3D Cell Culture Models Research Articles

Impact of Polymorphic Microfibers for Establishment of Neuronal Model

ABSTRACTThe biological and mechanical environment of cells is better mimicked in 3D compared to 2D cell cultures. However, creating accurate 3D cell culture models particularly for ultra‐soft tissues like brain or spinal cord is challenging since the hydrogels that match these properties are mechanically fragile. Therefore, implementing reinforcing structures, such as microfibers, is essential to provide the necessary support. Particularly, fibrous systems are of interest since they offer natural fibrillar structures similar to the extracellular matrix. This study focuses on exploring the interactions between a motor neuron‐like cell line and multiple microfiber‐morphologies and mechanics. Monitoring cell‐microfiber interactions over time we unveiled various dynamic undetected behaviors and interactions happening upon contact depending on the used microfiber properties. These highly defined microfiber fragments were fabricated using multiple processes—electrospinning, Melt Electrowriting, and microfluidic spinning—with properties differing in size, mechanics, and surface chemistry. The excellent control over our microfiber systems enabled the investigation of single parameters in an isolated manner. In addition, we quantified the observed varying movement modes of the monitored cell‐microfiber tandems. The study demonstrates the significance of microfiber design for biological applications and establishes methodological foundations for the implementation of customized microfiber systems in the field of biofabrication.

TMOD-28. MODELING MICROENVIRONMENTAL DYNAMICS: A NOVEL 3D GLIOMA-NEURON FUSION MODEL

Abstract Gliomas engage in bidirectional interactions with their microenvironment, wherein neuronal activity influences glioma proliferation and gliomas induce neuronal hyperexcitability. Although various models elucidate this complex interplay, a preference for a three-dimensional (3D) cell culture model has emerged to mimic intra-tumoral heterogeneity and microenvironmental interactions influencing glioma invasion, resistance, and recurrence. This study introduces an in vitro, self-assembled, scaffold-free, neuron-glioma spheroid fusion model to investigate the electrophysiological and functional interplay between glioma and its microenvironment. Primary mouse cortical neurons (MCN) were isolated from prenatal brain tissues and were cultured at 500,000 cells/spheroid on ultra-low attachment plates. Glioma organoids (WHO Grade 2-4) were developed from primary patient-derived tissue samples and were allowed to self-assemble for at least two weeks. After spontaneous spiking activity was detected in MCN spheroids, they were combined with glioma organoids in culture and allowed to fuse. A multielectrode array (MEA) was used to characterize the electrophysiological properties of the fusion model. Structural and functional characterizations were performed using immunofluorescence staining of proliferation, microglial, astrocytic, synaptic, and tumor markers. Additionally, the electrophysiological effects of the dual-IDH-mutant inhibitor, Vorasidenib, were assessed in WHO grade 2-4 IDH-mutant gliomas. Electrophysiological analysis of glioma-neuron co-cultures using MEA demonstrated a significantly increased network synchrony and firing rate in the fusion model when compared with the neuron-only condition, consistent with 2D models in the past. In correlation, the fusion model also demonstrated a significant increase in the Ki67 proliferation index across WHO grade 2-4 gliomas when compared to the glioma organoid-only condition. Vorasidenib treatment decreased neuronal firing rate and synchrony. Together, these results offer a high-throughput 3D model for recapitulating the tumor-brain microenvironment for both low and high-grade gliomas. Future studies will focus on the use of this model for drug screening and the exploration of malignant transformation in gliomas.

Leveraging Organ-on-Chip Models to Investigate Host-Microbiota Dynamics and Targeted Therapies for Inflammatory Bowel Disease.

Inflammatory bowel disease (IBD) is an idiopathic gastrointestinal disease with drastically increasing incidence rates. Due to its multifactorial etiology, a precise investigation of the pathogenesis is extremely difficult. Although reductionist cell culture models and more complex disease models in animals have clarified the understanding of individual disease mechanisms and contributing factors of IBD in the past, it remains challenging to bridge research and clinical practice. Conventional 2D cell culture models cannot replicate complex host-microbiota interactions and stable long-term microbial culture. Further, extrapolating data from animal models to patients remains challenging due to genetic and environmental diversity leading to differences in immune responses. Human intestine organ-on-chip (OoC) models have emerged as an alternative in vitro model approach to investigate IBD. OoC models not only recapitulate the human intestinal microenvironment more accurately than 2D cultures yet may also be advantageous for the identification of important disease-driving factors and pharmacological interventions targets due to the possibility of emulating different complexities. The predispositions and biological hallmarks of IBD focusing on host-microbiota interactions at the intestinal mucosal barrier are elucidated here. Additionally, the potential of OoCs to explore microbiota-related therapies and personalized medicine for IBD treatment is discussed.

The Impact of 3D Tumor Spheroid Maturity on Cell Migration and Invasion Dynamics

Cell motility is crucial in cancer metastasis, and understanding its regulation in tumor cells is vital for developing anti-metastatic therapies. Traditional 2D cell culture assays provide insights into cell migration but fail to replicate the complex 3D architecture of tissues in vivo. 3D cell culture models like tumor spheroids have been applied for cell migration tests. This study investigates the role of spheroid maturity in tumor cell motility, hypothesizing that spheroid maturity mirrors physiological conditions in solid tumors. Human prostate (DU145), breast (MCF-7), and murine breast (EMT-6) cancer cells were cultured into spheroids of varying ages (3, 7, and 11 days). The migration and invasion of these spheroids were analyzed, revealing that 11-day-old DU145 spheroids demonstrated the greatest horizontal migration, correlating with RNA-seq data showing increased cell adhesion, cytoskeleton dynamics, and motility pathways. Confocal microscopy and single-cell analysis indicated higher reactive oxygen species (ROS) levels in mature spheroids, potentially activating motility pathways. Additionally, DU 145 spheroids were treated with chemotherapy reagent Doxorubicin (DOX), and the results showed that spheroids culture for 7 and 11 days exhibited greater resistance to DOX compared to spheroids cultured for 3 days. These findings highlight the importance of considering spheroid maturity in cancer research and drug development, emphasizing the need for systematic analysis of spheroid growth conditions to ensure reproducible and reliable experimental settings.

PM2.5 Induces Developmental Neurotoxicity in Cortical Organoids

There is mounting evidence implicating the potential neurotoxic effects of PM2.5 during brain development, as it has been observed to traverse both the placental barrier and the fetal blood-brain barrier. However, the current utilization of 2D cell culture and animal models falls short in providing an accurate representation of human brain development. Consequently, the precise mechanisms underlying PM2.5-induced developmental neurotoxicity in humans remain obscure. To address this research gap, we constructed three-dimensional (3D) cortical organoids that faithfully recapitulate the initial stages of human cerebral cortex development. Our goal is to investigate the mechanisms of PM2.5-induced neurotoxicity using 3D brain organoids that express cortical layer proteins. Our findings demonstrate that exposure to PM2.5 concentrations of 5 μg/mL and 50 μg/mL induces neuronal apoptosis and disrupts normal neural differentiation, thereby suggesting a detrimental impact on neurodevelopment. Furthermore, transcriptomic analysis revealed PM2.5 exposure induced aberrations in mitochondrial complex I functionality, which is reminiscent of Parkinson's syndrome, potentially mediated by misguided axon guidance and compromised synaptic maintenance. This study is a pioneering assessment of the neurotoxicity of PM2.5 pollution on human brain tissues based on 3D cortical organoids, and the results are of great significance in guiding the formulation of the next air pollution prevention and control policies in China to achieve the sustainable improvement of air quality and to formulate pollution abatement strategies that can maximize the benefits to public health.

Tracking Small Extracellular Vesicles Using a Minimally Invasive PicoGreen Labeling Strategy.

Extracellular vesicles (EVs) are cell-secreted lipid bilayer delimited particles that mediate cellular communication. These tiny sacs of cellular information play an important role in cell communication and alter the physiological process under both normal and pathological conditions. As such, tracking EVs can provide valuable information regarding the basic understanding of cell communication, the onset of early malignancy, and biomarker discovery. Most of the current EV-tracking strategies are invasive, altering the natural characteristics of EVs by modifying the lipid bilayer with lipophilic dyes or surface proteins with fluorescent reporters. The invasive labeling strategies could alter the natural processes of EVs and thereby have major limitations for functional studies. Here, we report an alternative minimally invasive EV labeling strategy using PicoGreen (PG), a small molecule that fluoresces at 520 nm when bound to dsDNA. We show that PG binds to dsDNA associated with small EVs (50-200 nm), forming a stable and highly fluorescent PG-DNA complex in EVs (PG-EVs). In both 2D cell culture and 3D organoid models, PG-EV showed efficient tracking properties, including a high signal-to-noise ratio, time- and concentration-dependent uptake, and the ability to traverse a 3D environment. We further validated PG-EV tracking using dual-labeled EVs following two orthogonal labeling strategies: (1) Bioconjugation via surface amine labeling and (2) donor cell engineering via endogenously expressing mCherry-tetraspanin (CD9/CD63/CD81) reporter proteins. Our study has shown the feasibility of using PG-EV as an effective EV tracking strategy that can be applied for studying the functional role of EVs across multiple model systems.

Continuous Viscoelasticity Measurement of Cell Spheroids via Microfluidic Electrical Aspiration.

Measurement of viscoelastic characteristics of cells cultured in three-dimensional (3D) is critical to study many biological processes including tissue and organ growth. In this article, we present a unique electrical aspiration method to measure the viscoelastic properties of cell spheroids. A microfluidic sensor was created to demonstrate this method. Unlike the traditional optical aspiration method, the aspiration of the cell spheroids is monitored via monitoring the dynamic electrical resistance change of a symmetrical microfluidic aspiration channel. We first used the microfluidic device to measure the viscoelastic properties of two types of biological tissues derived from calfskin and porcine left ventricular myocardium. The equilibrium elastic modulus and creep time constants were measured to be 148.1 ± 24.1 kPa and 76.7 ± 3.5 s and 64.5 ± 7.7 kPa and 31.4 ± 2.7 s respectively, which matched well with reported data. The test validated the principle of the electrical aspiration method. Next, we applied the method for measuring cell spheroids made of human mesenchymal stem cells at different culture stages. The equilibrium elastic modulus and apparent viscosity decreased with increasing culture time. Compared to optical aspiration methods, this microfluidic electrical aspiration method has no reliance on transparent materials and image processing, which thus allows simple setup, fast data acquisition and analysis. The use of a symmetric aspiration channel and the linear half-space model enable measurements of a large number of viscoelastic properties via a single measurement with higher accuracy. This method will enable high throughput, continuous viscoelastic measurement of cell spheroids as well as other 3D cell culture models in flow conditions without the need for any optical measurements.

PNU-74654 Enhanced the Antiproliferative Activity of Gemcitabine by Targeting Wnt/β-Catenin Pathway in Pancreatic Cancer

Background: The Wnt/beta-catenin pathway is one of the pathways that is deregulated in pancreatic cancer and is reported to be associated with a poor prognosis. This indicates the need for the identification of novel agents to improve the efficacy of current therapy or have an improved efficacy. Therefore, in the present study, we explored the anticancer activity of PNU- 74654 alone or in combination with gemcitabine in 2 and 3-dimensional cell culture models of pancreatic cancer. Methods: The MTT assay was carried out to determine the viability of PC cancerous cells (PCCs), while the cytotoxicity of this agent was evaluated in a 3D cell culture model (spheroid). The effects of PNU-74654 were investigated in established cell migration/invasion assays. Results: The expression of candidate genes affecting the cell cycle, migration, and Wnt/bcatenin pathway was evaluated at mRNA and/or proteins by RT-PCR or Western blot. PNU- 74654 inhibited the cell growth at IC50 of 122 ± 0.4 umol/L and had a synergistic effect on the antiproliferative properties of gemcitabine by modulating the Wnt pathway. The PNU- 74654/gemcitabine combination reduced the migratory and invasiveness of PC cells, compared to control cells, through perturbation of E-cadherin. Conclusion: Our findings demonstrate the profound antitumor properties of PNU-74654 in in vitro models of pancreatic cancer, supporting further in vivo studies to evaluate the therapeutic impact of this novel therapy to target the Wnt pathway in the treatment of pancreatic cancer.

Inhibition of OXPHOS induces metabolic rewiring and reduces hypoxia in murine tumor models

IntroductionTumor hypoxia is a feature of many solid malignancies and is known to cause radio resistance. In recent years it has become clear that hypoxic tumor regions also foster an immunosuppressive phenotype and are involved in immunotherapy resistance. It has been proposed that reducing the tumors’ oxygen consumption will result in an increased oxygen concentration in the tissue and improve radio- and immunotherapy efficacy. The aim of this study is to investigate the metabolic rewiring of cancer cells by pharmacological attenuation of oxidative phosphorylation (OXPHOS) and subsequently reduce tumor hypoxia. Material and methodsThe metabolic effects of three OXPHOS inhibitors IACS-010759, atovaquone and metformin were explored by measuring oxygen consumption rate, extra cellular acidification rate, and [18F]FDG uptake in 2D and 3D cell culture. Tumor cell growth in 2D cell culture and hypoxia in 3D cell culture were analyzed by live cell imaging. Tumor hypoxia and [18F]FDG uptake in vivo following treatment with IACS-010759 was determined by immunohistochemistry and ex vivo biodistribution respectively. ResultsIn vitro experiments show that tumor cell metabolism is heterogeneous between different models. Upon OXPHOS inhibition, metabolism shifts from oxygen consumption through OXPHOS towards glycolysis, indicated by increased acidification and glucose uptake. Inhibition of OXPHOS by IACS-010759 treatment reduced diffusion limited tumor hypoxia in both 3D cell culture and in vivo. Although immune cell presence was lower in hypoxic areas compared with normoxic areas, it is not altered following short term OXPHOS inhibition. DiscussionThese results show that inhibition of OXPHOS causes a metabolic shift from OXPHOS towards increased glycolysis in 2D and 3D cell culture. Moreover, inhibition of OXPHOS reduces diffusion limited hypoxia in 3D cell culture and murine tumor models. Reduced hypoxia by OXPHOS inhibition might enhance therapy efficacy in future studies. However, caution is warranted as systemic metabolic rewiring can cause adverse effects.

3D Scaffold-Based Culture System Enhances Preclinical Evaluation of Natural Killer Cell Therapy in A549 Lung Cancer Cells.

Cell-based immunotherapies have emerged as promising cancer treatment modalities, demonstrating remarkable clinical efficacy. As interest in applying immune cell-based therapies to solid tumors has gained momentum, experimental models that enable long-term monitoring and mimic clinical administration are increasingly necessary. This study explores the potential of scaffold-based cell culture technologies, specifically three-dimensional (3D) extracellular matrix (ECM)-like frameworks, as promising solutions. These frameworks facilitate unhindered immune cell growth and enable continuous cancer cell culture. The three-dimensional (3D) cell culture model was developed using tailored scaffolds for natural killer (NK) cell culture. Within this framework, A549 lung cancer cells were cocultured with NK cells, allowing real-time monitoring for up to 28 days. The expression of critical markers associated with anticancer drug resistance and epithelial-mesenchymal transition (EMT) was evaluated in cancer cells within this 3D culture context. Compared to conventional 2D monolayer cultures, this 3D scaffold-based culture revealed that solid tumor cells, specifically A549 cells, exhibited heightened resistance to anticancer drugs. Additionally, the 3D culture environment upregulated the expression of EMT markers namely vimentin, N-cadherin, and fibronectin, while NK and zEGFR-CAR-NK cells displayed anticancer effects. In the two-dimensional (2D) coculture, only zEGFR-CAR-NK cells exhibited such effects in the 3D coculture system, highlighting an intriguing inconsistency with the 2D culture model, further confirmed by in vivo experiments. This in vitro 3D cell culture model reliably predicts outcomes in NK immunotherapy experiments. Thus, it represents a valuable tool for investigating drug resistance mechanisms and assessing the efficacy of immune cell-based therapies. By bridging the gap between in vitro and in vivo investigations, this model effectively translates potential treatments into animal models and facilitates rigorous preclinical evaluations.

8468 Development And Characterisation Of Three-Dimensional Cell Culture Models Of Adrenocortical Carcinoma

Abstract Disclosure: S. Feely: None. N. Mullen: None. P. Donlon: None. C. Hantel: None. M.C. dennedy: None. Adrenocortical carcinoma (ACC) is a rare malignancy carrying a poor prognosis and limited treatment options. Surgical resection is the only option for cure; but unsuitable for advanced disease. Mitotane is the approved medical therapy, but associated with treatment resistance and adverse effects. Newer therapies, such as immune checkpoint inhibitors, have limited success. Development of improved therapies is limited by current pre-clinical disease models. No available model reflects the tumour micro-environment. Three-dimensional (3D) cell culture models are increasingly used in cancer research (6), with a paucity of developed models in ACC. In the current study, we developed novel 3D models of ACC using a type-1 collagen matrix. We then characterised these as representative ACC disease models . 3D cell culture models of ACC were optimised by embedding ACC cell lines, MUC-1, HAC15 and H295R within a type-1 Collagen matrix at differing cell numbers. Expression of type-1 collagen in ACC primary tumours was determined using immunohistochemistry (n=4). Cell viability for each model was assessed by evaluating sytox blue staining by Flow Cytometry. Metabolic activity of ACC cells within the matrix was determined using AlamarBlue staining. Prolferative activity was assessed using Ki67. Steroidogenesis was evaluated by measuring cortisol, aldosterone and androstenedione using LC-MS/MS.. Expression of CYP11B1, CYP11B2 CYP17, and StAR was measured using RT-qPCR. Type 1 collagen is strongly expressed in ACC tumour microenvironment. MUC-1, H295R and HAC15 cells were successfully cultured in a type 1 collagen matrix. MUC-1 cells cultured in the collagen matrix remain viable throughout the period of experimentation with optimum viability between 14 and 21 days. H295R cells also remain viable with optimum viability at an earlier stage of 7-14 days. HAC15 cells demonstrate high resilience in 3D cell culture with maximum viability throughout the entire period of experimentation up to 21 days. All three models increase their metabolic and proliferative activity over time. All three steroids are expressed by all three models with an increase in aldosterone secretion observed in 3D HAC15 and H295R models compared to monolayer (p>0.001) at days 7 and 14. All models express key steroidogenic enzymes. 3D ACC cell culture models were developed successfully using collagen type 1 abundant in ACC. Typical characteristics of the ACC tumour environment included (i) the presence of live and necrotic cells (ii) high metabolic activity and proliferation, reflective of cell turnover.. Capacity of cells to spread and proliferate within the 3D cell culture models was limited by available collagen substrate. Steroidogenic capacity was demonstrated in 3D cell culture. This model therefore retained key characteristics of ACC and shows promise as an animal-sparing, pre-clinical model. Presentation: 6/1/2024

Construction of a lung cancer 3D culture model based on alginate/gelatin micro-beads for drug evaluation.

Lung cancer is one of the most common malignant tumors worldwide. Despite advances in lung cancer treatment, patients still face challenges related to drug resistance and recurrence. Current methods for evaluating anti-cancer drug activity are insufficient, as they rely on two-dimensional (2D) cell culture and animal models. Therefore, the development of an in vitro drug evaluation model capable of predicting individual sensitivity to anti-cancer drugs would greatly enhance the success rate of drug treatments for lung cancer patients. The purpose of this research is to utilise conditional reprogramming technology to cultivate patient-derived lung cancer cells and to construct an in vitro 3D culture model using sodium alginate (SA) and gelatin. The aim is to study the biological characteristics of cells in the 3D culture model and to further investigate the sensitivity of anti-cancer drugs based on the alginate-gelatin 3D culture model. This approach provides new means and insights for personalized precision anti-cancer therapy and the development of new anti-cancer drugs. Conditional reprogramming technology was used to generate conditionally reprogrammed lung adenocarcinoma cells (CRLCs). Alginate-gelatin hydrogel micro-beads were created to explore their potential use in the assessment of anti-cancer drugs. Cell proliferation was also examined using the MTS assay method. Live/dead staining was performed to estimate cell distribution and viability using calcein acetoxymethyl ester/propidium iodide (calcein-AM/PI) double staining. Protein expression was assessed by Western blot. The cells grown in the three-dimensional (3D) culture were in a state of continuous proliferation, and there was an obvious phenomenon of cell mass growth. The drug sensitivity assay results demonstrated that compared with the 2D-grown cells, the CRLCs grown in the alginate-gelatin hydrogel micro-beads exhibited more resistance to anti-cancer drugs. The results also showed that the 3D-cultured CRLCs showed greater protein expression levels of stem cell hallmarks, such as Nanog Homeobox (NANOG), SRY-Box Transcription Factor 2 (SOX-2), and aldehyde dehydrogenase 1 family member A1 (ALDH1A1), than the 2D-grown cells. These findings suggest that the 3D hydrogel cell culture models more closely mimicked the in vivo biological and clinical behavior of cells, and demonstrated higher innate resistance to anti-cancer drugs than the 2D cell culture models, and thus could serve as valuable tools for diagnosis, drug screening, and personalized medicine.

Protein absorption alters the cellular targeting of glycopolymeric nanoparticles

Glycopolymeric nanoparticles are widely employed in biomedical applications due to their high targeting ability, biocompatibility, and biodegradability. The pendant saccharides serve as targeting ligands to be explicitly coordinated to receptors on cell membrane surfaces. However, proteins in the blood often absorb onto the nanoparticle surface, potentially impacting the exposure and effectiveness of the saccharide ligands for targeted delivery. To elucidate the protein absorption effects, we prepared micelles decorated with different percentages of fructose moieties and evaluated the bioactivity of nanoparticles using 2D cell culture, tumor spheroid, liver organoid, and coculture models. The 2D cell culture model did not show a significant difference in activity between protein-coated and non-coated micelles. However, a dramatic decrease in cellular uptake and penetration ability of micelles was observed in 3D tumor spheroids after protein absorption. Additionally, protein absorption notably increased micelle uptake in liver organoids. In the coculture model, protein absorption reduced micelle uptake in tumor spheroids while favoring uptake in liver organoids. Our work suggests that decreasing non-specific protein absorption of glycopolymeric nanoparticles could enhance their delivery efficiency. These findings underscore the importance of understanding protein interactions in nanoparticle applications for drug delivery.

NK1 receptor blockade disrupts microtumor growth and aggregation in a three-dimensional syngeneic breast cancer model

Several data indicate that Substance P (SP) neurokinin type 1 receptor (NK1R) is at the center of the interaction between cancer cells and peripheral sensory neurons. Selecting the appropriate cancer cell line and its susceptibility to being modulated by NK1 antagonists are critical to studying this complex interaction. In the current study, we have focused on this selection by comparing several aspects of the allogeneic triple-negative breast cancer (TNBC) cell line (MDA-MB-231LUC+) with a modified syngeneic cell line (E0771LUC+), both expressing luciferase. This comparison was made using several methods, SP stimulation and 3D cell culture models, to better reproduce the heterogenous microenvironment of solid tumors observed in vivo. Furthermore, the susceptibility of the syngeneic cell line (E0771LUC+) to NK1R antagonist (Aprepitant) was tested. Our results indicate that E0771LUC+ recapitulates several essential aspects of the allogenic cell line, rendering this syngeneic line ideal to be used on immune-competent animals during in vivo studies. We have also found that both cell lines are susceptible to SP stimulation, and their proliferation is disrupted by NK1R antagonists (Aprepitant). Follow-up in vivo studies are required to verify and refine these findings.

3D cell culture models in research: applications to lung cancer pharmacology.

Lung cancer remains one of the leading causes of cancer-related mortality worldwide, necessitating innovative research methodologies to improve treatment outcomes and develop novel strategies. The advent of three-dimensional (3D) cell cultures has marked a significant advancement in lung cancer research, offering a more physiologically relevant model compared to traditional two-dimensional (2D) cultures. This review elucidates the various types of 3D cell culture models currently used in lung cancer pharmacology, including spheroids, organoids and engineered tissue models, having pivotal roles in enhancing our understanding of lung cancer biology, facilitating drug development, and advancing precision medicine. 3D cell culture systems mimic the complex spatial architecture and microenvironment of lung tumours, providing critical insights into the cellular and molecular mechanisms of tumour progression, metastasis and drug responses. Spheroids, derived from commercialized cell lines, effectively model the tumour microenvironment (TME), including the formation of hypoxic and nutrient gradients, crucial for evaluating the penetration and efficacy of anti-cancer therapeutics. Organoids and tumouroids, derived from primary tissues, recapitulate the heterogeneity of lung cancers and are instrumental in personalized medicine approaches, supporting the simulation of in vivo pharmacological responses in a patient-specific context. Moreover, these models have been co-cultured with various cell types and biomimicry extracellular matrix (ECM) components to further recapitulate the heterotypic cell-cell and cell-ECM interactions present within the lung TME. 3D cultures have been significantly contributing to the identification of novel therapeutic targets and the understanding of resistance mechanisms against conventional therapies. Therefore, this review summarizes the latest findings in drug research involving lung cancer 3D models, together with the common laboratory-based assays used to study drug effects. Additionally, the integration of 3D cell cultures into lung cancer drug development workflows and precision medicine is discussed. This integration is pivotal in accelerating the translation of laboratory findings into clinical applications, thereby advancing the landscape of lung cancer treatment. By closely mirroring human lung tumours, these models not only enhance our understanding of the disease but also pave the way for the development of more effective and personalized therapeutic strategies.

Development of two layer fibrous scaffolds for 3D in vitro modelling: Effects of morphology and surface properties on cell proliferation, adhesion and drug sensitivity

Three-dimensional (3D) in vitro cell models are gaining popularity in cancer research compared to two-dimensional (2D) cell culture models because of their ability to more accurately represent the tissue microenvironment. This study introduces a novel approach for the fabrication of 3D cell culture models by combining modified melt-electrospinning technology and solution electrospinning to create 3D two-layer fibrous scaffolds. By manipulating electrospinning parameters (voltage 11–12 kV and feed rate 0.85–1.55 mg/min), we engineered poly (ε-caprolactone) (PCL) scaffolds with varying fiber diameters (8.9 ± 2.2 to 23.1 ± 3.4 μm) and pore sizes (32.7 ± 12.8 to 122.5 ± 21.1 μm). Non-thermal plasma (NTP) treatment at energy density of 0.23 J/cm2 enhanced surface hydrophilicity, evident in a significant reduction in water contact angles from 113 ± 0° to 64 ± 0°. A comprehensive set of characterization techniques, including X-ray diffraction (XRD), differential scanning calorimetry (DSC), and thermogravimetric analysis (TGA) confirmed that plasma treatment altered the surface wettability without compromising the scaffold's chemical stability or crystalline structure. Biological assays involving human breast cancer MDA-MB-231 cells and human glioblastoma U-87 MG cells demonstrated high cell viability, indicating biocompatibility of the scaffolds. A pronounced difference in proliferation patterns was noted between MDA-MB-231 and U-87 MG, with the latter fully utilizing voids for the formation of cell aggregates, especially in the “fine” scaffold, indicating the dependence of cell behavior on scaffold architecture, which must be adjusted according to the modelled system and duration. Furthermore, investigation of the therapeutic efficacy of doxorubicin revealed a distinct drug response in 3D cultures, EC50 values of doxorubicin in 3D culture was approximately twice higher compared to 2D. These findings underscore the potential of tailored PCL scaffolds to create more physiologically relevant models for cancer research, tissue engineering, and drug screening applications, highlighting the necessity for careful consideration of scaffold design to achieve the desired in vitro model.

Development of Tissue-Engineered Model of Fibrotic Scarring after Spinal Cord Injury to Study Astrocyte Activation and Neurite Outgrowth In Vitro.

Traumatic spinal cord injuries (SCI) are debilitating injuries affecting twenty-seven million people worldwide and cause functional impairments. Despite decades of research and medical advancements, current treatment options for SCI remain limited, in part due to the complex pathophysiology of spinal cord lesions including cellular transformation and extracellular matrix (ECM) remodeling. Recent studies have increased focus on fibrotic scarring after SCI, and yet much remains unclear about the impact of fibrotic scarring on SCI lesion progression. Here, using collagen and decellularized spinal cord-based composite hydrogels, a three-dimensional (3D) cell culture model mimicking the fibrous core of spinal cord lesions was implemented to investigate its influence on the surrounding astrocytes. To mimic the fibrotic milieu, collagen fibril thickness was tuned using previously established temperature-controlled casting methods. In our platforms, astrocytes in fibro-mimetic hydrogels exhibited increased levels of activation markers such as glial fibrillary acidic protein and N-cadherin. Furthermore, astrocytes in fibro-mimetic hydrogels deposited more fibronectin and laminin, further hinting that astrocytes may also contribute to fibrotic scarring. These markers were decreased when Rho-ROCK and integrin β1 were inhibited via pharmacological inhibitors. Mechanistic analysis of Yes-associated protein reveals that blocking integrin β1 prevents mechanosensing of astrocytes, contributing to altered phenotypes in variable culture conditions. In the presence of these inhibitors, astrocytes increased the secretion of brain-derived neurotrophic factor, and a greater degree of dorsal root ganglia neurite infiltration into the underlying hydrogels was observed. Altogether, this study presents a novel tissue-engineered platform to study fibrotic scarring after SCI and may be a useful platform to advance our understanding of SCI lesion aggravation.

From in vitro to in silico: a pipeline for generating virtual tissue simulations from real image data.

3D cell culture models replicate tissue complexity and aim to study cellular interactions and responses in a more physiologically relevant environment compared to traditional 2D cultures. However, the spherical structure of these models makes it difficult to extract meaningful data, necessitating advanced techniques for proper analysis. In silico simulations enhance research by predicting cellular behaviors and therapeutic responses, providing a powerful tool to complement experimental approaches. Despite their potential, these simulations often require advanced computational skills and significant resources, which creates a barrier for many researchers. To address these challenges, we developed an accessible pipeline using open-source software to facilitate virtual tissue simulations. Our approach employs the Cellular Potts Model, a versatile framework for simulating cellular behaviors in tissues. The simulations are constructed from real world 3D image stacks of cancer spheroids, ensuring that the virtual models are rooted in experimental data. By introducing a new metric for parameter optimization, we enable the creation of realistic simulations without requiring extensive computational expertise. This pipeline benefits researchers wanting to incorporate computational biology into their methods, even if they do not possess extensive expertise in this area. By reducing the technical barriers associated with advanced computational modeling, our pipeline enables more researchers to utilize these powerful tools. Our approach aims to foster a broader use of in silico methods in disease research, contributing to a deeper understanding of disease biology and the refinement of therapeutic interventions.

Cellular and Microbial In Vitro Modelling of Gastrointestinal Cancer.

Human diseases are multifaceted, starting with alterations at the cellular level, damaging organs and their functions, and disturbing interactions and immune responses. In vitro systems offer clarity and standardisation, which are crucial for effectively modelling disease. These models aim not to replicate every disease aspect but to dissect specific ones with precision. Controlled environments allow researchers to isolate key variables, eliminate confounding factors and elucidate disease mechanisms more clearly. Technological progress has rapidly advanced model systems. Initially, 2D cell culture models explored fundamental cell interactions. The transition to 3D cell cultures and organoids enabled more life-like tissue architecture and enhanced intercellular interactions. Advanced bioreactor-based devices now recreate the physicochemical environments of specific organs, simulating features like perfusion and the gastrointestinal tract's mucus layer, enhancing physiological relevance. These systems have been simplified and adapted for high-throughput research, marking significant progress. This review focuses on in vitro systems for modelling gastrointestinal tract cancer and the side effects of cancer treatment. While cell cultures and in vivo models are invaluable, our main emphasis is on bioreactor-based in vitro modelling systems that include the gut microbiome.

Generation of an infantile GM1 gangliosidosis induced pluripotent stem cell line (CHOCi005-A) for disease modeling and therapeutic testing

GM1 gangliosidosis (GM1) is a rare autosomal recessive neurogenerative lysosomal storage disease characterized by deficiency of beta-galactosidase (β-gal) and intralysosomal accumulation of GM1 ganglioside and other glycoconjugates. Resources for GM1 disease modelling are limited, and access to relevant cell lines from human patients is not possible. Generation of iPSC lines from GM1 patient-derived dermal fibroblasts allows for disease modelling and therapeutic testing in 2D and 3D cell culture models relevant to CNS disorders, including various neuronal subtypes and cerebral organoids. The iPSC line described here will be critical to therapeutic development and set the foundation for translational gene therapy work.