acoustothermal‐integrated µ‐chips (constant frequency of 9.73 MHz and 60 Vpp, 60 min) based on the µ‐chips without applying acoustics (standard two‐tailed t ‐tests with a normal distribution). (q) The protein content analysis of the formulated AEVs using acoustothermal‐integrated µ‐chips at a constant frequency of 9.73 MHz and 60 Vpp for 60 min (one‐way ANOVA with the Tukey correction with a normal distribution). All bar graphs plot data as means ± SD of at least 3 independent experiments.

Article Snippet: The human glioblastoma U87 cell line (ATCC, Manassas, VA, U.S.)—as well as a stable GFP‐expressing U87 cell line [ ] (Note ) used as a control—were cultured in supplemented Dulbecco's Modified Eagle Medium (DMEM, Sigma–Aldrich, Burlington, MA, U.S.) with glucose (4 g/L glucose, Sigma–Aldrich).

Techniques: MANN-WHITNEY, Encapsulation, Two Tailed Test, Generated, Blocking Assay, Formulation

Journal: Advanced Materials (Deerfield Beach, Fla.)

Article Title: Multiphysics‐Driven Assembly of Biomimetic Vesicles

doi: 10.1002/adma.202518755

Figure Lengend Snippet: (a) The representative schematic of forming DOX‐AEVs (U87‐derived) using heater‐integrated µ‐chips connected to a peristaltic pump circulating the floating U87 cells (1 million/mL, 60 min circulation) with DOX (10 µM). The representative simulation of (b) the temperature profile of heater‐integrated µ‐chips at the constant temperature (60°C), and (c) the calculated temperature profile of the flow (2 µL/s) in the channel. (d) The effect of flow rate on the temperature profile and heat transfer of the channel (2 cm) related to heater‐integrated µ‐chips at the constant temperature of 60°C. The representative thermal images of (e) heater‐integrated µ‐chips (shown by a dashed rectangle) and (f) the sample reservoir at the end of the process (60 min). The entire formulation process was performed at 5°C. (g) The DOX loading ratio into the formulated U87‐AEVs using heater‐integrated µ‐chips (60°C for 60 min) compared to the µ‐chips without applying temperature (standard two‐tailed t ‐tests with a normal distribution). (h) The particle count analysis of the formulated U87‐AEVs using heater‐integrated µ‐chips at a constant temperature of 60°C for 60 min (standard two‐tailed t‐tests with a normal distribution). (i) The representative schematic shows the DOX‐loading procedure using ultrasonication to estimate and compare the DOX encapsulation rate in AEVs in situ. An intensive cell rupturing approach (control study) was employed using ultrasonication (30 kHz frequency, total duration of 1 and 5 min; 0.5 s of sonication and 0.5 s pause, 100% amplitude, performed in an ice‐cold box at 5°C. (j) The DOX loading ratio into the formulated U87‐AEVs using ultrasonication (one‐way ANOVA with a normal distribution) into a mixture of U87 cells (1 million per mL) and DOX (10 µM) in different processing conditions. (k) The particle count analysis and (l) average size of the formulated U87‐AEVs using ultrasonication (one‐way ANOVA with a normal distribution). All bar graphs plot data as means ± SD of 4 independent experiments.

Article Snippet: The human glioblastoma U87 cell line (ATCC, Manassas, VA, U.S.)—as well as a stable GFP‐expressing U87 cell line [ ] (Note ) used as a control—were cultured in supplemented Dulbecco's Modified Eagle Medium (DMEM, Sigma–Aldrich, Burlington, MA, U.S.) with glucose (4 g/L glucose, Sigma–Aldrich).

Techniques: Derivative Assay, Formulation, Two Tailed Test, Encapsulation, In Situ, Control, Sonication

Journal: Advanced Materials (Deerfield Beach, Fla.)

Article Title: Multiphysics‐Driven Assembly of Biomimetic Vesicles

doi: 10.1002/adma.202518755

Figure Lengend Snippet: (a) Representative analysis of expression of CD9, CD63, and CD81 on the original U87 cells, secreted‐/isolated NEVs, and formulated AEVs. (b) Representative Western blots of the original U87 cells (whole cell lysate: WCL) and the formulated AEVs for detecting the negative and positive exosome markers. (c) The representative analysis of the expression of cell‐membrane proteins on the original Raji cell and formulated Raji‐AEVs. (d) Representative immunoblots of the original Raji cells (whole cell lysate: WCL) and the formulated AEVs for detecting negative and positive exosome markers. See the whole WB membranes in Note . (e) The representative cryo‐EM micrographs of the formulated EVs (U87) determine their morphological characteristics and protein densities. The zoom‐in areas in the representative cryo‐EM image are indicated by squares and marked with (*) and (**). The protein densities on the outer and lumenal membrane sides of the EV's surface are shown by red arrows. The scale bars show 100 nm. The stability of U87‐AEVs over 42 days, continuously kept at 5°C, is shown by (f) AEV count per mL, (g) average size (peak), and (h) representative size distribution. All bar graphs represent means ± SD from 6 independent experiments.

Article Snippet: The human glioblastoma U87 cell line (ATCC, Manassas, VA, U.S.)—as well as a stable GFP‐expressing U87 cell line [ ] (Note ) used as a control—were cultured in supplemented Dulbecco's Modified Eagle Medium (DMEM, Sigma–Aldrich, Burlington, MA, U.S.) with glucose (4 g/L glucose, Sigma–Aldrich).

Techniques: Expressing, Isolation, Western Blot, Membrane, Cryo-EM Sample Prep

Journal: Advanced Materials (Deerfield Beach, Fla.)

Article Title: Multiphysics‐Driven Assembly of Biomimetic Vesicles

doi: 10.1002/adma.202518755

Figure Lengend Snippet: (a) The representative schematic of the immunomodulation of primary NK cells (PI‐gated CD3‐negative and CD56‐positive of the isolated human PBLs) by incubating with the AEVs and NEVs (derived from U87 and Raji cells). (b) The representative micrograph of the PBLs incubated with the PKH‐stained AEVs and NEVs (FITC‐label) for 24 h at 37°C. The scale bar presents 20 µm. The MFI fold change of activating and inhibitory NK cell receptors was analyzed following IL‐2 stimulation and incubation with (c) U87‐AEVs and ‐NEVs, as well as (d) Raji‐AEVs. The expression of cell surface receptors on the gated NK cell population was assessed after 72 h incubation at 37°C with IL2‐NK cells (IL‐2, 100 IU/mL) with or without EVs (10 µg/mL). Receptor expression fold change (relative to IL2‐NK cells without EV treatment) was determined via multiparameter flow cytometry using fluorochrome‐conjugated mAbs directed against activatory and inhibitory receptors. Cell counts of viable cells were obtained after incubating PBLs (3 × 10 5 cells/well, 96‐well plate) with or without treatment. Statistical analysis was conducted using one‐way ANOVA with Tukey correction (normal distribution) or standard two‐tailed t ‐tests (normal distribution). n ≥ 3 donors, mean ± SD. The gating strategy is illustrated in Figure . Representative images of the expression of these different receptors are presented in Figures and .

Article Snippet: The human glioblastoma U87 cell line (ATCC, Manassas, VA, U.S.)—as well as a stable GFP‐expressing U87 cell line [ ] (Note ) used as a control—were cultured in supplemented Dulbecco's Modified Eagle Medium (DMEM, Sigma–Aldrich, Burlington, MA, U.S.) with glucose (4 g/L glucose, Sigma–Aldrich).

Techniques: Isolation, Derivative Assay, Incubation, Staining, Expressing, Flow Cytometry, Two Tailed Test

Journal: Advanced Materials (Deerfield Beach, Fla.)

Article Title: Multiphysics‐Driven Assembly of Biomimetic Vesicles

doi: 10.1002/adma.202518755

Figure Lengend Snippet: (a) Immunoblot of U87 cells and U87‐EVs for detecting the expression of TGF‐β1 (Note ). The ELISA study determines the release of (b) Perforin and (c) GrB from the IL‐2‐stimulated PBLs treated with U87‐AEVs and ‐NEVs compared to the untreated IL‐2‐stimulated human PBLs. The study of n ≥ 3 independent donors, mean ± SD, one‐way ANOVA with the Tukey correction, with a normal distribution. (d) The representative schematic of the immunomodulation of primary NK cells (PI‐gated CD3‐negative and CD56‐positive of the isolated human PBLs) by incubating with the U87‐AEVs and NEVs. The representative analysis of (e) typical ligands of NK cells expressed on the target U87 cells. (f) The annexin V‐PI assay evaluates the performance of EV‐treated NK cells (IL2‐NK) against the target U87 cells (E:T ratio 1:1, incubation 8 h), assessing the viability of the target cells by gating CD45‐negative cells (n ≥ 3 independent donors, mean ± SD, one‐way ANOVA with the Tukey correction with a normal distribution).

Article Snippet: The human glioblastoma U87 cell line (ATCC, Manassas, VA, U.S.)—as well as a stable GFP‐expressing U87 cell line [ ] (Note ) used as a control—were cultured in supplemented Dulbecco's Modified Eagle Medium (DMEM, Sigma–Aldrich, Burlington, MA, U.S.) with glucose (4 g/L glucose, Sigma–Aldrich).

Techniques: Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Isolation, Incubation

Journal: Advanced Materials (Deerfield Beach, Fla.)

Article Title: Multiphysics‐Driven Assembly of Biomimetic Vesicles

doi: 10.1002/adma.202518755

Figure Lengend Snippet: (a) Representative analysis and expression of positive PKH67 staining of the formulated AEVs. (b) Representative homologous targeted binding and uptake of the formulated U87‐AEVs into the original U87 cells and non‐specific binding toward Raji cells. The U87‐AEVs, incubated with U87 (target) and Raji (control) cells, exhibit the highest interaction with the original cells, equipped with self‐identifying markers, facilitating the precise identification of these cells. (c) The representative confocal images indicate the translocation of the formulated U87‐AEVs (PKH67 shown in green) and their internalization into U87 cells (red), highlighting their ability to cross endothelial barriers, including those associated with the tumor blood‐brain barrier. (d) A representative confocal micrograph shows the diffusion assay of the formulated PKH‐AEVs into the dense U87 spheroids. Although the penetration into the spheroid core was limited due to low vascularity, the results suggest potential for deeper tissue access with surface modifications. (e) The endocytosis analysis of the formulated U87‐AEVs (PKH staining, shown in green) was performed using a pharmacological inhibition assay, which revealed primary uptake via clathrin‐ and macropinocytosis‐mediated endocytosis, with a lesser contribution from caveolae and pinocytosis. (f) The intercellular trafficking of internalized U87‐AEVs (green color) into intracellular compartments of U87 cells is demonstrated by representative confocal fluorescence micrographs of endosomes, mitochondria, and other organelles, attributed to their moderate negative surface charge. Pearson's correlation coefficient is shown in Figure . The analyzed expression of (g) the common vesicle's tetraspanins on the formulated DOX‐AEVs with and without implementing acoustics (Figure ), and (h) the proliferation rate of U87 cells incubated (72 h, 37°C) with the formulated DOX‐AEVs (20 µg/mL) (n = 5 independent samples, mean ± SD). The functionality of the therapeutic cargo and the biological integrity of AEVs are shown by the delivery of (i) GFP mRNA and (j) GFP Cas9 and their expression (see more micrographs and analyses in Figures and ).

Article Snippet: The human glioblastoma U87 cell line (ATCC, Manassas, VA, U.S.)—as well as a stable GFP‐expressing U87 cell line [ ] (Note ) used as a control—were cultured in supplemented Dulbecco's Modified Eagle Medium (DMEM, Sigma–Aldrich, Burlington, MA, U.S.) with glucose (4 g/L glucose, Sigma–Aldrich).

Techniques: Expressing, Staining, Binding Assay, Incubation, Control, Translocation Assay, Diffusion-based Assay, Inhibition, Fluorescence

Journal: MedComm

Article Title: Integrative Single‐Cell Analysis Reveals Targetable Vacuole Membrane Protein 1‐Mediated Mechanism of Tumor Angiogenesis in Glioblastoma

doi: 10.1002/mco2.70619

Figure Lengend Snippet: Overexpression of VMP1 promotes tumor growth. (A) Representative western blot image validating exogenous overexpression of VMP1 in U87 and U251 cell lines. (B) U87 subcutaneous xenografts of overexpressed VMP1 (VMP1‐OE) and control vector at Day 17 post‐injection ( n = 9). (C) The weight of the tumor grafts. (D) Quantification of normalized tumor weights of VMP1‐OE and vector (measured every 4 days from Day 3) ( n = 9). (E) Bioluminescence imaging of U87 VMP1‐OE and vector orthotopic xenografts at different time points from Day 3 to Day 17 ( n = 6). (F) Representative images of hematoxylin and eosin‐stained sections at Day 17 post‐injection. Tumor is indicated within the dashed line. Scale bar, 1000 µm. (G) Relative total photon flux of bioluminescence in mice with U87 VMP1‐OE and vector. (H) Kaplan–Meier survival analysis of mice with U87 VMP1‐OE and vector intracranial xenografts ( n = 6). (I) Top, immunohistochemical staining showing Ki67‐positive cells in subcutaneous and intracranial xenografts. Scale bar, 100 µm. Bottom, quantification of Ki67‐positive cells (%) in subcutaneous and intracranial models. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Human embryonic kidney cells 293T (293T) and human GBM cell lines U87 and U251 were purchased from the American Type Culture Collection (ATCC).

Techniques: Over Expression, Western Blot, Control, Plasmid Preparation, Injection, Imaging, Staining, Immunohistochemical staining

Journal: MedComm

Article Title: Integrative Single‐Cell Analysis Reveals Targetable Vacuole Membrane Protein 1‐Mediated Mechanism of Tumor Angiogenesis in Glioblastoma

doi: 10.1002/mco2.70619

Figure Lengend Snippet: VMP1 promoted tumor growth was independent of autophagy. (A) Representative western blot images of autophagy markers (p62 and LC3 I/II) in U87 and U251 cell lines with VMP1‐OE. (B) Representative transmission electron microscopy images of a cell in U87 and U251 with VMP1‐OE, showing no differences in autophagosome formation. Top: scale bar, 2 µm. Bottom: scale bar, 500 nm. (C) Western blot images of tissue samples from our glioma cohort (glioma) ( n = 47) and normal brain tissue (N), showing the protein expression of autophagy markers (p62, Beclin 1, and LC3 I/II). (D) Quantification of western blot images, patients were separated into two groups based on median VMP1 expression: VMP1 low glioma ( n = 23) and VMP1 high glioma ( n = 24). (E) Correlation analysis of western blot quantification value between VMP1 and autophagy markers (p62, Beclin 1, and LC3 I/II). (F) Confirmation of VMP1 knockdown in U87 and U251 cells using two different targeting sequences by western blot analysis. (G) U87 subcutaneous xenografts of VMP1 knockdown (shVMP1) and control vector (shNC) at Day 27 post‐injection ( n = 10) (left), and the tumor volume measured from Day 14 to Day 27 (right). (H) Bioluminescence imaging of U87 shVMP1 and vector orthotopic xenografts at Day 28. (I) Representative images of hematoxylin and eosin‐stained sections at Day 28 post‐injection. Scale bar, 2000 µm. (J) Kaplan–Meier survival analysis of mice with U87 shVMP1 and vector intracranial xenografts ( n = 5). (K) Representative western blot images of U87 shVMP1 and vector subcutaneous xenografts showing the expression of autophagy markers p62 and LC3 I/II (left). Quantification of band intensities normalized to GAPDH (right). ns, no statistical significance; * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Human embryonic kidney cells 293T (293T) and human GBM cell lines U87 and U251 were purchased from the American Type Culture Collection (ATCC).

Techniques: Western Blot, Transmission Assay, Electron Microscopy, Expressing, Knockdown, Control, Plasmid Preparation, Injection, Imaging, Staining

Journal: MedComm

Article Title: Integrative Single‐Cell Analysis Reveals Targetable Vacuole Membrane Protein 1‐Mediated Mechanism of Tumor Angiogenesis in Glioblastoma

doi: 10.1002/mco2.70619

Figure Lengend Snippet: VMP1 mediates angiogenesis and vascular permeability through activation of endothelial cells in the TME. (A) Representative western blot image (top) and quantification (bottom) of VEGFR2 expression in human primary endothelial cells (HUVEC) after culturing with conditioned medium (CM) collected from VMP1‐overexpressing glioblastoma cell lines U87 and U251. (B) Representative immunofluorescence image of VEGFR2 expression (red) and DAPI (blue) in HUVEC cultured with conditioned medium. Scale bar, 200 µm. (C) Representative immunofluorescence staining image of VE‐cadherin expression (red) and DAPI (blue) in HUVEC with CM. Scale bar, 200 µm. (D) Human protein angiogenesis array showing 55 angiogenesis‐related proteins in the CM collected. (E) Quantification of eight of the angiogenesis‐related proteins, including tissue factor (TF), granulocyte‐macrophage colony stimulating factor (GM‐CSF), macrophage inflammatory protein 1α (MIP1α), Serpin E1, Thrombospondin‐1 (THBS1), Angiogenin, tissue inhibitor of metalloproteinase 1 (TIMP‐1), and VEGF‐C. (F) Spatial distribution of spot degree between VMP1 high cancer cells and endothelial cells in the spatial mRNA dataset. (G–I) Spatial distribution of angiogenesis (G), Serpin E1 (H), and TIMP1 (I) expression in the spatial mRNA dataset. * p < 0.05; ** p < 0.01; *** p <0 .001.

Article Snippet: Human embryonic kidney cells 293T (293T) and human GBM cell lines U87 and U251 were purchased from the American Type Culture Collection (ATCC).

Techniques: Permeability, Activation Assay, Western Blot, Expressing, Immunofluorescence, Cell Culture, Staining

Journal: MedComm

Article Title: Integrative Single‐Cell Analysis Reveals Targetable Vacuole Membrane Protein 1‐Mediated Mechanism of Tumor Angiogenesis in Glioblastoma

doi: 10.1002/mco2.70619

Figure Lengend Snippet: Targeted inhibition of VEGFA represses VMP1‐mediated tumor growth. (A) The treatment timeline and bioluminescence detection of mice treated with bevacizumab (BEV) at different time points (Day 3, Day 7, and Day 13). (B) Bioluminescence detection of U87 tumor‐bearing mice treated with BEV and vehicle. (C) Hematoxylin and eosin staining of mice brain, dotted area indicates the tumor region. Scale bar, 1000 µm. (D) Relative total photon flux in orthotopic mice model after treatment. (E) Changes in body weight in mice after treatments. (F) Kaplan–Meier survival of mice after being treated with bevacizumab and vector control. ns, no statistical significance.

Article Snippet: Human embryonic kidney cells 293T (293T) and human GBM cell lines U87 and U251 were purchased from the American Type Culture Collection (ATCC).

Techniques: Inhibition, Staining, Plasmid Preparation, Control