High Cell Density Research Articles

Rapid and reproducible generation of glioblastoma spheroids for high-throughput drug screening

Identifying new substances that could potentially be used for tumor therapy and the precise analysis of their spectrum of action requires models that are as similar as possible to the tumor present in the patient. Traditionally, two-dimensional (2D) cell cultures are used. However, these only resemble solid tumors to a limited extent. More realistic in vivo models, such as tissue cultures, which are invaluable for the final analysis of the effect of new substances, are unsuitable for high-throughput screening (HTS), such as substance library screening. Therefore, we addressed which parameters need to be optimized to produce 3D cultures suitable for HTS using established tumor cell lines and ultra-low attachment plates, and we tested which experimental parameters need to be considered. In our studies, we have focused on cell lines from gliomas. Gliomas are incurable tumors of the central nervous system and are the subject of intensive research. Our studies used ten glioma cell lines from which we generated spheroids using ultra-low attachment plates. We then determined the spheroid size as a function of the initial cell number and the culture time. We analyzed cell viability using propidium iodide staining, evaluated the effects of temozolomide and radiation on spheroids, and compared the effect to that on 2D cultures. We found that spheroid size correlated linearly with the initial cell number. Fewer cells (250–500) generally resulted in better growth than a higher number. However, not all cell lines produced growing spheroids at all. The spheroids had an outer layer of living cells and an inner core of dead cells. The size of the inner core of dead cells was different in the various cell lines and developed differently during the incubation period. Radiation affected spheroids more than 2D cultures, especially at higher cell densities. Our results provide insight into using glioma cell lines to form spheroids as model systems. We have identified initial cell numbers as a critical parameter for their effective use in research, offering a hopeful outlook for tumor therapy research and drug development.

Increased Drug Resistance in Acute Lymphoblastic Leukemia Cells in Three-Dimensional High-Density Cell Cultures

Increased Drug Resistance in Acute Lymphoblastic Leukemia Cells in Three-Dimensional High-Density Cell Cultures

Interspecies Crosstalk via LuxI/LuxR-Type Quorum Sensing Pathways Contributes to Decreased Nematode Survival in Coinfections of Pseudomonas aeruginosa and Burkholderia multivorans.

Quorum sensing (QS) is a prominent chemical communication mechanism used by common bacteria to regulate group behaviors at high cell density, including many processes important in pathogenesis. There is growing evidence that certain bacteria can use QS to sense not only themselves but also other species and that this crosstalk could alter collective behaviors. In the current study, we report the results of culture-based and in vivo coinfection experiments that probe interspecies interactions between the opportunistic pathogens Pseudomonas aeruginosa and Burkholderia multivorans involving their LuxI/LuxR-type QS circuits. Using a Caenorhabditis elegans infection model, we show that infections with both species result in poorer host outcomes compared with monoinfections. We use genetic mutants and a transwell infection assay to establish that crosstalk via LuxR-type receptors and signals is important for this coinfection pathogenicity. Using laboratory cocultures with cell-based reporter systems, we show that the RhlR and CepR receptors in P. aeruginosa and B. multivorans, respectively, can each recognize a QS signal produced by the other species. Lastly, we apply chemical biology to complement our genetic approach and demonstrate the potential to regulate interspecies interactions between the wild-type strains of P. aeruginosa and B. multivorans through the application of synthetic compounds that modulate RhlR and CepR activities. Overall, this study reveals that interspecies interaction via QS networks is possible between P. aeruginosa and B. multivorans and that it can contribute to coinfection severity with these two species.

Preparation of PBAT/PLA Blend Microporous Foam With Excellent Resilience and Cushioning Properties by scCO2 Technology Through Improving Compatibility

ABSTRACTThe lightweight microcellular foams of polybutylene adipate terephthalate/polylactic acid (PBAT/PLA) with high mechanical strength and excellent resilience and buffering properties were reported using supercritical CO2 foaming technology. The effects of an epoxy chain extender (ST‐CE37B) on the mechanical properties, rheological properties, and dispersion morphology of PBAT/PLA blends were studied. The results showed that ST‐CE37B can act as compatibilizers and the PBAT/PLA blend with 0.5 phr ST‐CE37B exhibited the highest tensile strength (22 MPa). The PBAT/PLA foam with 0.7 phr ST‐CE37B, expansion ratio of 8.0 displayed the minor cell diameter (19.3 μm) and highest cell density (5.44 × 107 cells/cm3), the compressive strength was greater than that of pure PBAT/PLA foam, and the permanent strain was less than 15%. Therefore, compared with other foams, it can withstand larger stress according to the cushioning performance. In addition, this work also discussed the compression rebound performance (resilience) test of foam with the same blending sample, the same cell density, and different cell structures. It was found that foam with a larger cell size (49.2 μ m) showed lower permanent deformation and energy loss coefficient. This study provides a feasible method for preparing high microporous PBAT/PLA foam, expands the application range of biodegradable foam, and has research significance.

Germination of phytoplankton resting cells in surface sediments collected from the tributary and littoral zones of Lake Taihu, China

Phytoplankton responsible for harmful algal blooms (HABs) may originate from the seed bank present in lake and river sediments. In this study, we investigated the germination of phytoplankton in the laboratory from resting cells in sediments collected during spring from four tributary and littoral zones of Lake Taihu (east, south, west, and north), the season before HABs typically occur. We evaluated the potential for germinated phytoplankton to contribute to HABs. We found that 26 species from three phyla including Cyanobacteria, Bacillariophyta, and Chlorophyta germinated from the sediment during the 15-day incubation experiment. The germinated phytoplankton species from the sediments collected in most areas were moderately or very similar, but dominant species exhibited different temporal and spatial distributions. The densities of germinated cells for different phytoplankton species varied at different times during the incubation period. Cyanobacteria Microcystis spp. (1,511 cells g−1 wet weight) and Bacillariophyta Melosira sp. (2,865 cells g−1 wet weight) had the highest cell densities after 10-day incubation, whereas Chlorophyta had the highest cell density (54 cells g−1 wet weight) after 15-day incubation. There was a high number of Cyanobacteria resting cells that could germinate from the seed bank preserved in the sediments, suggesting potential risks of HABs throughout Lake Taihu.

Enhanced carbon dioxide bio-fixation and protein production of Chlorella pyrenoidosa through a highly efficient sequential heterotrophic and photoautotrophic culture strategy

Chlorella pyrenoidosa is one of the most promising microalgae for protein production with carbon dioxide (CO2) bio-fixation. However, the high production cost and low production capability under traditional photoautotrophic culture mode had severely limited its application. Here, a highly efficient sequential heterotrophic and photoautotrophic cultivation strategy was established. Firstly, the heterotrophic seeds culture conditions were optimized with glucose as the carbon source and 1 g L−1 of yeast extract was selected as a useful stimulus to boost cell growth. Then, the heterotrophic cells were transferred to photoautotrophic conditions directly and cultured under varied concentrations of CO2. Moreover, 8 % (v/v) of CO2, high light and high initial cell density were found to be beneficial for cell growth and CO2 sequestration. Finally, different initial nitrogen concentrations and nitrogen feeding strategies were evaluated to increase the protein content to 62.69 % of dry biomass. These results provide feasible strategies for efficient CO2 sequestration and protein production jointly in C. pyrenoidosa.

Measurement of optical density of microbes by multi‐light path transmission method

AbstractOptical density (OD) is an important indicator of microbial density, and a commonly used variable in growth curves to express the growth of microbial culture. However, OD values show a linear relationship with bacterial concentration only at low concentrations. When the cell density is high, the relationship loses linearity, and serial dilution is needed to obtain readings of better accuracy. Here, we show that measuring OD values using shorter light paths is in close equivalence to measuring OD values of the cell culture with corresponding dilution. By measuring three different light paths simultaneously, accurate OD values can be easily obtained from low to high cell density. Using this method, growth curves of Escherichia coli, Staphylococcus aureus, and Pichia pastoris are measured with higher accuracy. To further simplify the process, an L‐shaped cuvette and a corresponding turbidimeter are designed specifically for OD value measurement based on the multi‐light path transmission method.

Attenuating Cartilage Degeneration in a Low Mechanical Compression Rat Model Through Intra-Articular Injections of Allogeneic Bone Marrow-Derived Mesenchymal Stem Cells.

Mechanical stimulation significantly contributes to posttraumatic osteoarthritis (PTOA), a condition that impedes patient recovery following intra-articular injury. Effective treatment options for compression-induced injuries are limited. Bone marrow-derived mesenchymal stem cell (BMSC) implantation has emerged as a potential therapeutic breakthrough for joint diseases. The aim of this study was to attenuate the progression of PTOA induced by cyclic loading and demonstrate the potential effectiveness of BMSCs in a rat model of low mechanical compression. Using a rat model of compression-induced articular cartilage injury, assessments were conducted 2, 4, and 8 weeks after cyclic compressive loading. The expression of matrix metallopeptidase 13, transforming growth factor-beta 3 (TGF-β3), insulin-like growth factor 1 (IGF-1), and cleaved caspase-3 was evaluated through immunohistochemistry to investigate the mechanistic aspects underlying the prevention of compression-induced injury following BMSCs treatment. Intra-articular injections of BMSCs significantly improved scores in the OARSI (Osteoarthritis Research Society International) Osteoarthritis Cartilage Histopathology Assessment System and Histological-Histochemical Grading System. This treatment showed positive outcomes in maintaining high relative cell density and reducing proteoglycan loss after cyclic compression-induced injury. The expression patterns of IGF-1 and TGF-β3 provide valuable insights into the presence and distribution of these growth factors in healthy and injured cartilage. These findings highlight the efficacy of BMSCs treatment in attenuating the advancement of compression-induced injuries, albeit within a limited timeframe.

High cell density cultivation by anaerobic respiration

BackgroundOxygen provision is a bottleneck in conventional aerobic high cell density culturing (HCDC) of bacteria due to the low O2 solubility in water. An alternative could be denitrification: anaerobic respiration using nitrogen oxides as terminal electron acceptors. Denitrification is attractive because NO3− is soluble in water, the end-product (N2) is harmless, and denitrification is widespread among bacteria, hence suitable organisms for most purposes can be found. The pH must be controlled by injection of an inorganic acid to compensate for the pH increase by NO3−-consumption, resulting in salt accumulation if feeding the bioreactor with NO3− salt. We avoid this with our novel pH–stat approach, where the reactor is supplied with 5 M HNO3 to compensate for the alkalization, thus sustaining NO3−-concentration at a level determined by the pH setpoint. Here we present the first feasibility study of this method, growing the model strain Paracoccus denitrificans anaerobically to high densities with glucose as the sole C-source and NO3− as the N-source and electron acceptor.ResultsOur fed-batch culture reached 20 g cell dry weight L−1, albeit with slower growth rates than observed in low cell density batch cultures. We explored reasons for slow growth, and the measured trace element uptake indicates it is not a limiting factor. Bioassays with spent medium excluded accumulation of inhibitory compounds at high cell density as the reason for the slow growth. The most plausible reason is that high metabolic activity led to CO2/H2CO3 accumulation, thus suppressing pH, leading to a paucity in HNO3-feeding until N2-sparging had removed sufficient CO2. The three free intermediates in the denitrification pathway (NO3− → NO2− → NO → N2O → N2) can all reach toxic concentrations if the electron flow is unbalanced, and this did occur if cells were glucose-limited. On the other hand, accumulation of polyhydroxyalkanoates occurred if the cells were NO3−-limited. Carefully balancing glucose provision according to the HNO3 injected is thus crucial.ConclusionsThis work provides a proof of concept, while also identifying CO2/H2CO3 accumulation as a hurdle that must be overcome for further development and optimization of the method.

Comparative evaluation of collagen modifications in breast cancer in human and canine carcinomas

New diagnostic and therapeutic approaches have been increasingly demanded due to the high morbidity and mortality associated with breast cancer. Recently, changes in the collagen fibres in mammary neoplasms have been shown to provide information that can be helpful for more accurate diagnosis. We aimed to conduct a comparative analysis of the tumour stroma in human and canine mammary neoplasms to assess the relationship between collagen modifications and the behaviour of carcinomas in both species, by multiphoton microscopy. We present a retrospective study of 70 cases of human mammary tumour and 74 cases of canine mammary tumour. We analysed sections stained with haematoxylin and eosin from 1,200 representative areas of normal mammary tissue, fibroadenoma, grade I invasive carcinoma, grade III invasive carcinoma and invasive micropapillary carcinoma in human species and 1,304 representative areas of normal mammary tissue, benign mixed tumour, mixed carcinoma, carcinosarcoma, invasive micropapillary carcinoma and solid carcinoma in canine species. We obtained that both human and canine mammary carcinomas present lower density of collagen fibres, higher density of cells and the collagen fibres are more aligned than in normal tissue. For human mammary carcinomas, the collagen fibres are more linear as compared to normal tissue. In addition, we demonstrated that the carcinomas with unfavourable prognosis present shorter collagen fibres, and these collagen changes correlate with the clinical and pathological data in human and canine species. For dogs, there is a correlation between the mean fibre length with the specific survival times. Thus, we demonstrate that dogs provide an excellent comparative perspective for studying how changes in the tumour stroma affect patient survival.

The effects of Cefixime and Aspirin on phytoplankton community structure and dynamics: A Mesocosm approach

Pharmaceuticals are emerging contaminants of interest due to their ability to induce changes in the environment. These chemical compounds are discharged into aquatic ecosystems with a high likelihood of affecting non-target organisms such as phytoplankton. However, there is a gap in knowledge regarding the effects of pharmaceuticals on phytoplankton. This study used the mesocosm approach to investigate the effects of cefixime, a cephalosporin antibiotic and aspirin, a nonsteroidal anti-inflammatory drug on the community structure (species diversity, richness, and abundance) of phytoplankton. The mesocosm approach studied the effects of three treatments of pharmaceuticals: cefixime, aspirin and a combination of cefixime and aspirin, alongside a control experiment for 21 days. A total of 31 phytoplankton species belonging to five groups were identified; with diatoms and green algae having higher diversity compared to the other algae groups. Scenedesmus sp., Pediastrum sp. and Zygnema sp. were the top three recurring species in all the samples taken throughout the experimental period. Navicula sp. were the most recurring diatoms. The exposure of phytoplankton to cefixime, aspirin, and a combination of cefixime and aspirin significantly reduced the diversity, richness, and abundance of all species by the 21st day of the experiment. The control had the highest cell density (8,910 cells mL) of phytoplankton species on the 21st day, while the cefixime treatment had the lowest cell density (48 cells mL) f phytoplankton species on the seventh day of the study. This study demonstrated that cefixime and aspirin had major impacts on the phytoplankton community.

Enhancing Vaccine Efficacy and Accessibility through Microneedle Technology: A Review

Microneedling, a minimally invasive technique involving the creation of microchannels in the skin, has emerged as a promising platform for vaccine delivery. The use of microneedles for vaccination represents a new era in immunization techniques, wherein patients no longer dread sharp pricks. Their breakthrough potential lies in their ability to deliver vaccines directly into the epidermis or dermis where a high density of immune cells improve the particularly relevant effects of vaccines. Their intro- duction has met various challenges often experienced with traditional methods like bites caused by fear, pain from needles, or even that any medical doctor may take a long to administer them. Microneedles could improve stability and lessen the dependence on cold chain transport systems since they can be prepared in dry form. In addition to providing an al- ternative to syringes, they are also considered less painful than them. The review highlights the potential of mi- croneedles to improve vaccination rates, especially in un- derserved populations, and identifies key areas for future research and development.

Control of spatio-temporal patterning via cell growth in a multicellular synthetic gene circuit

A major goal in synthetic development is to build gene regulatory circuits that control patterning. In natural development, an interplay between mechanical and chemical communication shapes the dynamics of multicellular gene regulatory circuits. For synthetic circuits, how non-genetic properties of the growth environment impact circuit behavior remains poorly explored. Here, we first describe an occurrence of mechano-chemical coupling in synthetic Notch (synNotch) patterning circuits: high cell density decreases synNotch-gated gene expression in different cellular systems in vitro. We then construct, both in vitro and in silico, a synNotch-based signal propagation circuit whose outcome can be regulated by cell density. Spatial and temporal patterning outcomes of this circuit can be predicted and controlled via modulation of cell proliferation, initial cell density, and/or spatial distribution of cell density. Our work demonstrates that synthetic patterning circuit outcome can be controlled via cellular growth, providing a means for programming multicellular circuit patterning outcomes.

Optimizing the production of recombinant human papilloma virus type 52 major capsid protein L1 in Hansenula polymorpha

Cervical cancer is the fourth most prevalent cancer among women globally, with Thai women ranking it as the third most common. At present, a prophylactic vaccine, containing virus-like particles (VLPs) of HPV L1 capsid protein, is widely recognized as one of the major prevention strategies for cervical cancer. Unfortunately, due to a low cross-protection among subtypes, protection against each HPV subtype requires vaccination with VLPs of that specific subtype. This study aimed to optimize the cultivation medium and conditions to maximize the volumetric yield of HPV52 L1 protein produced by the recombinant H. polymorpha HPV52. The results revealed that supplementation of a complex organic nitrogen source HySoy (at 10 g/L) into SYN6 medium resulted in a significantly higher specific HPV52 L1 yield and the maximum specific and volumetric HPV52 L1 protein yield were obtained at 30℃. In this study, the volumetric yield of HPV52 L1 could be increased from 50.9 mg/L in flask-scale cultivation to 2728 mg/L in High Cell Density Cultivation or HCDC (53-folds) with a significant improvement in terms of productivity, from 0.85 mg/L.h in flask-scale cultivation to 22.7 mg/L.h in HCDC (27-folds).

Effect of Traditional and Non-Traditionally Processed Blue Corn Tortilla Consumption During the Gestation of Rats in the Dentate Gyrus of Pups.

The effect of consuming traditionally and non-traditionally processed blue corn tortillas on the dentate gyrus of rat pups during gestation was evaluated. Blue corn tortillas were made from grains steeped or not steeped in a solution of gallic acid and processed by traditional or microwave nixtamalization. Total polyphenol and total anthocyanin contents were analyzed. At day 20 of gestation, the pups were analyzed according to the diet administered to the pregnant rats, as follows: the control group fed with standard diet; the TN group = standard diet + blue corn tortilla by traditional nixtamalization; the TNGA group = standard diet + blue corn tortilla by traditional nixtamalization + gallic acid; the MN group = standard diet + blue corn tortilla by microwave nixtamalization; and the MNGA group = standard diet + blue corn tortilla by microwave nixtamalization + gallic acid. The cell density and soma size of the dentate gyrus in pups, along with the number of pups per litter and the litter weight, were recorded. The highest polyphenol and anthocyanin content were found in blue corn tortillas made from grains steeped in gallic acid and processed by microwave nixtamalization. The MNGA group showed larger litters as well as higher cell density (33%) and soma size (50% in the range of 30-50 μm2) in the dentate gyrus of pups as compared to the control.

Improving Chlorella protein production under heterotrophic high cell density fed-batch cultivation with a two-stage nitrogen nutrient supply strategy

Heterotrophic cultivation has been proved to be an effective and industrially scalable route for the economical production of Chlorella biomass. Nevertheless, the low protein content of heterotrophic Chlorella biomass hinders its utilization as a superior protein source. In this study, the influences of various C/N ratios on cell growth and protein production were evaluated for a previously isolated protein-rich alga Chlorella sorokiniana CMBB276. The results indicated that a high C/N ratio was favorable for cell growth, whereas a low C/N ratio was advantageous for protein synthesis. A proposed two-stage nitrogen supply strategy by shifting the C/N ratio from 18 to 6 was found to be capable of simultaneously improve the protein content and yield of C. sorokiniana CMBB276, achieving the maximum protein content of 58.6 % of dry cell weight (DCW) and a final protein yield of 87.0gL−1, respectively. We further verified that the fast accumulated ammonium in C. sorokiniana CMBB276 cultivated with the switched C/N ratio contributed to the remarkable enhancement of protein content, while concurrently causing cytotoxicity. Moreover, under high ammonium stress, while maintaining a high protein content, C. sorokiniana CMBB276 persisted in growing with a decreased level of cellular reactive oxygen species (ROS) and cell membrane damage through exogenous addition of acetate, which lays the foundation for further improvement of protein production by applying the two-stage nitrogen supply strategy in combination with acetate addition.

Perfusion Process Intensification for Lentivirus Production Using a Novel Scale-Down Model.

Process intensification has become an important strategy to lower production costs and increase manufacturing capacities for biopharmaceutical products. In particular for the production of viral vectors like lentiviruses (LVs), the transition from (fed-)batch to perfusion processes is a key strategy to meet the increasing demands for cell and gene therapy applications. However, perfusion processes are associated with higher medium consumption. Therefore, it is necessary to develop appropriate small-scale models to reduce development costs. In this work, we present the use of the acoustic wave separation technology in combination with the Ambr 250 high throughput bioreactor system for intensified perfusion process development using stable LV producer cells. The intensified perfusion process developed in the Ambr 250 model, performed at a harvest rate of 3 vessel volumes per day (VVD) and high cell densities, resulted in a 1.4-fold higher cell-specific functional virus yield and 2.8-fold higher volumetric virus yield compared to the control process at a harvest rate of 1 VVD. The findings were verified at bench scale after optimizing the bioreactor set-up, resulting in a 1.4-fold higher cell-specific functional virus yield and 3.1-fold higher volumetric virus yield.

De Novo Production of the Bioactive Phenylpropanoid Artepillin C Using Membrane-Bound Prenyltransferase in Komagataella phaffii.

Artepillin C is a diprenylated phenylpropanoid with various pharmacological benefits for human health. Its natural occurrence is limited to a few Asteraceae plants, such as Baccharis species, necessitating a stable supply through synthetic biology. In Saccharomyces cerevisiae, the utilization of aromatic substrates within the cell was limited, resulting in very low production of artepillin C. In this study, we used AcPT1, a p-coumaric acid (p-CA)-specific diprenyltransferase, in Komagataella phaffii to produce artepillin C. Detailed studies revealed that the critical bottleneck in K. phaffii was the supply of prenyl diphosphates, not phenylpropanoid flux. By enhancing the prenyl substrate pathway through overexpression of isopentenyl diphosphate isomerase and a truncated HMG-CoA reductase, we achieved a strong increase in artepillin C production. A major part of artepillin C was accumulated in yeast cells. One of the advantages of K. phaffii is its superior growth and ability to achieve high cell density cultivation compared to that of S. cerevisiae. Therefore, fed-batch cultivation with glycerol was performed. As a result, the dry cell weight (DCW) reached 61.0 g/L, and the intracellular amount of de novo produced artepillin C reached 187.3 μg/DCW. Analysis of intermediates revealed that the supply of p-CA constituted a bottleneck in artepillin C production in the engineered strain. By enhancing the p-CA supply, the intracellular accumulation of artepillin C reached 1200 μg/DCW even in batch cultivation. Moreover, the total intra- and extracellular amounts of artepillin C reached 12.5 mg/L, marking the highest de novo synthesis amount of artepillin C reported thus far, even under batch cultivation conditions.

Quorum sensing in Vibrio controls carbon metabolism to optimize growth in changing environmental conditions.

Bacteria sense population density via the cell-cell communication system called quorum sensing (QS). The evolution of QS and its maintenance or loss in mixed bacterial communities is highly relevant to understanding how cell-cell signaling impacts bacterial fitness and competition, particularly under varying environmental conditions such as nutrient availability. We uncovered a phenomenon in which Vibrio cells grown in minimal medium optimize expression of the methionine and tetrahydrofolate (THF) synthesis genes via QS. Strains that are genetically "locked" at high cell density grow slowly in minimal glucose media and suppressor mutants accumulate via inactivating-mutations in metF (methylenetetrahydrofolate reductase) and luxR (the master QS transcriptional regulator). In mixed cultures, QS mutant strains initially coexist with wild type, but as glucose is depleted, wild type outcompetes the QS mutants. Thus, QS regulation of methionine/THF synthesis is a fitness benefit that links nutrient availability and cell density, preventing accumulation of QS-defective mutants.

Molecular dynamics simulations of a multicellular model with cell-cell interactions and Hippo signaling pathway.

The transcriptional coactivator Yes-associated protein (YAP)/transcriptional co-activator with PDZ binding motif (TAZ) induces cell proliferation through nuclear localization at low cell density. Conversely, at extremely high cell density, the Hippo pathway, which regulates YAP/TAZ, is activated. This activation leads to the translocation of YAP/TAZ into the cytoplasm, resulting in cell cycle arrest. Various cancer cells have several times more YAP/TAZ than normal cells. However, it is not entirely clear whether this several-fold increase in YAP/TAZ alone is sufficient to overcome proliferation inhibition (contact inhibition) under high-density conditions, thereby allowing continuous proliferation. In this study, we construct a three-dimensional (3D) mathematical model of cell proliferation incorporating the Hippo-YAP/TAZ pathway. Herein, a significant innovation in our approach is the introduction of a novel modeling component that inputs cell density, which reflects cell dynamics, into the Hippo pathway and enables the simulation of cell proliferation as the output response. We assume such 3D model with cell-cell interactions by solving reaction and molecular dynamics (MD) equations by applying adhesion and repulsive forces that act between cells and frictional forces acting on each cell. We assume Lennard-Jones (12-6) potential with a softcore character so that each cell secures its exclusive domain. We set cell cycles composed of mitotic and cell growth phases in which cells divide and grow under the influence of cell kinetics. We perform mathematical simulations at various YAP/TAZ levels to investigate the extent of YAP/TAZ increase required for sustained proliferation at high density. The results show that a twofold increase in YAP/TAZ levels of cancer cells was sufficient to evade cell cycle arrest compared to normal cells, enabling cells to continue proliferating even under high-density conditions. Finally, this mathematical model, which incorporates cell-cell interactions and the Hippo-YAP/TAZ pathway, may be applicable for evaluating cancer malignancy based on YAP/TAZ levels, developing drugs to suppress the abnormal proliferation of cancer cells, and determining appropriate drug dosages. The source codes are freely available.