Uniform Spheroids Research Articles

Spherical Shell Bioprinting to Produce Uniform Spheroids with Controlled Sizes.

Conventional cell spheroid production methods are largely manual, leading to variations in size and shape that compromise consistency and reliability for use in cell-based therapeutic applications. To enhance spheroid production, a spherical shell bioprinting system was implemented, enabling the high-throughput generation of uniform cell spheroids with precisely controlled sizes. The system encapsulates cells within thin alginate hydrogel shells formed through bioprinting and ion crosslinking reactions. Alginate-calcium ion crosslinking created alginate shells that contained gelatin-based bioinks with embedded cells, facilitating spontaneous cell aggregation within the shells and eliminating the need for plastic wells. By adjusting cell concentrations in the alginate-gelatin bioink, we achieved precise control over spheroid size, maintaining a sphericity above 0.94 and size deviations within ±10 µm. This method has been successfully applied to various cell types including cancer cells, fibroblasts, chondrocytes, and epithelial cells, demonstrating its versatility. This scalable approach enhances the reliability of cell therapy and drug screening, offering a robust platform for future biomedical applications.

Massive fabrication of functional hepatic cancer spheroids by micropatterned GelMA hydrogel chip for drug screening

Since hepatic cancer incidence and mortality continue to grow worldwide, it is necessary to develop the biomimetic tumor models for drug development and tumor therapeutics. Cellular spheroids as an excellent simple 3D model can bridge the gap between 2D cell culture and live tissue. In this study, we proposed a biological methacrylated gelatin (GelMA) hydrogel-based microplatform for the massive generation of hepatocellular spheroids and downstream investigation of drug resistance. Micropatterned GelMA hydrogel microwell chip (GHM-chip) with tunable array was easily achieved in standard 24-culture well plates through the micro-molding fabrication strategy. The fabricated GHM-chip induced multicellular self-assembly behavior within the defined topography and further formed spheroidal structure. By regulating cell seeding density and designing microwell size, uniform hepatic cancer spheroids with tunable diameters were obtained in a simplicity, stability and controllable manner. In addition, the screening chemotherapy study of anti-cancer drug was completed through non-destructive recovery of spheroids from the GHM-chip. Beyond that, the recovered functional spheroids have potential application value in various biomedical fields such as tumor biology, pharmacology, and tissue microengineering. Finally, the proposed GHM-chip incorporated into standard cell culture plates with easy to manufacture and operate properties, may be an efficient culture microplatform for cancer research applications.

Automated Uniform Spheroid Generation Platform for High Throughput Drug Screening Process.

Three-dimensional (3D) spheroid models are crucial for cancer research, offering more accurate insights into tumour biology and drug responses than traditional 2D cell cultures. However, inconsistent and low-throughput spheroid production has hindered their application in drug screening. Here, we present an automated high-throughput platform for a spheroid selection, fabrication, and sorting system (SFSS) to produce uniform gelatine-encapsulated spheroids (GESs) with high efficiency. SFSS integrates advanced imaging, analysis, photo-triggered fabrication, and microfluidic sorting to precisely control spheroid size, shape, and viability. Our data demonstrate that our SFSS can produce over 50 GESs with consistent size and circularity in 30 min with over 97% sorting accuracy while maintaining cell viability and structural integrity. We demonstrated that the GESs can be used for drug screening and potentially for various assays. Thus, the SFSS could significantly enhance the efficiency of generating uniform spheroids, facilitating their application in drug development to investigate complex biological systems and drug responses in a more physiologically relevant context.

Formation and culture of cell spheroids by using magnetic nanostructures resembling a crown of thorns

In contrast to traditional two-dimensional cell-culture conditions, three-dimensional (3D) cell-culture models closely mimic complexin vivoconditions. However, constructing 3D cell culture models still faces challenges. In this paper, by using micro/nano fabrication method, including lithography, deposition, etching, and lift-off, we designed magnetic nanostructures resembling a crown of thorns. This magnetic crown of thorns (MCT) nanostructure enables the isolation of cells that have endocytosed magnetic particles. To assess the utility of this nanostructure, we used high-flux acquisition of Jurkat cells, an acute-leukemia cell line exhibiting the native phenotype, as an example. The novel structure enabled Jurkat cells to form spheroids within just 30 min by leveraging mild magnetic forces to bring together endocytosed magnetic particles. The size, volume, and arrangement of these spheroids were precisely regulated by the dimensions of the MCT nanostructure and the array configuration. The resulting magnetic cell clusters were uniform in size and reached saturation after 1400 s. Notably, these cell clusters could be easily separated from the MCT nanostructure through enzymatic digestion while maintaining their integrity. These clusters displayed a strong proliferation rate and survival capabilities, lasting for an impressive 96 h. Compared with existing 3D cell-culture models, the approach presented in this study offers the advantage of rapid formation of uniform spheroids that can mimicin vivomicroenvironments. These findings underscore the high potential of the MCT in cell-culture models and magnetic tissue enginerring.

Advanced Microfluidic Platform for Tumor Spheroid Formation and Cultivation Fabricated from OSTE+ Polymer

In the evolving landscape of cancer research, 3D cell cultures, particularly tumor cell spheroids, are increasingly preferred in drug screening due to their enhanced mimicry of in vivo tumor environments, especially in drug resistance aspects. However, the consistent formation of uniform spheroids and their precise manipulation remain complex challenges. Among various methodologies, droplet microfluidics emerges as a highly effective approach for tumor spheroid formation. This paper introduces a novel, multifaceted microfluidic system that streamlines the entire spheroid cultivation process: (i) generating tumor spheroids from cell suspensions within individual droplets, (ii) merging these droplets into a continuous aqueous phase once spheroid formation is complete, and (iii) transferring the spheroids to a specialized cultivation area within the chip, equipped with trapping elements for extended cultivation in perfusion mode. Remarkably, this process requires no hydrogel encapsulation or external handling, as all operations are conducted within the microfluidic chip. Fabricated from the innovative OSTE+ (off-stoichiometry thiol-ene epoxy) polymer, the chip is designed for repeated use. To show its efficacy, we successfully formed spheroids from MCF-7, GAMG, and U87 cell lines in our system and compared them with spheroids prepared by a traditional agarose microwell method. Additionally, our methodology has successfully enabled the in-chip release of spheroids from droplets, followed by their effective trapping for subsequent cultivation, a process we have exemplified with MCF-7 spheroids. To our knowledge, this research represents the first instance of a fully integrated droplet microfluidic platform achieving scaffoldless tumor spheroid formation and handling. Our method holds promise for improving high-throughput, automated procedures in the formation, transfer, and cultivation of tumor cell spheroids.Graphical abstract

Tailoring esophageal tumor spheroids on a chip with inverse opal scaffolds for drug screening

Esophageal cancer (EC) is characterized by high morbidity and mortality, and chemotherapy has become an indispensable means for comprehensive treatment. However, due to the limitation of the effective in vitro disease model, the development of chemotherapeutic agents still faces great challenges. In this paper, we present a novel tumor spheroid on a chip platform based on inverse opal hydrogel scaffolds to screen chemotherapeutic agents for EC treatment. With the microfluidic emulsion approach, the inverse opal hydrogel scaffolds were generated with tunable and organized pores, which could provide spatial confinement for cell growth. Thus, the suspended KYSE-70 cells could successfully form uniform cell spheroids on the inverse opal hydrogel scaffolds. It was demonstrated that the tumor cell spheroids could recapitulate 3D growth patterns in vivo and exhibited higher sensitivity to the chemotherapy agents compared with monolayer cells. Besides, by employing the scaffolds into a microfluidics to construct esophageal tumor on a chip, the device could realize high-throughput tumor cell spheroids generation and drug screening, indicating its promising role in chemotherapy drug development.

Diffusion-based culture and real-time impedance monitoring of tumor spheroids in hydrogel microwells of a suspended membrane under microfluidic conditions

Tumor spheroids are widely studied for in vitro modeling of tumor growth and responses to anticancer drugs. However, current methods are mostly limited to static and perfusion-based cultures, which can be improved by more accurately mimicking pathological conditions. Here, we developed a diffusion-based dynamic culture system for tumor spheroids studies using a thin membrane of hydrogel microwells and a microfluidic device. This allows for effective exchange of nutrients and metabolites between the tumors and the culture medium flowing underneath, resulting in uniform tumor spheroids. To monitor the growth and drug response of the spheroids in real-time, we performed spectroscopic analyses of the system's impedance, demonstrating a close correlation between the tumor size and the resistance and capacitance of the system. Our results also indicate an enhanced drug effect on the tumor spheroids in the presence of a low AC electric field, suggesting a weakening mechanism of the spheroids induced by external perturbation.

DIPG-08. PROBING THE BLOOD-BRAIN-TUMOR-BARRIER WITH A MICROVASCULAR MODEL OF DIFFUSE MIDLINE GLIOMA

Abstract BACKGROUND Pediatric diffuse midline glioma (DMG) is a uniformly fatal brain tumor in children, with most patients surviving less than 12 months. Current treatment for DMG is limited, and outcomes for this tumor have not improved. The blood-brain-barrier (BBB) remains a key challenge for DMG therapeutics, preventing most drugs from entering brain tissue. Functional characterization of the blood-brain-tumor-barrier (BBTB) – the interaction between tumors and BBB vasculature – remains largely unexplored for DMG. There is a need for a preclinical model of DMG that can probe the functional, physiological, and biological properties of the BBTB in vitro as a platform to evaluate therapeutic efficacy that is representative of human physiology. METHODS To generate an in vitro model of DMG with human microvasculature (DMG-BBTB model), we co-cultured DMG neurospheres, human brain microvascular endothelial cells, human brain vascular pericytes, and human astrocytes. Cells are simultaneously seeded into microfluidic devices at varied concentrations within a fibrin matrix. Self-assembled microvascular networks are formed within 7-8 days and used for downstream functional and phenotypic assays. RESULTS Vascular morphology and solute permeability in microvascular networks are influenced by cell identity, growth pattern, and relative concentration. We show that DMG neurospheres exhibit different patterns of growth when co-cultured as uniform spheroids versus single cells in suspension. Devices representing each growth pattern are further characterized for paracellular diffusion and permeability compared to devices without DMG cells through perfusion of high molecular weight dextran. Permeability values are correlated to biologic features such as the abundance and localization of junctional proteins and transporters. CONCLUSIONS We developed a platform to evaluate the microvasculature of the DMG BBTB as a tool to develop new therapies informed by vascular biology. We probed the functional, physiological, and biological properties of the human DMG BBTB in vitro, and in ongoing work are assessing approved and experimental therapeutics.

Generating human skeletal myoblast spheroids for vascular myogenic tissue engineering

Engineered myogenic microtissues derived from human skeletal myoblasts offer unique opportunities for varying skeletal muscle tissue engineering applications, such as in vitro drug-testing and disease modelling. However, more complex models require the incorporation of vascular structures, which remains to be challenging. In this study, myogenic spheroids were generated using a high-throughput, non-adhesive micropatterned surface. Since monoculture spheroids containing human skeletal myoblasts were unable to remain their integrity, co-culture spheroids combining human skeletal myoblasts and human adipose-derived stem cells were created. When using the optimal ratio, uniform and viable spheroids with enhanced myogenic properties were achieved. Applying a pre-vascularization strategy, through addition of endothelial cells, resulted in the formation of spheroids containing capillary-like networks, lumina and collagen in the extracellular matrix, whilst retaining myogenicity. Moreover, sprouting of endothelial cells from the spheroids when encapsulated in fibrin was allowed. The possibility of spheroids, from different maturation stages, to assemble into a more large construct was proven by doublet fusion experiments. The relevance of using three-dimensional microtissues with tissue-specific microarchitecture and increased complexity, together with the high-throughput generation approach, makes the generated spheroids a suitable tool for in vitro drug-testing and human disease modeling.

An inexpensive “do-it-yourself” device for rapid generation of uniform tumor spheroids

Three-dimensional (3D) cancer cell culture models such as tumor spheroids better recapitulate in vivo tumors than conventional two-dimensional (2D) models. However, two major challenges limit the routine use of 3D tumor spheroids. Firstly, most existing methods of generating tumor spheroids are not high-throughput. Secondly, tumor spheroids generated using current methods are highly variable in dimension. Here, we describe a simple 'Do-It-Yourself (DIY)' device that can be assembled for less than $7 of parts and generate uniform tumor spheroids in a high-throughput manner. We used a simple phone coin vibrating motor to superimpose the vibration for breaking a laminar jet of cell-loaded alginate solution into equally sized spherical beads. We generated 3,970 tumor spheroids/min, which exhibited a hypoxic core recapitulating in vivo tumors and could be used to test the diffusion efficacy of anticancer drugs. Such low-cost, easy-to-fabricate, simple-to-operate systems with high-throughput outcomes are essential to democratize and standardize cancer research.

In vitro vascularized liver tumor model based on a microfluidic inverse opal scaffold for immune cell recruitment investigation.

Liver cancer, characterized as a kind of malignant tumor within the digestive system, poses great health harm, and immune escape stands out as an important reason for its occurrence and development. Chemokines, pivotal in guiding immune cells' migration, is necessary to initiate and deliver an effective anti-tumor immune response. Therefore, understanding the chemotactic environment and identifying chemokines that regulate recruitment of immune cells to the tumor microenvironment (TME) are critical to improve current immunotherapy interventions. Herein, we report a well-defined inverse opal scaffold generated with a microfluidic emulsion template for the construction of a vascularized liver tumor model, offering insights into immune cells' recruitment. Due to the excellent 3D porous morphology of the inverse opal scaffold, human hepatocellular carcinoma cells can aggregate in the pores of the scaffold to form uniform multicellular tumor spheroids. More attractively, the vascularized liver tumor model can be achieved by constructing a 3D co-culture system involving endothelial cells and hepatocellular carcinoma cells. The results demonstrate that the 3D co-cultured tumor cells increase the neutrophil chemokines remarkably and recruit neutrophils to tumor tissues, then promote tumor progression. This approach opens a feasible avenue for realizing a vascularized liver tumor model with a reliable immune microenvironment close to that of a solid tumor of liver cancer.

Tumor Spheroid Fabrication and Encapsulation in Polyethylene Glycol Hydrogels for Studying Spheroid-Matrix Interactions.

Three-dimensional (3D) encapsulation of spheroids is crucial to adequately replicate the tumor microenvironment for optimal cell growth. Here, we designed an in vitro 3D glioblastoma model for spheroid encapsulation to mimic the tumor extracellular microenvironment. First, we formed square pyramidal microwell molds using polydimethylsiloxane. These microwell molds were then used to fabricate tumor spheroids with tightly controlled sizes from 50-500 μm. Once spheroids were formed, they were harvested and encapsulated in polyethylene glycol (PEG)-based hydrogels. PEG hydrogels are a versatile platform for spheroid encapsulation, as hydrogel properties such as stiffness, degradability, and cell adhesiveness can be tuned independently. Here, we used a representative soft (~8 kPa) hydrogel to encapsulate glioblastoma spheroids. Finally, a method to stain and image spheroids was developed to obtain high-quality images via confocal microscopy. Due to the dense spheroid core and relatively sparse periphery, imaging can be difficult, but using a clearing solution and confocal optical sectioning helps alleviate these imaging difficulties. In summary, we show a method to fabricate uniform spheroids, encapsulate them in PEG hydrogels and perform confocal microscopy on the encapsulated spheroids to study spheroid growth and various cell-matrix interactions.

Synthesis and evaluation of phenolic capsaicin-derived self-polymers for antibacterial activity

Capsaicin, as a low-toxic antibacterial bioactive substance, is recognized as an environmentally friendly antifouling agent. To obtain a naturally derived antifouling agent with stronger antibacterial ability, capsaicin-derived self-polymers (labeled POHABA, PPHABA, PAHMA, and PAMTHBA) containing phenols (catechol, hydroquinone, resorcinol, phloroglucinol) and N-hydroxymethyl acrylamide were synthesized by precipitation polymerization, which was uniform spheroids with folded surfaces (diameter 0.6–1.6 µm, except PAMTHBA diameter 200–400 nm). The antibacterial activity assay showed that they had a significant inhibitory effect on E. coli and S. aureus (MIC: 0.4 mg/mL against E. coli and 0.8 mg/mL against S. aureus; antibacterial rate: 99.99 % for E. coli and 98.18 % for S. aureus), which was related to the content of phenolic hydroxyl on the surface of the microspheres by comparison with the semi-quantitative analysis of EDS and XPS spectra. The antibacterial activity of self-polymers was significantly higher than that of the monomers, mainly because there were more and more uniform hydroxyl groups on the surface of polymer microspheres. These results provide a theoretical basis for capsaicin-derived self-polymers to be applied to environmentally friendly antifouling agents.

Droplet Microfluidic-Based Low-Cost and High-Speed Microsphere Array Direct Writing Technology and Its Applications.

Microsphere arrays have significant applications and broad development prospects in various fields and disciplines. The simple, efficient, low-cost, automatic, and controllable preparation of microsphere arrays in multiple dimensions and morphologies is still a significant challenge. Here, a novel microsphere array direct writing technology was developed using a low-cost portable droplet microfluidic device and a high-precision XY movable platform. The proposed technology provided a powerful platform for the direct-writing preparation of microsphere arrays and was successfully applied to the precise and controllable fabrication of microsphere arrays with different sizes, shapes, structures, and arrangements. Additionally, gel microsphere arrays with metal ion patterns were fabricated using the microsphere arrays as templates and exhibited excellent performance in the visual analytical detection of heavy metal ions. Moreover, the simulated microsphere arrays offer a promising platform for rapidly generating high-viability and uniform 3D tumor spheroids. Therefore, given the superiority of this technology and the great potential of microsphere arrays, this simple high-speed microsphere array direct writing technology has a promising application in the multidisciplinary intersection of chemical, biological, and material sciences.

Protocol development to further differentiate and transition stem cell-derived pancreatic progenitors from a monolayer into endocrine cells in suspension culture

The generation of functional β-cells from human pluripotent stem cells (hPSCs) for cell replacement therapy and disease modeling of diabetes is being investigated by many groups. We have developed a protocol to harvest and aggregate hPSC-derived pancreatic progenitors generated using a commercially available kit into near uniform spheroids and to further differentiate the cells toward an endocrine cell fate in suspension culture. Using a static suspension culture platform, we could generate a high percentage of insulin-expressing, glucose-responsive cells. We identified FGF7 as a soluble factor promoting aggregate survival with no inhibitory effect on endocrine gene expression. Notch inhibition of pancreatic progenitor cells during aggregation improved endocrine cell induction in vitro and improved graft function following implantation and further differentiation in mice. Thus we provide an approach to promote endocrine formation from kit-derived pancreatic progenitors, either through extended culture or post implant.

Robust formation of optimal single spheroids towards cost-effective in vitro three-dimensional tumor models.

While useful for fundamental in vitro studies, monolayer cell cultures are not physiologically relevant. Spheroids, a complex three-dimensional (3D) structure, more closely resemble in vivo tumor growth. Spheroids allow the results obtained relating to proliferation, cell death, differentiation, metabolism, and various antitumor therapies to be more predictive of in vivo outcomes. In the protocol herein, a rapid and high-throughput method is discussed for the generation of single spheroids using various cancer cell lines, including brain cancer cells (U87 MG, SEBTA-027, SF188), prostate cancer cells (DU-145, TRAMP-C1), and breast cancer cells (BT-549, Py230) in 96-round bottom-well plates. The proposed method is associated with significantly low costs per plate without requiring refining or transferring. Homogeneous compact spheroid morphology was evidenced as early as 1 day after following this protocol. Proliferating cells were traced in the rim, while dead cells were found to be located inside the core region of the spheroid using confocal microscopy and the Incucyte® live imaging system. H&E staining of spheroid sections was utilized to investigate the tightness of the cell packaging. Through western blotting analyses, it was revealed that a stem cell-like phenotype was adopted by these spheroids. This method was also used to obtain the EC50 of the anticancer dipeptide carnosine on U87 MG 3D culture. This affordable, easy-to-follow five-step protocol allows for the robust generation of various uniform spheroids with 3D morphology characteristics.

Abstract 5268: Drug screening of non-small cell lung cancer patient-derived organoids in a microwell platform

Abstract Patient-derived organoids (PDOs) have been widely accepted as ideal 3D tumor models for drug response prediction. However, research involving non-small cell lung cancer (NSCLC) PDOs is hampered by the compromised sample size and the lack of appropriate platforms tailored for scarce samples. Although BME2 domes show promising results in PDO maintenance and drug testing, the need for relatively large sample and the long incubation time hinder their application in drug response studies. Here, we report an agarose microwell (AMW) platform requiring miniscule sample volume for PDO rapid drug screening. The AMWs were formed by 3D-printed molds and attached to the bottom of 24-well plates. Each well has ~400 agarose microwells with 200μm diameter, 75μm depth and U-shaped bottom. Seeded cells migrate and form spheroids in each microwell. NSCLC spheroids of H358 (KRASG12C), A549 (KRASG12S) and PDOs CK7152 (KRASG12C, obtained from NCI PDMR) were cultured in arrays and treated by KRASG12C inhibitor adagrasib. PDOs normally grow in a BME2 dome, however large PDO size variability and consumption complicate drug evaluation. The AMWs are biocompatible and have a smooth surface that allows single cells or PDOs to migrate into microwells within 1d. The amount of PDOs in one dome is enough for 4 wells of 24-well plate. After 7d culture, PDOs formed in AMW exhibited high viability (89%±7% in AMW vs. 89%±11%, in dome) and better size uniformity (CV 29% in AMW vs. 44% in dome). AMWs can easily be implemented to standardize cytotoxicity evaluation. Following the exposure to adagrasib for 72h, we found that H358 spheroids were sensitive to KRASG12C specific inhibitor adagrasib in dose-dependent manner with an IC50 of 88nM; whereas A549 spheroids -without the target mutation- was not responsive. CK7152 PDOs were also exquisitely sensitive to adagrasib (34% and 21% viability reductions viability in AMWs and dome). Drug sensitivity can be affected by several factors that maintain proliferative signals active in tumor microenvironment. We have been able to simulate the effect of the tumor microenvironment, by culturing spheroids and PDOs with fibroblast supernatant in AMWs. The IC50 of adagrasib for H358 spheroids treated with WI-38 supernatant was about 3.5 × higher than that in normal medium (310nM vs. 88nM). We replicated these results with CK7152 PDOs treated with CAF supernatant that significantly decreased viability from 24% to 51% (p<0.001) following 72h treatment of 500nM adagrasib. Our AMW is a convenient 3D culture platform that generates uniform spheroids from a small sample volume for highly consistent drug screening results. The adagrasib drug response observed in AMW was comparable to that in conventional dome culture. Resistance due to tumor microenvironment can be readily assessed using fibroblast supernatant in AMW platform, promising for pre-clinical drug screening and personalized medicine. Citation Format: Qiyue Luan, Ines Pulido, Jian Zhou, Takeshi Shimamura, Ian Papautsky. Drug screening of non-small cell lung cancer patient-derived organoids in a microwell platform. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5268.

EMT and Tumor Turning Point Analysis in 3D Spheroid Culture of HNSCC and Mesenchymal Stem Cells.

The prognosis, metastasis, and behavior of head and neck squamous cancer cells are influenced by numerous factors concerning the tumor microenvironment, intercellular communication, and epithelial-to-mesenchymal transition (EMT). The aim of this study was to examine the codependent interaction of the mesenchymal stroma with head and neck squamous cell carcinoma (HNSCC) in a 3D spheroid structure. To simulate stroma-rich and -poor 3D tumor microenvironments, cells of the established cell SCC-040 were cultured with human mesenchymal stromal cells (MSCs), forming 3D stroma-tumor spheroids (STSs). STSs were compared to uniform spheroids of SCC-040 and MSC, respectively. The expressions of CD24, β-catenin, SNAI2, and ZEB2 were analyzed via RT-qPCR. The immunohistochemical expressions of E-cadherin, connexin 43, vimentin, and emmprin were analyzed, and protein expression pathways as well as Akt signaling were assessed via protein analysis. A promotive effect on the expressions of EMT markers ZEB2 (p = 0.0099), SNAI2 (p = 0.0352), and β-catenin (p = 0.0031) was demonstrated in STSs, as was the expression of Akt pathway proteins mTOR (p = 0.007), Erk1/2 (p = 0.0045), and p70 S6 Kinase (p = 0.0016). Our study demonstrated a change in genetic expression patterns early on in tumor development, indicating a tumor turning point.

Core–Shell Spheroid‐Laden Microgels Crosslinked under Biocompatible Conditions for Probing Cancer‐Stromal Communication

Multicellular cancer spheroids (MCSs) have emerged as a promising in vitro model that recaptures many features of solid tumours in vivo. To generate cancer spheroids, cells are encapsulated in microgels with high throughput. While the biophysical properties of the cancer spheroid and biomaterial influence the function and behavior of the cells, characterization of these properties remains largely unexplored. In addition, many existing techniques lack control over cell positioning, resulting in the formation of spheroids with large variability. Herein, a versatile, microfluidic platform for generating biocompatible core–shell microgels with uniform cancer spheroids is reported. In addition, an in situ micromechanics measuring device to determine the stiffness of individual artificial cancer niches is used. The power of the microfluidics‐based method by making MCS‐laden microgels to model the interactions of stromal cancer cells is demonstrated. Together with in vivo data, it is shown that tumor cell proliferation is promoted by cocultured fibroblasts.

3D Multicellular Tumor Spheroids in a Microfluidic Droplet System for Investigation of Drug Resistance.

A three-dimensional (3D) tumor spheroid model plays a critical role in mimicking tumor microenvironments in vivo. However, the conventional culture methods lack the ability to manipulate the 3D tumor spheroids in a homogeneous manner. To address this limitation, we developed a microfluidic-based droplet system for drug screening applications. We used a tree-shaped gradient generator to control the cell density and encapsulate the cells within uniform-sized droplets to generate a 3D gradient-sized tumor spheroid. Using this microfluidic-based droplet system, we demonstrated the high-throughput generation of uniform 3D tumor spheroids containing various cellular ratios for the analysis of the anti-cancer drug cytotoxicity. Consequently, this microfluidic-based gradient droplet generator could be a potentially powerful tool for anti-cancer drug screening applications.