3D Cell Culture Research Articles

Dorsomorphin inhibits AMPK, upregulates Wnt and Foxo genes and promotes the activation of dormant follicles

AMPK is a well-known energy sensor regulating cellular metabolism. Metabolic disorders such as obesity and diabetes are considered detrimental factors that reduce fecundity. Here, we show that pharmacologically induced in vitro activation (by metformin) or inhibition (by dorsomorphin) of the AMPK pathway inhibits or promotes activation of ovarian primordial follicles in cultured murine ovaries and human ovarian cortical chips. In mice, activation of primordial follicles in dorsomorphin in vitro-treated ovaries reduces AMPK activation and upregulates Wnt and FOXO genes, which, interestingly, is associated with decreased phosphorylation of β-catenin. The dorsomorphin-treated ovaries remain of high quality, with no detectable difference in reactive oxygen species production, apoptosis or mitochondrial cytochrome c oxidase activity, suggesting safe activation. Subsequent maturation of in vitro-treated follicles, using a 3D alginate cell culture system, results in mature metaphase eggs with protruding polar bodies. These findings demonstrate that the AMPK pathway can safely regulate primordial follicles by modulating Wnt and FOXO genes, and reduce β-catenin phosphorylation.

Viscoelasticity in 3D Cell Culture and Regenerative Medicine: Measurement Techniques and Biological Relevance.

The field of mechanobiology is gaining prominence due to recent findings that show cells sense and respond to the mechanical properties of their environment through a process called mechanotransduction. The mechanical properties of cells, cell organelles, and the extracellular matrix are understood to be viscoelastic. Various technologies have been researched and developed for measuring the viscoelasticity of biological materials, which may provide insight into both the cellular mechanisms and the biological functions of mechanotransduction. Here, we explain the concept of viscoelasticity and introduce the major techniques that have been used to measure the viscoelasticity of various soft materials in different length- and timescale frames. The topology of the material undergoing testing, the geometry of the probe, the magnitude of the exerted stress, and the resulting deformation should be carefully considered to choose a proper technique for each application. Lastly, we discuss several applications of viscoelasticity in 3D cell culture and tissue models for regenerative medicine, including organoids, organ-on-a-chip systems, engineered tissue constructs, and tunable viscoelastic hydrogels for 3D bioprinting and cell-based therapies.

Growing Role of 3D In Vitro Cell Cultures in the Study of Cellular and Molecular Mechanisms: Short Focus on Breast Cancer, Endometriosis, Liver and Infectious Diseases.

Over the past decade, the development of three-dimensional (3D) models has increased exponentially, facilitating the unravelling of fundamental and essential cellular mechanisms by which cells communicate with each other, assemble into tissues and organs and respond to biochemical and biophysical stimuli under both physiological and pathological conditions. This section presents a concise overview of the most recent updates on the significant contribution of different types of 3D cell cultures including spheroids, organoids and organ-on-chip and bio-printed tissues in advancing our understanding of cellular and molecular mechanisms. The case studies presented include the 3D cultures of breast cancer (BC), endometriosis, the liver microenvironment and infections. In BC, the establishment of 3D culture models has permitted the visualization of the role of cancer-associated fibroblasts in the delivery of exosomes, as well as the significance of the physical properties of the extracellular matrix in promoting cell proliferation and invasion. This approach has also become a valuable tool in gaining insight into general and specific mechanisms of drug resistance. Given the considerable heterogeneity of endometriosis, 3D models offer a more accurate representation of the in vivo microenvironment, thereby facilitating the identification and translation of novel targeted therapeutic strategies. The advantages provided by 3D models of the hepatic environment, in conjunction with the high throughput characterizing various platforms, have enabled the elucidation of complex molecular mechanisms underlying various threatening hepatic diseases. A limited number of 3D models for gut and skin infections have been developed. However, a more profound comprehension of the spatial and temporal interactions between microbes, the host and their environment may facilitate the advancement of in vitro, ex vivo and in vivo disease models. Additionally, it may pave the way for the development of novel therapeutic approaches in diverse research fields. The interested reader will also find concluding remarks on the challenges and prospects of using 3D cell cultures for discovering cellular and molecular mechanisms in the research areas covered in this review.

MDB-35. DEVELOPMENT OF DUAL LUCIFERASE ASSAY FOR 3D-COMPATIBLE HIGH THROUGHPUT SCREENING IN MEDULLOBLASTOMA

Abstract BACKGROUND Medulloblastoma is the most common malignant pediatric brain tumor, originating from the cerebellum, a part of the brain that controls balance, coordinated movement and other non-motor behaviors. 2D cell cultures are commonly used for testing of new compounds in high throughput screening (HTS) targeting this cancer, largely because there are numerous highly optimized assays available for analyzing this cell culture model. However, they have significant limitation in mimicking in vivo conditions of the tumor microenvironment. 3D cell-culture methods have been embraced to enhance the modeling of in vivo tissue, replicating the multicellular architecture and the characteristics of the extracellular matrix. Presently, there are efforts to integrate 3D cell-culture with HTS to yield more clinically relevant results. However, one of the requirements for this is a 3D-compatible cell-based assay suitable for HTS. We developed such an assay which measures the activity of YAP/TAZ, a central molecule of Hippo-signaling pathway, and has been implicated in medulloblastoma. It plays a crucial role in maintaining tissue balance by coordinating proliferation, apoptosis, and differentiation. METHODS We cloned 8xGTIIC-NlucP, a brighter and more sensitive luciferase (Nanoluciferase) reporter plasmid that detects YAP/TAZ activity. Responsiveness to YAP/TAZ activity, and its inhibition were detected using a dual luciferase assay in the medulloblastoma cell lines after transfection. A normalization plasmid with a constitutive promoter for firefly luciferase was used in this assay to differentiate compounds that affect YAP/TAZ activity specifically as opposed to those that are toxic to the cells. RESULTS A robust signal for YAP/TAZ activity, and its reduction when cells were treated with inhibitor, was seen in the three medulloblastoma cell lines used, which further validates the result. CONCLUSION In conclusion, we were able to develop a 3D-compatible cell-based assay with Nanoluciferase which can be a foundation for HTS targeting medulloblastoma using 3D cultures.

DIPG-27. MATCHED PRIMARY-RELAPSE PEDHGG PATIENT-DERIVED XENOGRAFT MODELS TO IDENTIFY THERAPY RESISTANCE PROFILES

Abstract BACKGROUND We established patient-derived xenografts (PDXs) from matched primary-relapse pairs of pediatric high-grade gliomas (pedHGGs) in collaboration with the Swedish Childhood Tumor Biobank. Further, we used human iPSC derived cortical organoids to evaluate interactions between glial and tumor cells under treatment pressure potentially lost in xenograft models. Using this setup, we hope to follow tumor relapse evolution and to identify novel mechanisms behind treatment evasion and relapse formation. METHODS Tumoral singel cell suspensions from matched primary-relapse pairs are injected orthotopically in immunodeficient NOG mice. We establish in vitro 2D models in stem cell conditions from the first generation of animals transplanted. Lentivirus-based labelling with GFP/Luciferase was done on PDX-derived cell lines to allow cell tracking and to quantify efficacy of drug screening efforts. Co-culture of these cells with normal cells in 3D assembloids were performed using 150-250 days old cortical organoids. Single-cell RNA sequencing and bulk RNA sequencing (RNA-seq) on patient biopsies, their corresponding PDXs from first generation of mice, and PDX-derived cell lines, was performed to track potential changes in vivo and in vitro and to further validate primary-relapse specific evolution. RESULTS 2D and 3D cell cultures offered efficient labeling and we quantified differences in sensitivity to standard therapy between primary and relapse samples. PDX models and assembloids highlighted pedHGG growth patterns, modes of invasion and cell interactions, especially astrocyte-tumor cell interactions. These 3D models also allow for further validation of targeted drug testing based on multiomics data (WGS, methylation and RNA-seq) from the patient biopsies. CONCLUSION PedHGG primary-relapse in vitro 2D cell line/ in vivo 3D assembloid models together with PDXs is a robust approach to study different aspects of relapse-specific tumor biology. Our results offer insights aiding the development of novel therapeutics inhibiting relapse mechanisms already in the first line of treatment.

Two-Dimensional and Spheroid-Based Three-Dimensional Cell Culture Systems: Implications for Drug Discovery in Cancer

The monolayer (two-dimensional or 2D) cell culture, while widely used, lacks fidelity in replicating vital cell interactions seen in vivo, leading to a shift toward three-dimensional (3D) models. Although monolayers offer simplicity and cost-effectiveness, spheroids mimic cellular environments better. This is due to its nutrient gradients, which influence drug penetration and provide a more accurate reflection of clinical scenarios than monolayers. Consequently, 3D models are crucial in drug development, especially for anti-cancer therapeutics, enabling the screening of cell cycle inhibitors and combination therapies vital for heterogeneous tumor populations. Inhibiting processes like migration and invasion often require drugs targeting the cytoskeleton, which can exhibit dual functionality with cell cycle inhibitors. Therapeutic approaches with promising anti-cancer potential often exhibit reduced efficacy in 3D cell culture compared to their performance in monolayer settings, primarily due to the heightened complexity inherent in this system. In the face of this scenario, this review aims to survey existing knowledge on compounds utilized in both 2D and 3D cell cultures, assessing their responses across different culture types and discerning the implications for drug screening, particularly those impacting the cell cycle and cytoskeletal dynamics.

Cytochrome P450 and UDP-Glucuronosyltransferase Expressions, Activities, and Induction Abilities in 3D-Cultured Human Renal Proximal Tubule Epithelial Cells.

The role of the kidney as an excretory organ for exogenous and endogenous compounds is well recognized, but there is a wealth of data demonstrating that the kidney has significant metabolizing capacity for a variety of exogenous and endogenous compounds that in some cases surpass the liver. The induction of drug-metabolizing enzymes by some chemicals can cause drug-drug interactions and intraindividual variability in drug clearance. In this study, we evaluated the expression and induction of cytochrome P450 (P450) and UDP-glucuronosyltransferase (UGT) isoforms in 3D-cultured primary human renal proximal tubule epithelial cells (RPTEC) to elucidate their utility as models of renal drug metabolism. CYP2B6, CYP2E1, CYP3A4, CYP3A5, and all detected UGTs (UGT1A1, UGT1A4, UGT1A6, UGT1A9, and UGT2B7) mRNA levels in 3D-RPTEC were significantly higher than those in 2D-RPTEC and HK-2 cells and were close to the levels in the human kidney cortex. CYP1B1 and CYP2J2 mRNA levels in 3D-RPTEC were comparable to those in 2D-RPTEC, HK-2 cells, and the human kidney cortex. Midazolam 1'-hydroxylation, trifluoperazine N-glucuronidation, serotonin O-glucuronidation, propofol O-glucuronidation, and morphine 3-glucuronidation in the 3D-RPTEC were significantly higher than the 2D-RPTEC and comparable to those in the HepaRG cells, although bupropion, ebastine, and calcitriol hydroxylations were not different between the 2D- and 3D-RPTEC. Treatment with ligands of the aryl hydrocarbon receptor and farnesoid X receptor induced CYP1A1 and UGT2B4 expression, respectively, in 3D-RPTEC compared with 2D-RPTEC. We provided information on the expression, activity, and induction abilities of P450s and UGTs in 3D-RPTEC as an in vitro human renal metabolism model. SIGNIFICANCE STATEMENT: This study demonstrated that the expression of cytochrome P450s (P450s) and UDP-glucuronosyltransferases (UGTs) in 3D-cultured primary human renal proximal tubule epithelial cells (3D-RPTEC) was higher than those in 2D-cultured primary human renal proximal tubule epithelial cells and HK-2 cells. The results were comparable to that in the human kidney cortex. 3D-RPTEC are useful for evaluating the induction of kidney P450s, UDP-glucuronosyltransferases, and human renal drug metabolism in cellulo.

Naphthoquinone-Quinolone Hybrids with Antitumor Effects on Breast Cancer Cell Lines-From the Synthesis to 3D-Cell Culture Effects.

Breast cancer stands as one of the foremost cause of cancer-related deaths globally, characterized by its varied molecular subtypes. Each subtype requires a distinct therapeutic strategy. Although advancements in treatment have enhanced patient outcomes, significant hurdles remain, including treatment toxicity and restricted effectiveness. Here, we explore the anticancer potential of novel 1,4-naphthoquinone/4-quinolone hybrids on breast cancer cell lines. The synthesized compounds demonstrated selective cytotoxicity against Luminal and triple-negative breast cancer (TNBC) cells, which represent the two main molecular types of breast cancer that depend most on cytotoxic chemotherapy, with potency comparable to doxorubicin, a standard chemotherapeutic widely used in breast cancer treatment. Notably, these derivatives exhibited superior selectivity indices (SI) when compared to doxorubicin, indicating lower toxicity towards non-tumor MCF10A cells. Compounds 11a and 11b displayed an improvement in IC50 values when compared to their precursor, 1,4-naphthoquinone, for both MCF-7 and MDA-MB-231 and a comparable value to doxorubicin for MCF-7 cells. Also, their SI values were superior to those seen for the two reference compounds for both cell lines tested. Mechanistic studies revealed the ability of the compounds to induce apoptosis and inhibit clonogenic potential. Additionally, the irreversibility of their effects on cell viability underscores their promising therapeutic utility. In 3D-cell culture models, the compounds induced morphological changes indicative of reduced viability, supporting their efficacy in a more physiologically relevant model of study. The pharmacokinetics of the synthesized compounds were predicted using the SwissADME webserver, indicating that these compounds exhibit favorable drug-likeness properties and potential as antitumor agents. Overall, our findings underscore the promise of these hybrid compounds as potential candidates for breast cancer chemotherapy, emphasizing their selectivity and efficacy.

Characterization of ERα Signaling to Cell Proliferation Induced by Chronic and Pulsatile E2 Stimulation in 2D and 3D Cell Cultures.

17β-estradiol is a hormone that plays a vital role in human physiology. It acts through estrogen receptors, specifically estrogen receptor α and estrogen receptor β, and its action is determined by the pulsatile secretion in the bloodstream. 17β-estradiol affects cell proliferation, and dysregulation of 17β-estradiol:estrogen receptor α signaling contribute to the development of breast cancer. Previous research on 17β-estradiol:estrogen receptor α signaling has primarily used two-dimensional cell cultures, which do not fully recapitulate the complexity of tumors that exist in a three-dimensional environment and do not consider the pulsatile nature of this hormone. To address these limitations, we studied 17β-estradiol:estrogen receptor α signaling in cell proliferation using both two-dimensional and three-dimensional breast cancer cell culture models under continuous and pulsatile stimulation conditions. Results revealed that breast cancer cells grown in an alginate-based three-dimensional matrix exhibited similar responsiveness to 17β-estradiol compared with cells grown in conventional two-dimensional culture plates. 17β-estradiol induced the expression of proteins containing estrogen response element in the three-dimensional model. The efficacy of the antiestrogen drugs fulvestrant (ICI182,280) and 4OH-tamoxifen was also demonstrated in the three-dimensional model. These results support the use of the three-dimensional culture model for studying tumor response to drugs and provide a more realistic microenvironment for such studies. Furthermore, the study revealed that a brief 5-min exposure to 17β-estradiol triggered a physiological response comparable with continuous hormone exposure, suggesting that the cellular response to 17β-estradiol is more important than the continuous presence of the hormone. In conclusion, the study demonstrates that the alginate-based three-dimensional culture model is suitable for studying the effects of 17β-estradiol and antiestrogen drugs on breast cancer cells, offering a more realistic representation of tumor-microenvironment interactions. The results also highlight the importance of considering the physiological importance of the temporal dynamics in studying 17β-estradiol signaling and cellular responses.

Real-time label-free three-dimensional invasion assay for anti-metastatic drug screening using impedance sensing.

Tumor metastasis presents a formidable challenge in cancer treatment, necessitating effective tools for anti-cancer drug development. Conventional 2D cell culture methods, while considered the "gold standard" for invasive studies, exhibit limitations in representing cancer hallmarks and phenotypes. This study proposes an innovative approach that combines the advantages of 3D tumor spheroid culture with impedance-based biosensing technologies to establish a high-throughput 3D cell invasion assay for anti-metastasis drug screening through multicellular tumor spheroids. In addition, the xCELLigence device is employed to monitor the time-dependent kinetics of cell behavior, including attachment and invasion out of the 3D matrix. Moreover, an iron chelator (deferoxamine) is employed to monitor the inhibition of epithelial-mesenchymal transition in 3D spheroids across different tumor cell types. The above results indicate that our integrated 3D cell invasion assay with impedance-based sensing could be a promising tool for enhancing the quality of the drug development pipeline by providing a robust platform for predicting the efficacy and safety of anti-metastatic drugs before advancing into preclinical or clinical trials.

Cellpose as a reliable method for single-cell segmentation of autofluorescence microscopy images.

Autofluorescence microscopy uses intrinsic sources of molecular contrast to provide cellular-level information without extrinsic labels. However, traditional cell segmentation tools are often optimized for high signal-to-noise ratio (SNR) images, such as fluorescently labeled cells, and unsurprisingly perform poorly on low SNR autofluorescence images. Therefore, new cell segmentation tools are needed for autofluorescence microscopy. Cellpose is a deep learning network that is generalizable across diverse cell microscopy images and automatically segments single cells to improve throughput and reduce inter-human biases. This study aims to validate Cellpose for autofluorescence imaging, specifically from multiphoton intensity images of NAD(P)H. Manually segmented nuclear masks of NAD(P)H images were used to train new Cellpose models. These models were applied to PANC-1 cells treated with metabolic inhibitors and patient-derived cancer organoids (across 9 patients) treated with chemotherapies. These datasets include co-registered fluorescence lifetime imaging microscopy (FLIM) of NAD(P)H and FAD, so fluorescence decay parameters and the optical redox ratio (ORR) were compared between masks generated by the new Cellpose model and manual segmentation. The Dice score between repeated manually segmented masks was significantly lower than that of repeated Cellpose masks (p<0.0001) indicating greater reproducibility between Cellpose masks. There was also a high correlation (R 2 >0.9) between Cellpose and manually segmented masks for the ORR, mean NAD(P)H lifetime, and mean FAD lifetime across 2D and 3D cell culture treatment conditions. Masks generated from Cellpose and manual segmentation also maintain similar means, variances, and effect sizes between treatments for the ORR and FLIM parameters. Overall, Cellpose provides a fast, reliable, reproducible, and accurate method to segment single cells in autofluorescence microscopy images such that functional changes in cells are accurately captured in both 2D and 3D culture.

Assessing the impact of green and roasted coffee extracts on colorectal cancer cells in a 3D cell culture model

BackgroundColorectal cancer (CRC) is one of the most common and deadly malignant neoplasms worldwide. It is associated with multiple lifestyle risk factors such as poor diet, physical inactivity, alcohol and red meat consumption, smoking, and obesity. Consequently, studies have been conducted on the protective activity of bioactive compounds present in some foods against CRC, making it necessary to understand how diet can affect human health. In this sense, coffee, the second most consumed beverage in the world, is a major source of phytochemicals associated with a possible antitumor effect on CRC cells. PurposeThis study aimed to analyze the chemopreventive potential of green and roasted coffee extracts on HT-29 CRC cells cultured in 2D and 3D models. MethodsFlow cytometry was employed to quantify propidium iodide and DiOC6 uptake, ROS production, phosphatidylserine exposure, and cell cycle progression. In addition, cells and spheroids were analyzed by light microscopy to identify morphological changes. ResultsThe results showed that, under the employed conditions, treatment with both extracts had a concentration-dependent cytotoxic effect. Decreased cell viability was associated with mitochondrial membrane hyperpolarization, prooxidant effect, increased phosphatidylserine exposure, and changes in the G1 phase of the cell cycle. ConclusionFor the evaluated cell markers, 3D cultures required higher doses to achieve significant cytotoxic effects when compared to monolayer cultures. Since 3D cultures favor the formation of complex cell–cell interactions, this model offers a closer look at in vivo conditions for evaluating the toxicity and preclinical efficacy of potential chemopreventive agents for CRC. The experimental evidence from this study supports the 3R principles of replacement, reduction, and refinement for toxicological research in the pharmaceutical industry.

Reconfigurable Hanging Drop Microarray Platform for On-Demand Preparation and Analysis of Spheroid Array.

In response to the increasing demand for spheroid-based cancer research, the importance of developing integrated platforms that can simultaneously facilitate high-throughput spheroid production and multiplexed analysis is emphasized. In addition, the understanding of how the size and cellular composition of tumors directly influence their internal structures and functionalities underlines the critical need to produce spheroids of diverse sizes and compositions on a large scale. To address this rising demand, this work presents a configurable and linkable in vitro three-dimensional (3D) cell culture kit (CLiCK) for spheroids, termed CLiCK-Spheroid. This platform consists of three primary components: a hanging drop microarray (HDMA), a concave pillar microarray (CPMA), and gradient blocks. The HDMA alone produces a homogeneous spheroid array, while its combination with the gradient block enables one-step generation of a size-gradient spheroid array. Using the size-gradient spheroid arrays, the occurrence of necrotic cores based on spheroid size is demonstrated. Additionally, spheroids in a single batch can be conveniently compartmentalized and regrouped using a CPMA, enhancing the versatility of spheroid arrays and enabling multiplexed drug treatments. By combining the different assembly methods, this work has achieved high-throughput production of cell composition-gradient spheroid arrays, with noticeable variations in morphology and vascularization based on cell compositions.

Engineering free-standing electrospun PLLCL fibers on microfluidic platform for cell alignment

Here, a PLLCL-on-chip platform was developed by direct electrospinning of poly (L-lactide-co-ε-caprolactone) (PLLCL) on polymethyl methacrylate (PMMA) microfluidic chips. Designed microchip provides the electrospinning of free-standing aligned PLLCL fibers which eliminates limitations of conventional electrospinning. Besides, aligned fiber structure favors cell alignment through contactless manipulation. Average fiber diameter, and fiber alignment was evaluated by SEM analyses, then, leakage profile of microchip was investigated. 3D cell culture studies were conducted using HeLa and NIH-3T3 cells, and nearly 85% cell viability was observed in PLLCL-on-chip for 15 days, while cell viability of 2D control started to decrease after 7 days based on Live dead and Alamar Blue analyses. These findings emphasize biocompatibility of PLLCL-on-chip platform for 3D cell culture and its ability to mimic extracellular matrix (ECM). Immunostaining results prove that PLLCL-on-chip platform favors the secretion of ECM proteins compared to control groups, and cytoskeletons of cells were in aligned orientation in PLLCL-on-chip, while they were in random orientation in control groups. Overall, these results demonstrate that the developed platform is suitable for the formation of various 3D cell culture models and a potential candidate for cell alignment studies.Graphical

Development and Optimization of a Lactate Dehydrogenase Assay Adapted to 3D Cell Cultures

In recent years, 3D cell culture systems have emerged as sophisticated in vitro models, providing valuable insights into human physiology and diseases. The transition from traditional 2D to advanced 3D cultures has introduced novel obstacles, complicating the characterization and analysis of these models. While the lactate dehydrogenase (LDH) activity assay has long been a standard readout for viability and cytotoxicity assessments in 2D cultures, its applicability in long-term 3D cultures is hindered by inappropriate normalization and low LDH stability over time. In response to these challenges, we propose an optimization of LDH assays, including a crucial normalization step based on total protein quantification and a storage method using an LDH preservation buffer. We applied it to compare unexposed cerebral organoids with organoids exposed to a toxic dose of valproic acid, and showed efficient normalization of cellular viability as well as enhanced LDH stability within the buffer. Importantly, normalized LDH activity results obtained were independent of organoid dimension and cell density. This refined LDH assay, tailored to address 3D culture constraints, allows for the transposition of this routine test from 2D to 3D cultures.

Deformable microlaser force sensing

Mechanical forces are key regulators of cellular behavior and function, affecting many fundamental biological processes such as cell migration, embryogenesis, immunological responses, and pathological states. Specialized force sensors and imaging techniques have been developed to quantify these otherwise invisible forces in single cells and in vivo. However, current techniques rely heavily on high-resolution microscopy and do not allow interrogation of optically dense tissue, reducing their application to 2D cell cultures and highly transparent biological tissue. Here, we introduce DEFORM, deformable microlaser force sensing, a spectroscopic technique that detects sub-nanonewton forces with unprecedented spatio-temporal resolution. DEFORM is based on the spectral analysis of laser emission from dye-doped oil microdroplets and uses the force-induced lifting of laser mode degeneracy in these droplets to detect nanometer deformations. Following validation by atomic force microscopy and development of a model that links changes in laser spectrum to applied force, DEFORM is used to measure forces in 3D and at depths of hundreds of microns within tumor spheroids and late-stage Drosophila larva. We furthermore show continuous force sensing with single-cell spatial and millisecond temporal resolution, thus paving the way for non-invasive studies of biomechanical forces in advanced stages of embryogenesis, tissue remodeling, and tumor invasion.

Electrofluidic control for textile-based cell culture: Identification of appropriate conditions required to integrate cell culture with electrofluidics.

Electric field-driven microfluidics, known as electrofluidics, is a novel attractive analytical tool when it is integrated with low-cost textile substrate. Textile-based electrofluidics, primarily explored on yarn substrates, is in its early stages, with few studies on 3D structures. Further, textile structures have rarely been used in cellular analysis as a low-cost alternative. Herein, we investigated novel 3D textile structures and develop optimal electrophoretic designs and conditions that are favourable for direct 3D cell culture integration, developing an integrated cell culture textile-based electrofluidic platform that was optimised to balance electrokinetic performance and cell viability requirements. Significantly, there were contrasting electrolyte compositional conditions that were required to satisfy cell viability and electrophoretic mobility requiring the development of and electrolyte that satisfied the minimum requirements of both these components within the one platform. Human dermal fibroblast cell cultures were successfully integrated with gelatine methacryloyl (GelMA) hydrogel-coated electrofluidic platform and studied under different electric fields using 5mM TRIS/HEPES/300mM glucose. Higher analyte mobility was observed on 2.5% GelMA-coated textile which also facilitated excellent cell attachment, viability and proliferation. Cell viability also increased by decreasing the magnitude and time duration of applied electric field with good cell viability at field of up to 20Vcm-1.

Cytotoxic and molecular differences of anticancer agents on 2D and 3D cell culture.

Cancer and multidrug resistance are regarded as concerns related to poor health outcomes. It was found that the monolayer of 2D cancer cell cultures lacks many important features compared to Multicellular Tumor Spheroids (MCTS) or 3D cell cultures which instead have the ability to mimic more closely the in vivo tumor microenvironment. This study aimed to produce 3D cell cultures from different cancer cell lines and to examine the cytotoxic activity of anticancer medications on both 2D and 3D systems, as well as to detect alterations in the expression of certain genes levels. 3D cell culture was produced using 3D microtissue molds. The cytotoxic activities of colchicine, cisplatin, doxorubicin, and paclitaxel were tested on 2D and 3D cell culture systems obtained from different cell lines (A549, H1299, MCF-7, and DU-145). IC50 values were determined by MTT assay. In addition, gene expression levels of PIK3CA, AKT1, and PTEN were evaluated by qPCR. Similar cytotoxic activities were observed on both 3D and 2D cell cultures, however, higher concentrations of anticancer medications were needed for the 3D system. For instance, paclitaxel showed an IC50 of 6.234µM and of 13.87µM on 2D and 3D H1299 cell cultures, respectively. Gene expression of PIK3CA in H1299 cells also showed a higher fold change in 3D cell culture compared to 2D system upon treatment with doxorubicin. When compared to 2D cell cultures, the behavior of cells in the 3D system showed to be more resistant to anticancer treatments. Due to their shape, growth pattern, hypoxic core features, interaction between cells, biomarkers synthesis, and resistance to treatment penetration, the MCTS have the advantage of better simulating the in vivo tumor conditions. As a result, it is reasonable to conclude that 3D cell cultures may be a more promising model than the traditional 2D system, offering a better understanding of the in vivo molecular changes in response to different potential treatments and multidrug resistance development.

Reflected shear wave computed tomography for elasticity imaging of 3D cell cultures using a single-element transducer

Shear wave elasticity imaging (SWEI) has long been used to quantify tissue stiffness in clinical diagnoses. In comparison with conventional bulk-based measurement methods, SWEI offers the distinct advantage of nondestructive sampling, thereby enabling the spatiotemporal monitoring of stiffness variations. However, applying SWEI to assessing millimeter-scale three-dimensional (3D) cell environments has faced limitations despite its potential in mechanobiology, and the existing techniques are insufficient for imaging inhomogeneous media environments. In this study, we investigated a computed tomography technique specifically designed for reflected SWEI (called R-SWCT) by rotational scanning in a sample using a 20-MHz ultrasound single-element transducer. We focused on samples containing a single inclusion with higher stiffness than the surrounding medium, mimicking the situation of a tumor sphere embedded in a 3D gel. Our method reconstructs the stiffness distribution using a curve-fitting method, wherein coefficients of Gaussian curves are fitted to the wavefronts of simulated signals. This reconstruction method yielded coefficients that closely approximated the wavefronts obtained experimentally, with a maximum difference between the measured and predicted shear wave speeds of only 0.1 m/s for phantom samples and 0.2 m/s for cell samples. The system and methodologies proposed in this research have demonstrated the feasibility of employing R-SWCT to study the remodeling dynamics of a cell group within its surrounding matrix in an in vitro setting. This noninvasive method also facilitates an exploration of how irradiation dosage used in radiation therapy can induce temporal alterations in the shear wave speed in 3D cancer cell cultures.

Nanoporous, Gas Permeable PEGDA Ink for 3D Printing Organ‐on‐a‐Chip Devices

AbstractPolydimethylsiloxane (PDMS), commonly used in organ‐on‐a‐chip (OoC) systems, faces limitations in replicating complex geometries, hindering its effectiveness in creating 3D OoC models. In contrast, poly(ethylene glycol)diacrylate (PEGDA‐250), favored for its fabrication ease and resistance to small molecule absorption, is increasingly used for 3D printing microfluidic devices. However, applications in cell culture have been limited due to poor cell adhesion. Here, a nanoporous PEGDA ink (P‐PEGDA) is introduced to enhance cell adhesion. P‐PEGDA is formulated with a porogen, photopolymerized, followed by the porogen removal. Utilizing P‐PEGDA, complex microstructures, and membranes as thin as 27 µm are 3D‐printed. Porogen concentrations from 10 to 30% are tested yielding constructs with increasing porosity and oxygen permeability surpassing PDMS, without compromising printing resolution. Tests across four cell lines show &gt;80% cell viability, with a notable 77‐fold increase in MDA‐MB‐231 cell coverage on the porous scaffolds. Finally, an OoC model comprising a gyroid scaffold with a central opening filled with a cancer spheroid is introduced. This setup, after a 14 days co‐culture, demonstrates significant endothelial sprouting and integration within the spheroid. The P‐PEGDA formulation is suitable for high‐resolution 3D printing of constructs for 3D cell culture and OoC owing to its printability, gas permeability, biocompatibility, and cell adhesion.