Traditional monolayer cell cultures, while simple, inaccurately predict *in vivo* drug behavior due to the lack of cell-cell and cell-matrix interactions. Three-dimensional (3D) cell spheroids offer a more accurate representation of these interactions. However, current methods for spheroid generation (pellet culture, liquid overlay, hanging drop, spinning flask, magnetic levitation) are often labor-intensive, slow, and result in inconsistent spheroid sizes. Microfluidic approaches, particularly droplet-based methods, provide a solution to these limitations by enabling automated, high-throughput generation of homogenous spheroids with controlled size. This study focuses on a droplet-based microfluidic device to address these challenges. The introduction also highlights the use of nanomaterials, specifically reduced graphene oxide (rGO) nanocomposites, in cancer therapy, particularly photothermal therapy (PTT), emphasizing the need for a robust 3D cell culture model for evaluating their efficacy. The research aims to develop a microfluidic system capable of producing uniform spheroids for drug testing and PTT applications, using rGO-BPEI-PEG nanocomposites as a model PTT agent and demonstrating its applicability with neural stem cell (NSC)-derived neurospheres.

The literature review discusses existing methods for generating cell spheroids, including their limitations. It categorizes microfluidic approaches into microwell, microtrap, and droplet-based methods, highlighting the advantages and disadvantages of each. Existing droplet-based methods are reviewed, with specific attention to techniques involving alginate, magnetic beads, and cell cluster breakup to enhance homogeneity. The review also covers the use of graphene oxide (GO) and its derivatives in photothermal therapy (PTT), including the modification of GO with polyethylene glycol (PEG) and branched polyethyleneimine (BPEI) to improve stability and therapeutic efficacy. The literature establishes the need for a high-throughput, highly reproducible method for generating uniform spheroids for evaluating the effectiveness of nanomaterials in PTT and other therapeutic applications.

The authors designed a droplet-based microfluidic device with two inlets for oil and cell-containing aqueous phases. Droplets were generated at a microfluidic junction using shear forces. The droplet size was controlled by varying the oil flow rate, achieving diameters between 180.8 ± 2.9 and 305.9 ± 8.1 µm. Spheroid size was further controlled by adjusting the cell concentration in the aqueous phase and the droplet volume. The generated droplets were cultured for 2 days to allow spheroid formation. The spheroids' size and shape were analyzed, demonstrating high homogeneity with approximately 80% of droplets containing a single spheroid. The generation frequency was ~70 Hz, resulting in high throughput. rGO-BPEI-PEG nanocomposites were synthesized, characterized using AFM and FT-IR spectroscopy to confirm their size, morphology, and chemical composition. UV-Vis spectroscopy and zeta potential analysis were used to study their optical properties and surface charge. The cytotoxicity of the nanocomposites was evaluated using a CCK-8 assay. The photothermal effects of the rGO-BPEI-PEG nanocomposites on U87MG brain tumor spheroids were assessed by exposing them to NIR laser irradiation. Immunostaining was performed using E-cadherin to confirm spheroid formation and cell-cell interactions. Finally, NSC-derived neurospheres were generated using the device to assess the effects of neurotoxins on neurite outgrowth.

The droplet-based microfluidic device successfully generated homogenous tumor spheroids with diameters ranging from 98.6 ± 1.0 to 126.4 ± 4.9 µm by controlling cell concentration and droplet volume. The system achieved a high droplet generation frequency of ~70 Hz. The synthesized rGO-BPEI-PEG nanocomposites exhibited desirable optical properties for PTT and showed a size reduction compared to unmodified GO. The nanocomposites demonstrated photothermal effects, reducing the viability of U87MG brain tumor spheroids upon NIR laser irradiation. Immunostaining confirmed the formation of well-defined spheroids with significant E-cadherin expression, indicating proper cell-cell junctions. The device also produced uniformly sized NSC-derived neurospheres suitable for drug screening, and the study demonstrated that the neurites were regulated by neurotoxins. The study demonstrates the advantages of the droplet-based microfluidic system over previous methods, as shown in Supplementary Table S1.

The results demonstrate the effectiveness of the droplet-based microfluidic device for generating homogenous, uniform-sized spheroids suitable for drug screening and PTT studies. The high-throughput nature of the device allows for large-scale generation of spheroids, overcoming the limitations of conventional methods. The successful application of the device with both brain tumor spheroids and NSC-derived neurospheres highlights its versatility. The rGO-BPEI-PEG nanocomposites show promise as effective PTT agents, and the study's findings provide valuable data for further development and optimization of PTT therapies. The consistent and well-defined nature of the spheroids generated by the microfluidic device enhances the reliability and reproducibility of experimental results. The combined results showcase the potential of this platform to accelerate drug development and evaluation, particularly for cancer therapies and neurobiological studies.

This study successfully developed a high-throughput, droplet-based microfluidic device for generating homogenous cell spheroids with controlled size. The device was successfully utilized to evaluate the efficacy of rGO-BPEI-PEG nanocomposites in photothermal therapy and to assess the effect of neurotoxins on neurite outgrowth from NSC-derived neurospheres. The platform is highly versatile and shows great promise for various applications in drug screening and cancer research. Future research could explore further optimization of the device for different cell types and applications, as well as investigating the use of other nanomaterials for targeted therapies.

The study primarily focused on U87MG glioblastoma cells and NSC-derived neurospheres. Further investigation is needed to determine the device's effectiveness with other cell types and spheroid sizes. While the rGO-BPEI-PEG nanocomposites demonstrated photothermal effects, further studies are necessary to evaluate their long-term effects and potential toxicity *in vivo*. The current system needs further scaling up for high throughput industrial application.