Investigation of the Anticancer Effect of S-Allyl-L Cysteine on the C6 Rat Glioma Cell Line in vitro in 2D- and 3D-Cell Culture Models.

AIM: To investigate the antiproliferative and apoptotic effects of S-allyl cysteine (SAC) on C6 glioblastoma cells using two- and three-dimensional (2D and 3D) cell culture systems. MATERIAL AND METHODS: Three groups of rat glioma cell line C6 were prepared: 2D-Control, 2D-SAC, 3D-CMC-Control, and 3D-CMC-SAC. The control cells were incubated under standard culture conditions, the SAC cells were incubated in a culture medium supplemented with the IC50 dose (50 ?M for both the 2D-SAC C6 and 3D-CMC-SAC groups) of SAC for 24 and 48 h. All experimental cells were stained with antibodies recognizing NOTCH1 and JAGGED1, and the mRNA expression levels of NOTCH1 and JAGGED1 were evaluated by qRT-PCR. RESULTS: Increasing doses of SAC were administered for 24 h to the C6 glioma cell line. The concentration of 50 ?M was selected as the most suitable dose for administration. The gene expression profiles differed between these two cell culture types. We found that the expression levels of NOTCH1 receptor mRNA were lower in cells exposed to 50-?M SAC for 24 h than those of control cells in both 2D and 3D cell cultures. The immunoreactivities of both the biomarkers JAGGED1 and NOTCH1 in the glioma cells decreased significantly in the SAC group. CONCLUSION: These findings indicate that SAC is a potential drug candidate for human use, as indicated by its nontoxic nature. In addition, SAC was found to exert an anticancer effect, which is associated with the modulation of JAGGED1 and NOTCH1 signaling pathways in glioma cancer cells.

Application of three-dimensional cell culture technology in screening anticancer drugs.

The drug development process involves a variety of drug activity evaluations, which can determine drug efficacy, strictly analyze the biological indicators after the drug action, and use these indicators as the preclinical drug evaluation criteria. At present, most of the screening of preclinical anticancer drugs mainly relies on traditional 2D cell culture. However, this traditional technology cannot simulate the tumor microenvironment in vivo, let alone reflect the characteristics of solid tumors in vivo, and has a relatively poor ability to predict drug activity. 3D cell culture is a technology between 2D cell culture and animal experiments, which can better reflect the biological state in vivo and reduce the consumption of animal experiments. 3D cell culture can link the individual study of cells with the study of the whole organism, reproduce in vitro the biological phenotype of cells in vivo more greatly, and thus predict the activity and resistance of anti-tumor drugs more accurately. In this paper, the common techniques of 3D cell culture are discussed, with emphasis on its main advantages and application in the evaluation of anti-tumor resistance, which can provide strategies for the screening of anti-tumor drugs.

Role of three-dimensional cell culture in therapeutics and diagnostics: an updated review.

Drug development and testing are a tedious and expensive process with a high degree of uncertainty in the clinical success and preclinical validation of manufactured therapeutic agents. Currently, to understand the drug action, disease mechanism, and drug testing, most therapeutic drug manufacturers use 2D cell culture models to validate the drug action. However, there are many uncertainties and limitations with the conventional use of 2D (monolayer) cell culture models for drug testing that are primarily attributed due to poor mimicking of cellular mechanisms, disturbance in environmental interaction, and changes in structural morphology. To overcome such odds and difficulties in the preclinical validation of therapeutic medications, newer in vivo drug testing cell culture models with higher screening efficiencies are required. One such promising and advanced cell culture model reported recently is the "three-dimensional cell culture model." The 3D cell culture models are reported to show evident benefits over conventional 2D cell models. This review article outlines and describes the current advancement in cell culture models, their types, significance in high-throughput screening, limitations, applications in drug toxicity screening, and preclinical testing methodologies to predict in vivo efficacy.

Vascularization Strategies in 3D Cell Culture Models: From Scaffold-Free Models to 3D Bioprinting.

The discrepancies between the findings in preclinical studies, and in vivo testing and clinical trials have resulted in the gradual decline in drug approval rates over the past decades. Conventional in vitro drug screening platforms employ two-dimensional (2D) cell culture models, which demonstrate inaccurate drug responses by failing to capture the three-dimensional (3D) tissue microenvironment in vivo. Recent advancements in the field of tissue engineering have made possible the creation of 3D cell culture systems that can accurately recapitulate the cell-cell and cell-extracellular matrix interactions, as well as replicate the intricate microarchitectures observed in native tissues. However, the lack of a perfusion system in 3D cell cultures hinders the establishment of the models as potential drug screening platforms. Over the years, multiple techniques have successfully demonstrated vascularization in 3D cell cultures, simulating in vivo-like drug interactions, proposing the use of 3D systems as drug screening platforms to eliminate the deviations between preclinical and in vivo testing. In this review, the basic principles of 3D cell culture systems are briefly introduced, and current research demonstrating the development of vascularization in 3D cell cultures is discussed, with a particular focus on the potential of these models as the future of drug screening platforms.

3D cell cultures toward quantitative high-throughput drug screening.

3D cell cultures are being utilized for drug discovery and development. However, there are still challenges to implementing them generally in quantitative high-throughput screening (HTS) due to the complexity of the 3D architecture, the time- and labor-consuming process, and the lack of compatibility with traditional screening protocols. Therefore, there is a great need for the integration of microfabrication techniques, automation systems, and high-throughput analytical tools that reveal the pharmacological and toxicological effects of therapeutics using 3D cultures. We first review the current advances in 3D culture models and discuss their key challenges in HTS. Last, we review recent progress and breakthroughs in the automation and high-throughput imaging of 3D culture models, which can be integrated with machine-learning (ML) tools to aid quantitative HTS for drug discovery and development.

Recapitulating Tumor Microenvironment Using AXTEX-4DTM for Accelerating Cancer Research and Drug Screening.

OBJECTIVE: The formation of three-dimensional spheroid tumor model using the scaffold-based platforms has been demonstrated over many years now. 3D tumor models are generated mainly in non-scalable culture systems, using synthetic and biological scaffolds. Many of these models fail to reflect the complex tumor microenvironment and do not allow long-term monitoring of tumor progression. This has resulted in inconsistent data in drug testing assays during preclinical and clinical studies. METHODS: To overcome these limitations, we have developed 3D tissueoids model by using novel AXTEX-4D platform. RESULTS: Cancer 3D tissueoids demonstrated the basic features of 3D cell culture with rapid attachment, proliferation, and longevity with contiguous cytoskeleton and hypoxic core. This study also demonstrated greater drug resistance in 3D-MCF-7 tissueoids in comparison to 2D monolayer cell culture. CONCLUSION: In conclusion, 3D-tissueoids are more responsive than 2D-cultured cells in simulating important tumor characteristics, anti-apoptotic features, and their resulting drug resistance.

Fabrication and Characterization of a Three-Dimensional Fibrin Gel Model to Evaluate Anti-Proliferative Effects of Astragalus hamosus Plant Extract on Breast Cancer Cells.

BACKGROUND: Breast Cancer (BC) is a malignancy with high mortality among women. Recently, scaffold-based three-dimensional (3D) models have been developed for anti-cancer drug research. The present study aimed to investigate the anti-proliferative effects of Astragalus hamosus (A. hamosus) in 3D fibrin gel against MCF-7 cell line. We have also evaluated anti-proliferative effect of A. hamosus differences between 3D and 2D cultures. METHODS: The fibrin gel formulation was first optimized by testing the structural and mechanical properties. Then the cytotoxic effect of A. hamosus extract was assessed on MCF-7 cells by MTT assay. Cell apoptosis was evaluated using TUNEL method and flow cytometry. Cell cycle and proliferation were analyzed by flow cytometry. Apoptosis-related gene expression such as Bcl-2, caspase-3, -8 and -9 were quantified by real time-PCR. RESULTS: TUNEL staining showed a significant damage accompanied with cell apoptosis. Flow cytometry analysis revealed that apoptosis increased after treatment with A. hamosus extract in 3D culture model compared to 2D culture. The A. hamosus extract arrested cell cycle in the S and G2/M phases in 3D model while in the 2D culture G0/G1 phase was affected. Treatment with A. hamosus extract led to upregulation of the caspase-3, -8 and -9 genes and downregulation of the Ki-67 in the 3D-culture compared with the 2D culture. CONCLUSION: These results indicated that A. hamosus extract could be used as a therapeutic candidate for BC due to its anti-proliferative effects. Furthermore, 3D fibrin gel could be better than 2D-cultured cells in simulating important tumor characteristics in vivo, namely, anti-proliferative and anti-apoptotic features.

Advances in microfluidic 3D cell culture for preclinical drug development.

Drug development is often a very long, costly, and risky process due to the lack of reliability in the preclinical studies. Traditional current preclinical models, mostly based on 2D cell culture and animal testing, are not full representatives of the complex in vivo microenvironments and often fail. In order to reduce the enormous costs, both financial and general well-being, a more predictive preclinical model is needed. In this chapter, we review recent advances in microfluidic 3D cell culture showing how its development has allowed the introduction of in vitro microphysiological systems, laying the foundation for organ-on-a-chip technology. These findings provide the basis for numerous preclinical drug discovery assays, which raise the possibility of using micro-engineered systems as emerging alternatives to traditional models, based on 2D cell culture and animals.

Effect of NMDA receptor agonist and antagonist on spermatogonial stem cells proliferation in 2- and 3- dimensional culture systems.

BACKGROUND: The main purpose of this study was to investigate the effect of D-serine (DS) and Dizocilpine (MK-801) on the proliferation of spermatogonial stem cells (SSCs) in two-dimensional (2D) and three-dimensional (3D) culture systems. METHODS AND RESULTS: The SSCs of male NMRI mice were isolated by enzymatic digestion and cultured for two weeks. Then, the identity of SSCs was validated by anti-Plzf and anti-GFR-α1 antibodies via immunocytochemistry (ICC). The proliferation capacity of SSCs was evaluated by their culture on a layer of the decellularized testicular matrix (DTM) prepared from mouse testis, as well as two-dimensional (2D) with different mediums. After two weeks of the initiation of proliferation culture on 3D and 2D medium, the pre-meiotic at the mRNA and protein levels were evaluated via qRT-PCR and flow cytometry methods, respectively. The results showed that the proliferation rate of SSCs in 3D culture with 50 mM glutamic acid and 20 mM D-serine was significantly different from other groups after 14 days treatment. mRNA expression levels of promyelocytic leukemia zinc finger (Plzf) in 3D cultures supplemented by 20 mM D-serine and 50 mM glutamic acid were considerably higher than the 3D control group (p < 0.001). The flow cytometry analysis revealed that the amount of Plzf in the 2D-culture groups of SSCs with 20 mM MK-801 was considerably lower compared to the 2D-culture control group (p < 0.001). CONCLUSIONS: This study indicated that decellularized testicular matrix supplemented with D-serine and glutamic acid could be considered a promising vehicle to support cells and provide an appropriate niche for the proliferation of SSCs.

Microfluidic organ-on-chip system for multi-analyte monitoring of metabolites in 3D cell cultures.

Three-dimensional cell cultures using patient-derived stem cells are essential in vitro models for a more efficient and individualized cancer therapy. Currently, culture conditions and metabolite concentrations, especially hypoxia, are often not accessible continuously and in situ within microphysiological systems. However, understanding and standardizing the cellular microenvironment are the key to successful in vitro models. We developed a microfluidic organ-on-chip platform for matrix-based, heterogeneous 3D cultures with fully integrated electrochemical chemo- and biosensor arrays for the energy metabolites oxygen, lactate, and glucose. Advanced microstructures allow straightforward cell matrix integration with standard laboratory equipment, compartmentalization, and microfluidic access. Single, patient-derived, triple-negative breast cancer stem cells develop into tumour organoids in a heterogeneous spheroid culture on-chip. Our system allows unprecedented control of culture conditions, including hypoxia, and simultaneous verification by integrated sensors. Beyond previous works, our results demonstrate precise and reproducible on-chip multi-analyte metabolite monitoring under dynamic conditions from a matrix-based culture over more than one week. Responses to alterations in culture conditions and cancer drug exposure, such as metabolite consumption and production rates, could be accessed quantitatively and in real-time, in contrast to endpoint analyses. Our approach highlights the importance of continuous, in situ metabolite monitoring in 3D cell cultures regarding the standardization and control of culture conditions, and drug screening in cancer research. Overall, the results underline the potential of microsensors in organ-on-chip systems for successful application, e.g. in personalized medicine.

An Immunocompetent Microphysiological System to Simultaneously Investigate Effects of Anti-Tumor Natural Killer Cells on Tumor and Cardiac Microtissues.

Existing first-line cancer therapies often fail to cope with the heterogeneity and complexity of cancers, so that new therapeutic approaches are urgently needed. Among novel alternative therapies, adoptive cell therapy (ACT) has emerged as a promising cancer treatment in recent years. The limited clinical applications of ACT, despite its advantages over standard-of-care therapies, can be attributed to (i) time-consuming and cost-intensive procedures to screen for potent anti-tumor immune cells and the corresponding targets, (ii) difficulties to translate in-vitro and animal-derived in-vivo efficacies to clinical efficacy in humans, and (iii) the lack of systemic methods for the safety assessment of ACT. Suitable experimental models and testing platforms have the potential to accelerate the development of ACT. Immunocompetent microphysiological systems (iMPS) are microfluidic platforms that enable complex interactions of advanced tissue models with different immune cell types, bridging the gap between in-vitro and in-vivo studies. Here, we present a proof-of-concept iMPS that supports a triple culture of three-dimensional (3D) colorectal tumor microtissues, 3D cardiac microtissues, and human-derived natural killer (NK) cells in the same microfluidic network. Different aspects of tumor-NK cell interactions were characterized using this iMPS including: (i) direct interaction and NK cell-mediated tumor killing, (ii) the development of an inflammatory milieu through enrichment of soluble pro-inflammatory chemokines and cytokines, and (iii) secondary effects on healthy cardiac microtissues. We found a specific NK cell-mediated tumor-killing activity and elevated levels of tumor- and NK cell-derived chemokines and cytokines, indicating crosstalk and development of an inflammatory milieu. While viability and morphological integrity of cardiac microtissues remained mostly unaffected, we were able to detect alterations in their beating behavior, which shows the potential of iMPS for both, efficacy and early safety testing of new candidate ACTs.

Ciprofloxacin and Levofloxacin as Potential Drugs in Genitourinary Cancer Treatment-The Effect of Dose-Response on 2D and 3D Cell Cultures.

INTRODUCTION: Introducing new drugs for clinical application is a very difficult, long, drawn-out, and costly process, which is why drug repositioning is increasingly gaining in importance. The aim of this study was to analyze the cytotoxic properties of ciprofloxacin and levofloxacin on bladder and prostate cell lines in vitro. METHODS: Bladder and prostate cancer cell lines together with their non-malignant counterparts were used in this study. In order to evaluate the cytotoxic effect of both drugs on tested cell lines, MTT assay, real-time cell growth analysis, apoptosis detection, cell cycle changes, molecular analysis, and 3D cultures were examined. RESULTS: Both fluoroquinolones exhibited a toxic effect on all of the tested cell lines. In the case of non-malignant cell lines, the cytotoxic effect was weaker, which was especially pronounced in the bladder cell line. A comparison of both fluoroquinolones showed the advantage of ciprofloxacin (lower doses of drug caused a stronger cytotoxic effect). Both fluoroquinolones led to an increase in late apoptotic cells and an inhibition of cell cycle mainly in the S phase. Molecular analysis showed changes in BAX, BCL2, TP53, and CDKN1 expression in tested cell lines following incubation with ciprofloxacin and levofloxacin. The downregulation of topoisomerase II genes (TOP2A and TOP2B) was noticed. Three-dimensional (3D) cell culture analysis confirmed the higher cytotoxic effect of tested fluoroquinolone against cancer cell lines. CONCLUSIONS: Our results suggest that both ciprofloxacin and levofloxacin may have great potential, especially in the supportive therapy of bladder cancer treatment. Taking into account the low costs of such therapy, fluoroquinolones seem to be ideal candidates for repositioning into bladder cancer therapeutics.

Novel Human Placenta-Based Extract for Vascularization Strategies in Tissue Engineering.

There is critical unmet need for new vascularized tissues to support or replace injured tissues and organs. Various synthetic and natural materials were already established for use of two-dimensional (2D) and three-dimensional (3D) in vitro neovascularization assays, however, they still cannot mimic the complex functions of the sum of the extracellular matrix (ECM) in native intact tissue. Currently, this issue is only addressed by artificial products such as Matrigel™, which comprises a complex mixture of ECM proteins, extracted from animal tumor tissue. Despite its outstanding bioactivity, the isolation from tumor tissue hinders its translation into clinical applications. Since nonhuman ECM proteins may cause immune reactions, as are frequently observed in clinical trials, human ECM proteins represent the best option when aiming for clinical applications. Here, we describe an effective method of isolating a human placenta substrate (hpS) that induces the spontaneous formation of an interconnected network of green fluorescence-labeled human umbilical vein endothelial cells (gfpHUVECs) in vitro. The substrate was biochemically characterized by using a combination of bicinchoninic acid (BCA) assay, DNA, and glycosaminoglycan (GAG) content assays, sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) analysis and Western blot, angiogenesis arrays, chromatographic thrombin detection, high performance liquid chromatography (HPLC)-based amino acid quantification analysis, and assessment of antimicrobial properties. 2D in vitro cell culture experiments have been performed to determine the vasculogenic potential of hpS, which demonstrated that cell networks developed on hpS show a significantly higher degree of complexity (number of tubules/junctions; total/mean tube length) when compared with Matrigel. As 3D cell culture techniques represent a more accurate representation of the in vivo condition, the substrate was 3D solidified using various natural polymers. 3D in vitro vasculogenesis assays have been performed by seeding gfpHUVECs in an hpS-fibrinogen clot. In conclusion, hpS provides a potent human/material-based alternative to xenogenic-material-based biomaterials for vascularization strategies in tissue engineering.

High-throughput screening on cochlear organoids identifies VEGFR-MEK-TGFB1 signaling promoting hair cell reprogramming.

Hair cell degeneration is a major cause of sensorineural hearing loss. Hair cells in mammalian cochlea do not spontaneously regenerate, posing a great challenge for restoration of hearing. Here, we establish a robust, high-throughput cochlear organoid platform that facilitates 3D expansion of cochlear progenitor cells and differentiation of hair cells in a temporally regulated manner. High-throughput screening of the FDA-approved drug library identified regorafenib, a VEGFR inhibitor, as a potent small molecule for hair cell differentiation. Regorafenib also promotes reprogramming and maturation of hair cells in both normal and neomycin-damaged cochlear explants. Mechanistically, inhibition of VEGFR suppresses TGFB1 expression via the MEK pathway and TGFB1 downregulation directly mediates the effect of regorafenib on hair cell reprogramming. Our study not only demonstrates the power of a cochlear organoid platform in high-throughput analyses of hair cell physiology but also highlights VEGFR-MEK-TGFB1 signaling crosstalk as a potential target for hair cell regeneration and hearing restoration.

Patient derived organoids in prostate cancer: improving therapeutic efficacy in precision medicine.

With advances in the discovery of the clinical and molecular landscapes of prostate cancer (PCa), implementation of precision medicine-guided therapeutic testing in the clinic has become a priority. Patient derived organoids (PDOs) are three-dimensional (3D) tissue cultures that promise to enable the validation of preclinical drug testing in precision medicine and coclinical trials by modeling PCa for predicting therapeutic responses with a reliable efficacy. We evaluate the advances in 3D culture and PDO use to model clonal heterogeneity and screen for effective targeted therapies, with a focus on the technological advances in generating PDOs. Recent innovations include the utilization of PDOs both in original research and/or correlative studies in clinical trials to examine drug effects within the PCa tumor microenvironment (TME). There has also been a significant improvement with the utilization of various extracellular matrices and single cell assays for the generation and long-term propagation of PDOs. Single cell derived PDOs could faithfully recapitulate the original tumor and reflect the heterogeneity features. While most PDO use for precision medicine understandably involved tissues derived from metastatic patients, we envision that the generation of PDOs from localized PCa along with the incorporation of cells of the TME in tissue models would fulfill the great potential of PDOs in predicting drug clinical benefits. We conclude that single cell derived PDOs reiterate the molecular features of the original tumor and represent a reliable pre-clinical PCa model to understand individual tumors and design tailored targeted therapies.

3D cell culture models: Drug pharmacokinetics, safety assessment, and regulatory consideration.

Nonclinical testing has served as a foundation for evaluating potential risks and effectiveness of investigational new drugs in humans. However, the current two-dimensional (2D) in vitro cell culture systems cannot accurately depict and simulate the rich environment and complex processes observed in vivo, whereas animal studies present significant drawbacks with inherited species-specific differences and low throughput for increased demands. To improve the nonclinical prediction of drug safety and efficacy, researchers continue to develop novel models to evaluate and promote the use of improved cell- and organ-based assays for more accurate representation of human susceptibility to drug response. Among others, the three-dimensional (3D) cell culture models present physiologically relevant cellular microenvironment and offer great promise for assessing drug disposition and pharmacokinetics (PKs) that influence drug safety and efficacy from an early stage of drug development. Currently, there are numerous different types of 3D culture systems, from simple spheroids to more complicated organoids and organs-on-chips, and from single-cell type static 3D models to cell co-culture 3D models equipped with microfluidic flow control as well as hybrid 3D systems that combine 2D culture with biomedical microelectromechanical systems. This article reviews the current application and challenges of 3D culture systems in drug PKs, safety, and efficacy assessment, and provides a focused discussion and regulatory perspectives on the liver-, intestine-, kidney-, and neuron-based 3D cellular models.

Organoid systems to study the human female reproductive tract and pregnancy.

Both the proper functioning of the female reproductive tract (FRT) and normal placental development are essential for women's health, wellbeing, and pregnancy outcome. The study of the FRT in humans has been challenging due to limitations in the in vitro and in vivo tools available. Recent developments in 3D organoid technology that model the different regions of the FRT include organoids of the ovaries, fallopian tubes, endometrium and cervix, as well as placental trophoblast. These models are opening up new avenues to investigate the normal biology and pathology of the FRT. In this review, we discuss the advances, potential, and limitations of organoid cultures of the human FRT.