1. Introduction
Gliomas are the most prevalent primary malignant tumors in the adult central nervous system and present major clinical challenges [
1], [
2]. Despite advancements in standard treatment protocols, the median survival time for glioma patients remains very short [
3]. This underscores the urgent need for innovative approaches to better understand glioma biology and accelerate the development of effective therapeutic strategies. In particular, robust glioma models that enable rapid and reliable drug screening are essential for identifying more effective treatments and improving clinical outcomes [
4].
The glioma microenvironment comprises not only glioma cells but also non-tumor components, such as endothelial cells and macrophages, and various soluble cytokines [
5] (
Fig. 1(a)). Among these components, endothelial tissue plays a pivotal role in glioma progression by promoting angiogenesis, mediating intercellular communication, and supporting tumorigenesis [
6]. Additionally, tumor-associated macrophages are also key contributors to glioma pathogenesis due to their plasticity, enabling polarization into distinct phenotypes depending on local cues. M1 macrophages are characterized by their pro-inflammatory and anti-tumor properties, whereas M2 macrophages suppress the immune response, enhance glioma growth and invasiveness, and promote tumor-associated angiogenesis [
7].
To replicate this complexity, considerable efforts have been made to develop various
in vitro models, yet achieving biologically relevant systems that are high-throughput, simple to fabricate, and broadly accessible remains challenging. Early two-dimensional (2D) co-culture systems, such as Transwell assays, captured some aspects of heterocellular interactions but were limited by monolayer cell growth without extracellular matrix (ECM) encapsulation, thereby failing to mimic realistic tumor microenvironments and compromising biological relevance [
8], [
9]. Recent three-dimensional (3D) models have addressed certain limitations by enhancing clinical fidelity through structural and functional integration—such as establishing 3D cellular connections and remodeling ECM components [
10]. Notably, microfluidic-based models offer improved simulation and control over the tumor microenvironment, enabling dynamic intercellular interactions and physiological processes [
11]. Fabricated using soft-lithography microfabrication and biomaterials, these models typically consist of multicompartment units connected by microchannels, effectively mimicking key features of the tumor niche [
12], [
13]. However, their intricate fabrication processes, low throughput, and restricted accessibility—often limited to specialized laboratories—hindered their practicality for high-scale drug screening and widespread use.
Hydrogel microsphere-based organ mimetics, constructed from biocompatible materials capable of forming 3D porous scaffolds, have recently shown great promise due to their high-throughput production and physiological similarity [
14], [
15], [
16], [
17]. These systems serve as a critical bridge between microscale biomimetic constructs and macroscale functional representations, providing a low-toxicity and stable 3D microenvironment with high physiological fidelity and controllable transmission of biochemical signals [
18], [
19]. In this context, advanced microfabrication techniques, including photolithography- or 3D printing-based microfluidic chips, have enabled precise fabrication of cell-laden hydrogel models [
20], [
21], [
22]. Such models have successfully replicated functions of organs like the heart, liver, and pancreatic islets, demonstrating broad potential in biomedical research [
23], [
24], [
25]. Nevertheless, developing proficient methods for achieving precise spatial organization of different cell types within hydrogels, as well as recapitulating the critical structural and functional features of biological barriers, remains a key direction for future development [
26], [
27]. Such models facilitate the observation of processes—including growth, proliferation, migration, substance exchange, and signal transduction—within the co-culture models, enabling the simulation of tumor cell invasion pathways, immune cell trafficking, and endothelial barrier function.
Herein, we present, for the first time, a glioma model based on compartmentalized hydrogel microspheres with engineered rough surfaces fabricated via a biocompatible aerosol microfluidic approach. During microsphere fabrication, microfluidic was employed to precisely control the internal compartmentalization. Leveraging the known property that rough surfaces enhance protein adsorption [
28], aerosol technology was utilized to generate customized surface roughness, promoting Matrigel modification (
Fig. 1(b)). Glioma-associated cells (U251 cells and M2-polarized THP-1 cells) were spatially organized into addressable compartments, while human umbilical vein endothelial cells (HUVECs) formed a functional endothelial barrier on the Matrigel-coated surface. The structural and functional integrity of the microsphere model was systematically validated. Subsequently, chlorogenic acid (CGA), a compound with documented therapeutic potential in gliomas, was applied to stimulate the model and evaluate drug responses (
Fig. 1(c)). It was demonstrated that each microsphere functions as a microtissue mimicking key structural and functional characteristics of gliomas, enabling rapid assessment of drug efficacy, and accelerating the translation from discovery to clinical application. Collectively, this glioma microsphere model provides a promising
in vitro platform, at the single-microsphere level, for mechanistic investigations and high-throughput preclinical screening of glioma therapeutics.
2. Materials and methods
2.1. Materials
Polydimethylsiloxane (PDMS) prepolymers and initiators were purchased from Dow Corning (USA). Sodium alginate (medium viscosity) was obtained from Sigma-Aldrich (USA). Matrigel was acquired from Corning (USA). The 0.9% normal saline, 1 mol∙L-1 CaCl2 solution, Dulbecco’s Modified Eagle Medium (DMEM), Roswell Park Memorial Institute (RPMI)-1640 medium, penicillin-streptomycin, trypsin, and phosphate-buffered saline (PBS) were sourced from Solarbio (China). Fetal bovine serum (FBS) was purchased from ExCell Bio (China). Distributed application program interface (DAPI) staining solution and cell counting kit-8 (CCK-8) were obtained from Beyotime (China). Live/dead assay kit (Calcein-acetoxymethyl ester (AM)/propidium iodide (PI)), CD31 monoclonal antibody, the secondary antibody labeled with Alexa Fluor 488, the secondary antibody labeled with Alexa Fluor 555, and fluorescent microspheres were acquired from Thermo Fisher Scientific (USA). CellTracker fluorescent probes, phycoerythrin (PE) CD163 monoclonal antibody, and vascular endothelial (VE)-cadherin monoclonal antibody were obtained from Invitrogen (USA). Anti-zonula occluden-1 (ZO-1) antibody was purchased from Boster Biological Technology (China). PE anti-human CD206 antibody was obtained from BioLegend (USA). Human interleukin (IL)-6 and IL-10 enzyme-linked immunosorbent assay (ELISA) kits were purchased from BioDee Biotechnology (China). Fluorescein isothiocyanate (FITC)-dextran with different molecular weights (MWs, 3-5/40/150 kDa) was purchased from MedChemExpress (USA). Acetonitrile was obtained from Vokai Biotechnology Co., Ltd. (China). Water was purified by the Milli-Q ultrapure water system of Millipore. CGA (purity > 99%) was supplied by Chengdu Refmedic Technology Co., Ltd. (China).
2.2. Generation of the six-compartmental microspheres with rough surfaces
The six-compartmental microspheres were fabricated using a six-channel integrated PDMS microfluidic device. The six dispersed phases were delivered at a total volumetric flow rate of 2500 μL∙h
-1 and an electric field intensity of 6000 V. Each independent dispersed phase was prepared by mixing 1.5% alginate solution with normal saline containing red or green fluorescent microspheres at a volume ratio of 9:1. The microsphere production throughput reached approximately 3 microspheres∙s
-1, with real-time monitoring performed using a high-speed microscope (VW-9000; Keyence, Japan). Concurrently, CaCl
2 mist was generated via aerosol ejectors positioned on both sides of the droplet trajectory, ensuring uniform coverage of the microsphere surface and facilitating the formation of a roughened texture [
29]. The resulting microspheres with rough surfaces were collected in a CaCl
2-containing bath through the aerosol stream.
2.3. Fabrication of a glioma module and an endothelial barrier in alginate microspheres
A 1.5% alginate solution containing U251 cells or M2-polarized THP-1 cells (5 × 10
6 cells∙mL
-1) was introduced into the microfluidic system [
30]. Meanwhile, an equal volume of magnetic particles and normal saline mixed with alginate in the same proportion was introduced into the system. Magnetic nanoparticles were used for spatial localization, with U251 cells on both sides and THP-1 cells on the opposite side. CaCl
2 aerosol ejectors were then activated along the droplet trajectory to generate surface roughness. To modify the microsphere surfaces, they were transferred to a solution containing 0.5 mg∙mL
-1 Matrigel for cell adhesion experiments. HUVECs (2 × 10
7 cells∙mL
-1) were seeded onto the microsphere surface and co-incubated for approximately 1 h. Fresh culture medium supplemented with 5 mmol∙L
-1 CaCl
2 was subsequently added, and the cell-laden microspheres were further incubated at 37 °C and 5% CO
2 for subsequent analyses.
2.4. Viability assay
On days 1, 4, and 7 after the generation of the glioma microspheres, cell viability was assessed using the live/dead assay kit. Microspheres were incubated with Calcein-AM/PI working solution for 20 min, followed by fluorescence imaging via confocal microscopy. Live cells are stained green (Calcein-AM), while dead cells are stained red (PI). Cell viability was quantified using ImageJ software and defined as: Viability (%) = Number of live cells/(Number of live cells + Number of dead cells).
2.5. RNA extraction and quantitative polymerase chain reaction (qPCR)
Total RNA was isolated from U251 cells and glioma microspheres using the tissue/cell RNA extraction kit (ABclonal, China). Nanodrop 2000 (Thermo Fisher Scientific) was utilized for the determination of RNA density. Complementary DNA (cDNA) was synthesized from messenger RNA (mRNA) using a reverse transcription kit for qPCR. Amplification was performed using SYBR green fast qPCR mix on a T100™ thermal cycler. Gene expression levels were normalized to the internal reference glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The primer sequences used were as follows: Notch1 (forward: 5′-AGCTACTCCTCGCCTGTGGACAA-3′, reverse: 5′-AGCTACTCCTCGCCTGTGGACAA-3′); matrix metalloproteinase 9 (MMP9; forward: 5′-GCACGACGTCTTCCAGTACC-3′, reverse: 5′-CAGGATGTCATAGGTCACGTAGC-3′); GAPDH (forward: 5′-CCTGGTATGACAACGAATTTG-3′, reverse: 5′-CAGTGAGGGTCTCTCTCTTCC-3′).
2.6. Endothelial barrier functionality assay
To visualize and quantify the endothelial barrier function, tight junction (TJ) proteins between adjacent endothelial cells—VE-cadherin and ZO-1—were analyzed by immunofluorescence staining. After three days of culture, glioma microspheres were collected and washed three times with 0.1 mol∙L-1 CaCl2-containing saline, then fixed in 100 μL of 4% paraformaldehyde for 15 min. After removal of the supernatant and three additional washes, permeabilization was performed with 100 μL of 0.5% Triton X-100 for 10 min. Microspheres washed three times were then incubated with 100 μL of blocking solution at room temperature for 1 h. Primary antibodies against VE-cadherin, ZO-1, and CD31 were diluted (1:100), and then incubated with microspheres overnight at 4 °C overnight. Following washing, secondary antibodies—goat anti-rabbit immunoglobulin G (IgG) H&L (Alexa Fluor 488) and goat anti-mouse IgG H&L (Alexa Fluor 555) were diluted at 1:500 and incubated at 37 °C for 1 h. Finally, DAPI was applied for nuclear staining (10 min), and samples were imaged using confocal microscopy. Concurrently, solutions containing FITC, 40 kDa FITC-dextran, or 150 kDa FITC-dextran were prepared at 25 μg∙mL-1 in the DMEM. Microsphere reservoirs with and without the HUVEC monolayer were exposed to the solutions cultured for 2 h. Diffusional permeability was evaluated by monitoring FITC probe diffusion into the hydrogel core. The penetration efficiency = FIinside/FIoutside, where FIinside is the fluorescence intensity of fluorescent molecules diffused inside the microsphere and FIoutside is the fluorescence intensity outside the microsphere. All images were all processed using ZEN software (Zeiss, Germany).
2.7. ELISA assay of IL-6 and IL-10 after drug treatment
The expressions of cytokines associated with types of macrophages were quantitatively measured through the ELISA method. After incubation and CGA stimulation for 24 h, the supernatants of glioma microspheres were collected to assess the levels of IL-6 and IL-10. Protein concentrations were determined based on standard curves derived from optical density (OD) values measured using a microplate reader.
2.8. Chromatographic and electrospray ionization mass spectrometry conditions
Each sample was analyzed using the supernatant collected from ten microspheres. In this experiment, a C18 column (100 mm × 2.1 mm, 3 μm; Shimadzu, Japan) was loaded on the liquid chromatography triple quadrupole tandem mass spectrometry (LC-QqQ-MS/MS; Shimadzu). The column temperature was maintained at 40 °C. The mobile phase consisted of 0.1% (v/v) aqueous formic acid (solvent A) and 0.1% (v/v) formic acid in acetonitrile (solvent B) at a flow rate of 0.3 mL∙min-1. Gradient elution conditions were as follows: 5% B (0-2.5 min), 5%-70% B (2.5-2.7 min), 70% B (2.7-3.7 min), 70%-95% B (3.7-3.8 min), 95% B (3.8-4.3 min), 95%-5% B (4.3-4.5 min), and 5% B (4.5-7.0 min). The supernatant was collected from glioma microspheres treated with varying concentrations of CGA for 24 h and extracted using acetonitrile with the volume ratio of 1:2. Then the mixture was vortexed and centrifuged at 10 000 r∙min-1 for 10 min to achieve desalting and protein precipitation. The resulting supernatant was stored in an autosampler at 4 °C with an injection volume of 1 μL. Mass spectrometric parameters were maintained as follows: interface temperature, 300 °C; heating block temperature, 250 °C; desolvation line temperature, 150 °C; spray voltage, 3.00 kV; nebulizer gas (N2) flow rate, 3 L∙min-1; and heating gas (N2) and drying gas (N2) flow rate, 10 L∙min-1. Each metabolite was detected in positive or negative ion mode using multiple reaction monitoring (MRM). The MS/MS transitions of each compound are shown in Table S1 in Appendix A. The standard substance stock solution of cell metabolites (10 mmol∙L-1) was diluted to a series of concentrations to construct the standard curve, ranging from 1 to 500 μmol∙L-1 in tryptophan, 0.1 to 2.5 μmol∙L-1 in kynurenine, 5 to 100 μmol∙L-1 in glutamate, 1 to 50 μmol∙L-1 in glutamine, and 25 to 500 μmol∙L-1 in lactate.
2.9. Data and statistical analysis
Quantitative data are presented as mean ± standard deviation (s.d.) from at least three independent experiments. One-way analysis of variance (ANOVA) test and Tukey’s multiple comparisons test were performed using GraphPad Prism 9.5.0. Statistical significance was defined as P < 0.05.
3. Results and discussion
3.1. Fabrication of multicompartmental microspheres with rough surfaces
The microfluidic device for generating multicompartmental microspheres consists of an upstream microfluidic chip, a downstream conductive needle, and an aerosol generator (Fig. S1 in Appendix A). In representative experiments, a six-channel chip was employed to fabricate six-compartment microspheres with rough surfaces. Multiple alginate solutions containing red or green fluorescent nanoparticles were introduced into the multicompartment chip as the dispersed phase. Within the chip, laminar flow was maintained, enabling the formation of a stable six-compartment fluid configuration. This structured fluid stream then entered a conductive needle directly connected to the chip outlet. A high-voltage electric field was simultaneously applied, enabling the continuous detachment of alginate droplets when the combined forces of electric stress and gravity overcame the surface tension. Below the device, a CaCl
2 aerosol atmosphere was created by an aerosol generator. As the falling alginate droplets collided with these aerosols [
29], localized and inhomogeneous crosslinking occurred between Ca
2+ ions and alginate, leading to the formation of irregular solidified domains on the microsphere surface. Subsequently, the microspheres fell into a collecting bath containing Ca
2+, where they underwent complete gelation to form structurally stabilized alginate microspheres.
To assess the stability of the proposed methods, fluorescence microscopy was used to examine the internal architecture of the microspheres. All fabricated microspheres exhibited highly consistent internal structures, characterized by uniform spatial distribution of six compartments and dimensional homogeneity (
Fig. 2(a), Fig. S2 in Appendix A). Fluorescence signals showed even distribution of red and green channels, indicating minimal signal crosstalk and consistent compartmental occupancy (
Fig. 2(b)), thereby confirming the internal structural stability of the compartmentalized microspheres. Additionally, microsphere diameter distribution—a key factor for model standardization—was analyzed. As shown in
Fig. 2(c), the microspheres displayed low size variability, with a coefficient of variation (CV) below 4%, demonstrating successful generation of monodisperse microspheres. Furthermore, microsphere size could be precisely tuned by adjusting fabrication parameters: increasing the electric field strength or decreasing the alginate flow rate resulted in smaller diameters, highlighting the tunability and controllability of the system (
Fig. 2(d)).
To better showcase surface topography, microspheres were fabricated using alginate solutions devoid of fluorescent nanoparticles. As shown in
Fig. 2(e), the microspheres exhibited a well-defined rough surface texture. This surface roughness enhances the capacity for functional molecule adsorption [
29], enabling multifunctional modifications such as surface coating with FITC-labeled Matrigel (
Fig. 2(f)).
3.2. Fabrication of endothelialized glioma microspheres
Following the proposed protocol, glioma models were constructed by incorporating glioma-relevant cells: U251 cells, M2-polarized THP-1 cells, and HUVECs. Successful polarization of THP-1 cells to the M2 phenotype was confirmed by detection of characteristic marker proteins (Fig. S3 in Appendix A). For microsphere assembly, one compartment was loaded with magnetic nanoparticles to serve as a positional marker, while adjacent compartments were loaded with U251 cells [
31], [
32]. The compartment opposite was loaded with M2-polarized THP-1 cells, while the remaining two compartments were filled with plain alginate solution (
Fig. 3(a), Fig. S4(a) in Appendix A). This configuration formed the basis for fabricating cell-laden microspheres with rough surfaces, demonstrating that six-compartment structure not only enables encapsulation of cells within designated compartments but also allows spatial segregation of different cell types by incorporating magnetic nanoparticle-loaded and empty compartments—thereby facilitating clear identification of individual cell populations.
Owing to the non-adhesive properties of the alginate material and the protein-binding capacity of the rough alginate surface, the cell-laden microspheres were immersed in a Matrigel solution for surface functionalization [
33], [
34]. Matrigel supports cell adhesion through receptor-protein interactions [
35], promoting optimal cell attachment on the microsphere surface. Following incubation with Matrigel, the microspheres were exposed to a HUVEC suspension, resulting in firm cellular adhesion. Within three days, HUVECs formed a confluent and uniform monolayer on the microsphere surface (Fig. S4(b) in Appendix A).
To validate the spatial organization of the glioma microspheres, cell tracker staining was used to label HUVEC, U251, and M2-polarized THP-1 cells with blue, red, and green fluorescence, respectively. Fluorescence imaging confirmed precise localization of each cell type within their preassigned compartments, achieving the intended architectural design (
Fig. 3(a)). For microspheres without fluorescent labeling, cell identity was determined based on the position of the magnetic nanoparticle-containing compartment as a spatial reference. Cell viability was assessed under both static and flow culture conditions over 24 h. The results showed significantly higher viability in microspheres cultured under flow conditions compared to those in static culture (Fig. S5 in Appendix A), leading to the adoption of dynamic flow culture in all subsequent experiments.
3.3. Evaluation of the endothelialized glioma microspheres
Cell viability was evaluated using live/dead staining, which revealed high cell viability (> 95%) throughout a 7-day culture period (
Figs. 3(b) and
(c)), indicating microspheres produced could perform long-term cultivation. Immunostaining was conducted to verify the formation of an endothelial barrier on the microsphere surface. Regulation of interendothelial adherent junctions (AJs) and TJs plays a critical role in controlling endothelial permeability [
36]. VE-cadherin and ZO-1, which are key components of AJs and TJs, respectively, were evaluated for their role in regulating permeability [
37], [
38]. As shown in
Figs. 3(d-i) and
(d-ii), continuous fluorescence signals for VE-cadherin and ZO-1 were observed across the microsphere surface, confirming well-formed cell-cell junctions and structural continuity of the endothelial layer. Additionally, strong expression of CD31, a canonical endothelial marker, indicated the active and healthy state of the endothelial cells (
Fig. 3(d-iii)).
The permeability of the endothelial barrier was further evaluated by exposing the microspheres with and without HUVECs monolayer to solutions of FITC or FITC-dextran (40 or 150 kDa;
Fig. 3(e)). Microspheres lacking HUVEC coverage served as the control group. Fluorescence intensity inside and outside the microspheres was measured at 0 and 2 h to quantify barrier function [
39]. After 2 h of diffusion, the endothelial layer significantly reduced the influx of free FITC compared to the control. Similarly, the penetration of 40 and 150 kDa FITC-dextrans into the hydrogel core was markedly slowed. At the 2-h time point, endothelialized microspheres exhibited significantly lower intramicrosphere fluorescence accumulation than non-endothelialized controls. These findings confirm that the engineered endothelial barrier exerts size-selective regulation over molecular permeation.
To further characterize the glioma microspheres, qPCR assays were performed to compare the expression levels of key glioma-related genes, including
Notch1 and
MMP9, between the glioma microspheres and conventional 2D U251 cell cultures. These genes play pivotal roles in the pathogenesis and progression of gliomas. Increased expression of
Notch1 and
MMP9 is positively correlated with enhanced tumor malignancy, invasiveness, and proliferative capacity.
GAPDH was utilized as the internal reference gene for normalization.
Notch1 and
MMP9 are widely recognized markers for evaluating glioma aggressiveness and prognosis [
40]. RNA was extracted from crushed microspheres. For each sample, RNA was pooled from ten individual microspheres. RNA concentration was quantified prior to reverse transcription into cDNA. Target gene amplification was carried out using gene-specific primers, with
GAPDH serving as the internal control. qPCR results revealed a significant upregulation of
Notch1 mRNA levels and markedly increased
MMP9 expression in glioma microspheres compared to 2D U251 cell cultures (
Fig. 3(f)). These findings indicate that the glioma microspheres, incorporating multiple cell types, enhance the expression of glioma-characteristic genes, likely due to cell-cell interactions, activated signaling pathways, and the replication of the glioma microenvironment. Thus, the glioma microspheres provide a more effective and biologically representative platform for investigating glioma initiation and progression, offering a substantial improvement over conventional 2D monolayer cultures.
3.4. Drug treatment on endothelialized glioma microspheres
CGA has recently emerged as a promising therapeutic candidate for glioma, with a Phase II clinical trial nearing completion in China [
41]. It has been reported that CGA can cross the blood-brain barrier and exert effects on glioma tissue (
Fig. 4(a)) [
42], [
43]. In this study, CGA was employed as a model drug to evaluate the functional responsiveness and pharmacological relevance of the glioma microsphere system.
The cytotoxicity of CGA on U251, HUVEC, and M2-polarized THP-1 cells was assessed using the CCK-8 assay, with absorbance measured spectrophotometrically using a microplate reader. Treatment with CGA at 100, 400, and 1200 μmol∙L-1 for 24 h significantly reduced cell viability of U251, HUVEC, and M2-polarized THP-1 cells respectively in a concentration-dependent manner (Fig. S6 in Appendix A). At 200 μmol∙L-1, U251 cell viability decreased to approximately 70%, while no significant reduction was observed in HUVEC and M2-polarized THP-1 cells, indicating selective toxicity toward glioma cells. Therefore, 200 μmol∙L-1 CGA was selected for subsequent experiments.
qPCR was performed first. It was observed that, compared to the control group, treatment with 200 μmol∙L
-1 CGA led to reduced
Notch1 expression and a significant downregulation of
MMP9 mRNA levels (
Fig. 4(b)), confirming the pharmacological activity of CGA within the 3D glioma microsphere model.
CGA has also been shown to modulate tumor-associated macrophages by promoting their transition from the pro-tumorigenic M2 phenotype to the anti-tumor M1 phenotype [
44]. To assess this effect in the context of the glioma microspheres, the levels of proinflammatory cytokine IL-6 and anti-inflammatory cytokine IL-10 were quantified following CGA treatment (
Fig. 4(c)). ELISA results showed a significant increase in IL-6 levels and a marked decrease in IL-10 levels after CGA treatment for 24 h, suggesting a shift toward M1-like macrophage polarization.
Metabolic reprogramming is a hallmark of glioma progression. A total of 45 metabolites were profiled in microspheres with or without CGA treatment using LC-QqQ-MS/MS (Tables S2 and S3 in Appendix A). Following semi-quantitative analysis, a heatmap of metabolic differences between the control and treated groups was generated in
Fig. 4(d).
Z-score normalization was applied, with red-to-green color gradients reflecting decreasing metabolite abundance, enabling clear visualization of changes induced by CGA. Principal component analysis (PCA) results showed distinct clustering and significant separation between groups (Fig. S7 in Appendix A), indicating profound metabolic alterations upon CGA exposure. These results suggest that CGA exerts its therapeutic effects, at least in part, through modulation of metabolic pathways in the glioma microsphere model. Specifically, decreased tryptophan and elevated kynurenine levels are linked to malignant transformation and immune evasion [
45], while increased glutamine uptake, extracellular glutamate accumulation, and lactate production support glioma bioenergetics and tumor progression [
46], [
47]. To further validate the glioma model, key metabolites—tryptophan, kynurenine, glutamate, glutamine, and lactate—were selected as target metabolites for targeted quantification. After 24 h of treatment, tryptophan and glutamine levels were significantly increased, whereas kynurenine, glutamate, and lactate levels were markedly reduced compared to the control group (
Fig. 4(e), Figs. S8-S10 in Appendix A). Collectively, these data demonstrate that the glioma microsphere model recapitulates CGA-induced metabolic responses observed
in vivo, highlighting its potential as an advanced
in vitro platform for disease modeling and therapeutic evaluation [
48].
4. Conclusions
In this study, we developed a hydrogel microsphere-based glioma (glioma-on-a-microsphere) model that integrates multicompartmental architecture, an endothelial barrier and glioma-associated cell types to mimic the structural and functional complexities of the glioma microenvironment. A custom-designed microfluidic device enabled the fabrication of uniform microspheres with rough surfaces, allowing precise compartmentalization and versatile surface functionalization. Cells were accurately localized within designated compartments, maintaining high viability throughout a 7-day culture period. Moreover, the endothelial monolayer formed on the microsphere surface expressed characteristic markers and effectively regulated the permeability of small molecules, demonstrating well-defined spatial organization, sustained cellular viability, and a complete endothelial barrier capable of achieving selective permeation function was established. Using CGA as a therapeutic model, this platform demonstrated the ability to evaluate drug efficacy and mechanisms utilizing qPCR quantification, ELISA assay, and mass spectrometry analysis, covering effects on gene expression, immune modulation, and metabolic changes. These meticulously designed microspheres hold considerable potential for high-throughput drug screening, disease modeling, and personalized medicine, providing a powerful tool for bridging the gap between in vitro models and physiological relevance.
It is certain that this model retains substantial scope for refinement. In the future, the integration of stem cell-derived cells or tissues into hydrogel system should be able to better capture cell-cell and cell-matrix interactions, thereby further enhancing the physiological relevance of
in vitro models for biomedical research and therapeutic applications [
49].
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