Three-dimensional Cell Research Articles

Noncontact elasticity measurement of hydrogels in a culture dish using reverberant optical coherence elastography

Estimating the elasticity of hydrogel phantoms in a cell culture plane is important for understanding the cell behavior in response to various types of mechanical stimuli. Hence, a noncontact tool for measuring the elastic properties of hydrogel phantoms in such three-dimensional cell cultures is required. A well-known method to determine the mechanical properties of hydrogels is the transient wave method. However, due to the multiple reflections of waves from the boundaries, a bigger cell culture plane or multiple directional filters may be required. In this study, we utilized reverberant shear wave elastography, which is based on the autocorrelation principle, to evaluate the shear wave speed in hydrogel samples within a culture dish. Numerical simulations were performed first to confirm the validity of the reverberant elastography method. Subsequently, we used this method to measure the wave speeds in hydrogel phantoms with different concentrations. Shear rheology tests were also performed, and their results were found to be in good agreement with the measured shear wave speeds. The proposed method could be useful for measuring the elasticity of tissues in tissue engineering applications in an inexpensive and noncontact manner.

Contribution to the world of 3D cell products created by bio 3D printing technology and to the life science field

We have been making 3D tissues consist of cells only, based on the corporate philosophy of "contributing to dramatic advances in medical care through the practical application of innovative 3D cell stacking technology." Currently, in the field of regenerative medicine, we are working toward obtaining approval from the Ministry of Health, Labor and Welfare and commercializing large artificial organs that are made from patients' own cells and have functions such as nerve regeneration, osteochondral regeneration, and blood vessels. On the other hand, this three-dimensional cell stacking technology can be extended to technology for culturing cells in an environment similar to the human body, and is expected to serve as a new methodology for evaluating the effects of new products in various fields on living organisms. Therefore, we are planning a business to provide developers of pharmaceuticals, foods, cosmetics, etc. with a small device called "Functional Cell Device (FCD)" that reproduces some of the functions of human organs outside the body. As the first step, we have developed a three-dimensional liver construct (3D mini-liver). The in vitro human liver model has a wide range of usage, such as evaluation of hepatotoxicity of drugs, elucidation of drug metabolism mechanism, and model of liver disease. In this report, we will outline it together with actual examples in regenerative medicine.

A cellular tilting mechanism important for dynamic tissue shape changes and cell differentiation in Drosophila

Dynamic changes in three-dimensional cell shape are important for tissue form and function. In the developing Drosophila eye, photoreceptor differentiation requires the progression across the tissue of an epithelial fold known as the morphogenetic furrow. Morphogenetic furrow progression involves apical cell constriction and movement of apical cell edges. Here, we show that cells progressing through the morphogenetic furrow move their basal edges in opposite direction to their apical edges, resulting in a cellular tilting movement. We further demonstrate that cells generate, at their basal side, oriented, force-generating protrusions. Knockdown of the protein kinase Src42A or photoactivation of a dominant-negative form of the small GTPase Rac1 reduces protrusion formation. Impaired protrusion formation stalls basal cell movement and slows down morphogenetic furrow progression and photoreceptor differentiation. This work identifies a cellular tilting mechanism important for the generation of dynamic tissue shape changes and cell differentiation.

Energy absorption characteristics and multi-objective optimization of 3D bionic negative Poisson’s ratio honeycomb

This paper introduces a new bionic cell element with a negative Poisson’s ratio, which is derived from the traditional re-entrant hexagonal cell element. By incorporating rotation into the two-dimensional (2D) cell element, a three-dimensional (3D) cell element is formed, which exhibits enhanced mechanical properties. The 3D honeycomb is obtained by arraying the 3D cell elements and is enhanced by adding longitudinal ribs to the honeycomb. The present study employs the ABAQUS/EXPLICIT software to investigate the correlation between various compression velocities and the crashworthiness and energy absorption capacity of a given system. The effects of different compression velocities on the 3D honeycomb deformation pattern and energy absorption performance are analyzed. The simulation results show that the new bionic negative Poisson’s ratio honeycomb has better crashworthiness and energy absorption capacity than the traditional re-entrant hexagonal honeycomb. The nominal stress-strain curves of different honeycombs and the energy absorbed per unit mass of honeycomb are compared and analyzed, and the NSGA-II genetic algorithm is used to perform a multi-objective optimization analysis of the peak stress and energy absorbed per unit mass of honeycomb. The results show that the optimized honeycomb can increase the absorbed energy per unit mass by 7.5 % when the peak stress is the same as the initial honeycomb, while the peak stress can be reduced by 9.6 % when the absorbed energy per unit mass is the same.

Multi-objective optimization strategy for multipolar magnesium electrolysis cell based on thermal-electric model

In the operation of industrial multipolar magnesium electrolysis cell, cell voltage and current density are important indicators of the production performance of the cell, while thermal balance is a key factor for normal operation. This paper utilizes the finite element method to establish a three-dimensional full cell thermal-electric model, studying the thermal field distribution and current density distribution in a 165kA multipolar magnesium electrolysis cell. Based on the heat balance of the multipolar cell, the influence of structural parameters on current intensity, average current density at the anode surface and resistance voltage is assessed. Dimensionless analysis yielded optimization criteria, and the Non-dominated Sorting Genetic Algorithm II (NSGA-II) was employed for multi-objective optimization problems. When stringent optimization is executed, significant performance improvements can be achieved in terms of the electrolysis cell's resistance voltage and current density. Moreover, under the optimization criteria, the relative error of the 265kA multipolar magnesium electrolysis cell model is less than 1 %. The results indicate that the workflow combining the finite element method with multi-objective optimization algorithms can be applied to the optimization and scale-up design of high-current multipolar magnesium electrolysis cell.

Real-time 3D tracking of swimming microbes using digital holographic microscopy and deep learning.

The three-dimensional swimming tracks of motile microorganisms can be used to identify their species, which holds promise for the rapid identification of bacterial pathogens. The tracks also provide detailed information on the cells' responses to external stimuli such as chemical gradients and physical objects. Digital holographic microscopy (DHM) is a well-established, but computationally intensive method for obtaining three-dimensional cell tracks from video microscopy data. We demonstrate that a common neural network (NN) accelerates the analysis of holographic data by an order of magnitude, enabling its use on single-board computers and in real time. We establish a heuristic relationship between the distance of a cell from the focal plane and the size of the bounding box assigned to it by the NN, allowing us to rapidly localise cells in three dimensions as they swim. This technique opens the possibility of providing real-time feedback in experiments, for example by monitoring and adapting the supply of nutrients to a microbial bioreactor in response to changes in the swimming phenotype of microbes, or for rapid identification of bacterial pathogens in drinking water or clinical samples.

Nobiletin Inhibits Breast Cancer Stem Cell by Regulating the Cell Cycle: A Comprehensive Bioinformatics Analysis and In Vitro Experiments

Inhibiting breast cancer stem cell (BCSC) signaling pathways is a strategic method for successfully treating breast cancer. Nobiletin (NOB) is a compound widely found in orange peel that exhibits a toxic effect on various types of cancer cells, and inhibits the signaling pathways that regulate the properties of BCSCs; however, the effects of NOB on BCSCs remain elusive. The purpose of this study was to determine the target genes of NOB for inhibiting BCSCs using in vitro three-dimensional breast cancer cell culture (mammospheres) and in silico approaches. We combined in vitro experiments to develop mammospheres and conducted cytotoxicity, next-generation sequencing, and bioinformatics analyses, such as gene ontology, the Reactome pathway enrichment, network topology, gene set enrichment analysis, hub genes selection, genetic alterations, prognostic value related to the mRNA expression, and mRNA and protein expression of potential NOB target genes that inhibit BCSCs. Here, we show that NOB inhibited BCSCs in mammospheres from MCF-7 cells. We also identified CDC6, CHEK1, BRCA1, UCHL5, TOP2A, MTMR4, and EXO1 as potential NOB targets inhibiting BCSCs. NOB decreased G0/G1, but increased the G2/M cell population. These findings showed that NOB is a potential therapeutic candidate for BCSCs treatment by regulating cell cycle.

Characterization of TGFβ1-induced tendon-like structure in the scaffold-free three-dimensional tendon cell culture system

The biological mechanisms regulating tenocyte differentiation and morphological maturation have not been well-established, partly due to the lack of reliable in vitro systems that produce highly aligned collagenous tissues. In this study, we developed a scaffold-free, three-dimensional (3D) tendon culture system using mouse tendon cells in a differentially adherent growth channel. Transforming Growth Factor-β (TGFβ) signaling is involved in various biological processes in the tendon, regulating tendon cell fate, recruitment and maintenance of tenocytes, and matrix organization. This known function of TGFβ signaling in tendon prompted us to utilize TGFβ1 to induce tendon-like structures in 3D tendon constructs. TGFβ1 treatment promoted a tendon-like structure in the peripheral layer of the constructs characterized by increased thickness with a gradual decrease in cell density and highly aligned collagen matrix. TGFβ1 also enhanced cell proliferation, matrix production, and morphological maturation of cells in the peripheral layer compared to vehicle treatment. TGFβ1 treatment also induced early tenogenic differentiation and resulted in sufficient mechanical integrity, allowing biomechanical testing. The current study suggests that this scaffold-free 3D tendon cell culture system could be an in vitro platform to investigate underlying biological mechanisms that regulate tenogenic cell differentiation and matrix organization.

Electrophysiological properties of dorsal root ganglion neurons cultured on 3D silicon micro-pillar substrates

BackgroundSilicon-based micro-pillar substrates (MPS), as three-dimensional cell culture platforms with vertically aligned micro-patterned scaffolding structures, are known to facilitate high-quality growth and morphology of dorsal root ganglion (DRG) sensory neurons, promote neurite outgrowth and enhance neurite alignment. However, the electrophysiological aspects of DRG neurons cultured on silicon MPSs have not been thoroughly investigated, which is of greatest importance to ensure that such substrates do not disrupt neuronal homeostasis and function before their widespread adoption in diverse biomedical applications. New methodWe conducted whole-cell patch-clamp recordings to explore the electrophysiological properties of DRG neurons cultured on MPS arrays, utilizing a custom-made upright patch-clamp setup. ResultsOur findings revealed that DRG neurons exhibited similar electrophysiological responses on patterned MPS samples when compared to the control planar glass surfaces. Notably, there were no significant differences observed in the action potential parameters or firing patterns of action potentials between neurons grown on either substrate. Comparison with existing methodsIn the current study we for the first time confirmed that successful electrophysiological recordings can be obtained from the cells grown on MPS. ConclusionOur results imply that, despite the potential alterations caused by the cumulative trauma of tissue harvest and cell dissociation, essential functional cell properties of DRG neurons appear to be relatively maintained on MPS surfaces. Therefore, vertically aligned silicon MPSs could be considered as a potentially effective three-dimensional system for supporting a controlled cellular environment in culture.

Genome-scale model of Rothia mucilaginosa predicts gene essentialities and reveals metabolic capabilities.

Cystic fibrosis (CF), an inherited genetic disorder caused by mutations in the cystic fibrosis transmembrane conductance regulator gene, results in sticky and thick mucosal fluids. This environment facilitates the colonization of various microorganisms, some of which can cause acute and chronic lung infections, while others may positively impact the disease. Rothia mucilaginosa, an oral commensal, is relatively abundant in the lungs of CF patients. Recent studies have unveiled its anti-inflammatory properties using in vitro three-dimensional lung epithelial cell cultures and in vivo mouse models relevant to chronic lung diseases. Apart from this, R. mucilaginosa has been associated with severe infections. However, its metabolic capabilities and genotype-phenotype relationships remain largely unknown. To gain insights into its cellular metabolism and genetic content, we developed the first manually curated genome-scale metabolic model, iRM23NL. Through growth kinetics and high-throughput phenotypic microarray testings, we defined its complete catabolic phenome. Subsequently, we assessed the model's effectiveness in accurately predicting growth behaviors and utilizing multiple substrates. We used constraint-based modeling techniques to formulate novel hypotheses that could expedite the development of antimicrobial strategies. More specifically, we detected putative essential genes and assessed their effect on metabolism under varying nutritional conditions. These predictions could offer novel potential antimicrobial targets without laborious large-scale screening of knockouts and mutant transposon libraries. Overall, iRM23NL demonstrates a solid capability to predict cellular phenotypes and holds immense potential as a valuable resource for accurate predictions in advancing antimicrobial therapies. Moreover, it can guide metabolic engineering to tailor R. mucilaginosa's metabolism for desired performance.IMPORTANCECystic fibrosis (CF) is a genetic disorder characterized by thick mucosal secretions, leading to chronic lung infections. Rothia mucilaginosa is a common bacterium found in various parts of the human body, acting as a normal part of the flora. In people with weakened immune systems, it can become an opportunistic pathogen, while it is prevalent and active in CF airways. Recent studies have highlighted its anti-inflammatory properties in the lower pulmonary system, indicating the intricate relationship between microbes and human health. Herein, we have developed the first manually curated metabolic model of R. mucilaginosa. Our study examined the previously unknown relationships between the bacterium's genotype and phenotype and identified essential genes that impact the metabolism under various conditions. With this, we opt for paving the way for developing new strategies in antimicrobial therapy and metabolic engineering, leading to enhanced therapeutic outcomes in cystic fibrosis and related conditions.

Theoretical Analysis of Thermophysical Properties of 3D Carbon/Epoxy Braided Composites with Varying Temperature.

A three-dimensional helix geometry unit cell is established to simulate the complex spatial configuration of 3D braided composites. Initially, different types of yarn factors, such as yarn path, cross-sectional shape, properties, and braid direction, are explained. Then, the multiphase finite element method is used to develop a new theoretical calculation procedure based on the unit cell for predicting the impacts of environmental temperature on the thermophysical properties of 3D four-direction carbon/epoxy braided composites. The changing rule and distribution characteristics of the thermophysical properties for 3D four-direction carbon/epoxy braided composites are obtained at temperatures ranging from room temperature to 200 °C. The influences of environmental temperature on the coefficients of thermal expansion (CTE) and the coefficients of thermal conduction (CTC) are evaluated, by which some important conclusions are drawn. A comparison is conducted between theoretical and experimental results, revealing that variations in temperature exert a notable influence on the thermophysical characteristics of 3D four-directional carbon/epoxy braided composites. The theoretical calculation procedure is an effective tool for the mechanical property analysis of composite materials with complex geometries.

Evaluation puramatrix as a 3D microenvironment for neural differentiation of human breastmilk stem cells

The extracellular matrix is recognized as an efficient and determining component in the growth, proliferation, and differentiation of cells due to its ability to perceive and respond to environmental signals. Applying three-dimensional scaffolds can create conditions similar to the extracellular matrix and provide an opportunity to investigate cell fate. In this study, we employed the PuraMatrix hydrogel scaffold as an advanced cell culture platform for the neural differentiation of stem cells derived from human breastmilk to design an opportune model for tissue engineering.Isolated stem cells from breastmilk were cultured and differentiated into neural-like cells on PuraMatrix peptide hydrogel and in the two-dimensional system. The compatibility of breastmilk-derived stem cells with PuraMatrix and cell viability was evaluated by scanning electron microscopy and MTT assay, respectively. Induction of differentiation was achieved by exposing cells to the neurogenic medium. After 21 days of the initial differentiation process, the expression levels of glial fibrillary acidic protein (GFAP), microtubule-associated protein (MAP2), β-tubulin III, and neuronal nuclear antigen (NeuN) were analyzed using the immunostaining technique.The results illustrated a notable expression of MAP2, β-tubulin-III, and NeuN in the three-dimensional cell culture in comparison to the two-dimensional system, indicating the beneficial effect of PuraMatrix scaffolds in the process of differentiating breastmilk-derived stem cells into neural-like cells.In view of the obtained results, the combination of breastmilk-derived stem cells and PuraMatrix hydrogel scaffold could be an advisable preference for neural tissue regeneration and cell therapy.

Photocatalytic manipulation of Ca2+ signaling for regulating cellular and animal behaviors via MOF-enabled H2O2 generation.

The in situ generation of H2O2 in cells in response to external stimulation has exceptional advantages in modulating intracellular Ca2+ dynamics, including high controllability and biological safety, but has been rarely explored. Here, we develop photocatalyst-based metal-organic frameworks (DCSA-MOFs) to modulate Ca2+ responses in cells, multicellular spheroids, and organs. By virtue of the efficient photocatalytic oxygen reduction to H2O2 without sacrificial agents, photoexcited DCSA-MOFs can rapidly trigger Ca2+ outflow from the endoplasmic reticulum with single-cell precision in a repeatable and controllable manner, enabling the propagation of intercellular Ca2+ waves (ICW) over long distances in two-dimensional and three-dimensional cell cultures. After photoexcitation, ICWs induced by DCSA-MOFs can activate neural activities in the optical tectum of tadpoles and thighs of spinal frogs, eliciting the corresponding motor behaviors. Our study offers a versatile optical nongenetic modulation technique that enables remote, repeatable, and controlled manipulation of cellular and animal behaviors.

A Scaffold-Assisted 3D Cancer Cell Model for Surface-Enhanced Raman Scattering-Based Real-Time Sensing and Imaging.

Despite recent advances in the development of scaffold-based three-dimensional (3D) cell models, challenges persist in imaging and monitoring cell behavior within these complex structures due to their heterogeneous cell distribution and geometries. Incorporating sensors into 3D scaffolds provides a potential solution for real-time, in situ sensing and imaging of biological processes such as cell growth and disease development. We introduce a 3D printed hydrogel-based scaffold capable of supporting both surface-enhanced Raman scattering (SERS) biosensing and imaging of 3D breast cancer cell models. The scaffold incorporates plasmonic nanoparticles and SERS tags, for sensing and imaging, respectively. We demonstrate the scaffold's adaptability and modularity in supporting breast cancer spheroids, thereby enabling spatial and temporal monitoring of tumor evolution.

Osteogenic differentiation of mesenchymal stem cell spheroids: A microfluidic device and electrochemiluminescence imaging study

Human mesenchymal stem cells (MSCs) have attracted attention as a major cellular source for regenerative medicine and preclinical studies. Three-dimensional (3D) cell culture systems can be used to construct physiologically relevant MSC spheroids with enhanced tissue regeneration capacity. However, what aspect of the MSC spheroids retains the regeneration capacity is not yet fully understood. Here, we induced osteoblast differentiation of MSC spheroids and investigated their respiratory activity using electrochemiluminescence (ECL) imaging developed in our previous study. In parallel, the ability of MSC spheroids to induce angiogenesis was investigated in a microfluidic device with a co-culture of endothelial cells. We found that the osteogenic differentiation of MSCs induced changes in their metabolic activity (respiratory activity of the spheroids decreased during differentiation) and lead to the loss of their angiogenesis-inducing ability. In other words, it is suggested that maintaining the stem cell nature with in the MSC spheroids is an important factor for tissue regeneration. In this study, we demonstrated the effective use of ECL imaging and microfluidic systems for the evaluation of MSC spheroids, providing new insights into stem cell tissue engineering.

Modular Multiwell Viscoelastic Hydrogel Platform for Two- and Three-Dimensional Cell Culture Applications.

Hydrogels have gained significant popularity as model platforms to study reciprocal interactions between cells and their microenvironment. While hydrogel tools to probe many characteristics of the extracellular space have been developed, fabrication approaches remain challenging and time-consuming, limiting multiplexing or widespread adoption. Thus, we have developed a modular fabrication approach to generate distinct hydrogel microenvironments within the same 96-well plate for increased throughput of fabrication as well as integration with existing high-throughput assay technologies. This approach enables in situ hydrogel mechanical characterization and is used to generate both elastic and viscoelastic hydrogels across a range of stiffnesses. Additionally, this fabrication method enabled a 3-fold reduction in polymer and up to an 8-fold reduction in fabrication time required per hydrogel replicate. The feasibility of this platform for two-dimensional (2D) cell culture applications was demonstrated by measuring both population-level and single-cell-level metrics via microplate reader and high-content imaging. Finally, a 96-well hydrogel array was utilized for three-dimensional (3D) cell culture, demonstrating the ability to support high cell viability. Together, this work demonstrates a versatile and easily adaptable fabrication approach that can support the ever-expanding tool kit of hydrogel technologies for cell culture applications.

Colon cancer inhibitory properties of Caulerpa lentillifera polysaccharide and its molecular mechanisms based on three-dimensional cell culture model

Caulerpa lentillifera is rich in polysaccharides, and its polysaccharides show a significant effect in different biological activities including anti-cancer activity. As an edible algae-derived polysaccharide, exploring the role of colon cancer can better develop the application from a dietary therapy perspective. However, more in-depth studies of C. lentillifera polysaccharide on anti-colon cancer activity and mechanism are needed. In this study, we found that Caulerpa lentillifera polysaccharides (CLP) showed potential anti-colon cancer effect on human colon cancer cell HT29 in monolayer (IC50 = 1.954 mg/mL) and spheroid (IC50 = 0.402 mg/mL). Transcriptomics and metabolomics analyses revealed that CLP had an inhibitory effect on HT29 3D spheroid cells by activating aminoacyl-tRNA biosynthesis as well as arginine and proline metabolism pathways. Furthermore, the anti-colon cancer effects of CLP were confirmed through other human colon cancer cell HCT116 and LoVo in monolayer cells (IC50 = 1.890 mg/mL and 1.437 mg/mL, respectively) and 3D spheroid cells (IC50 = 0.344 mg/mL and 0.975 mg/mL, respectively), and three patient-derived organoids with IC50 values of 6.333–8.780 mg/mL. This study provided basic data for the potential application of CLP in adjuvant therapeutic food for colon cancer on multiple levels, while further investigation of detailed mechanism in vivo was still required.

Optimizing ground improvement with encased stone columns: a 3D numerical analysis in very soft clay

Purpose This study aims to investigate the behavior of different types of stone columns, including the short and floating columns, as well as the ordinary and the geosynthetic encased stone columns (OSC and GESC). The effectiveness of the geosynthetic encasement and the impact of the installation using the lateral expansion method on the column performance is evaluated through a three-dimensional (3D) unit cell numerical analysis. Design/methodology/approach A full 3D numerical analysis is carried out using the explicit finite element code PLAXIS 3D to examine the installation influence on settlement reduction (ß), lateral displacement (Ux) and vertical displacement (Uz) relative to different values of lateral expansion of the column (0% to 15%). Findings The findings demonstrate the superior performance of GESC, particularly short columns outperforming floating counterparts. This enhanced performance is attributed to the combined effects of geosynthetic encasement and increased lateral expansion. Notably, these strategies contribute significantly to decreasing lateral displacement (Ux) at the column’s edge and reducing vertical displacement (Uz) under the rigid footing. Originality/value In contrast to previous studies that examined the installation effect of OSC contexts, this paper presents a comprehensive investigation into the effect of geosynthetic encasement and the installation effects using the lateral expansion method in very soft soil, using 3D numerical simulation. The study emphasizes the significance of the consideration of geosynthetic encasement and lateral expansion of the column during the design process to enhance column performance.

Cell Membrane Fragment-Wrapped Parenteral Nanoemulsions: A New Drug Delivery Tool to Target Gliomas.

Poor prognosis in high-grade gliomas is mainly due to fatal relapse after surgical resection in the absence of efficient chemotherapy, which is severely hampered by the blood-brain barrier. However, the leaky blood-brain-tumour barrier forms upon tumour growth and vascularization, allowing targeted nanocarrier-mediated drug delivery. The homotypic targeting ability of cell-membrane fragments obtained from cancer cells means that these fragments can be exploited to this aim. In this experimental work, injectable nanoemulsions, which have a long history of safe clinic usage, have been wrapped in glioma-cell membrane fragments via co-extrusion to give targeted, homogeneously sized, sterile formulations. These systems were then loaded with three different chemotherapeutics, in the form of hydrophobic ion pairs that can be released into the target site thanks to interactions with physiological components. The numerous assays performed in two-dimensional (2D) and three-dimensional (3D) cell models demonstrate that the proposed approach is a versatile drug-delivery platform with chemo-tactic properties towards glioma cells, with adhesive interactions between the target cell and the cell membrane fragments most likely being responsible for the effect. This approach's promising translational perspectives towards personalized nanomedicine mean that further in vivo studies are foreseen for the future.

Simple and Rapid Method of Microwell Array Fabrication for Drug Testing on 3D Cancer Spheroids.

Three-dimensional (3D) cell culture systems are becoming increasingly popular due to their ability to mimic the complex process of angiogenesis in cancer, providing more accurate and physiologically relevant data than traditional two-dimensional (2D) cell culture systems. Microwell systems are particularly useful in this context as they provide a microenvironment that more closely resembles the in vivo environment than traditional microwells. Poly(ethylene glycol) (PEG) microwells are particularly advantageous due to their bio-inertness and the ability to tailor their material characteristics depending on the PEG molecular weight. Although there are several methods available for microwell fabrication, most of them are time-consuming and expensive. The current study utilizes a low-cost laser etching technique on poly(methyl methacrylate) materials followed by molding with PDMS to produce microwells. The optimal conditions for making concave microwells are an engraving parameter speed of 600 mm/s, power of 20%, and a design diameter of the microwell of 0.4 mm. The artificial tumor achieved its full size after 7 days of cell growth in a microwell system, and the cells developed drugs through a live/dead assay test. The results of the drug testing revealed that the IC50 value of zerumbone-loaded liposomes in HepG2 was 4.53 pM, which is greater than the IC50 value of zerumbone. The HepG2 cancer sphere's 3D platform for medication testing revealed that zerumbone-loaded liposomes were very effective at high doses. These findings generally imply that zerumbone-loaded liposomes have the capacity to target the liver and maintain medication delivery.