Conventional 2D Cell Culture Research Articles

Therapeutic enhancement of a cytotoxic agent using photochemical internalisation in 3D compressed collagen constructs of ovarian cancer

Photochemical internalisation (PCI) is a method for enhancing delivery of drugs to their intracellular target sites of action. In this study we investigated the efficacy of PCI using a porphyrin photosensitiser and a cytotoxic agent on spheroid and non-spheroid compressed collagen 3D constructs of ovarian cancer versus conventional 2D culture. The therapeutic responses of two human carcinoma cell lines (SKOV3 and HEY) were compared using a range of assays including optical imaging. The treatment was shown to be effective in non-spheroid constructs of both cell lines causing a significant and synergistic reduction in cell viability measured at 48 or 96 h post-illumination. In the larger spheroid constructs, PCI was still effective but required higher saporin and photosensitiser doses. Moreover, in contrast to the 2D and non-spheroid experiments, where comparable efficacy was found for the two cell lines, HEY spheroid constructs were found to be more susceptible to PCI and a lower dose of saporin could be used. PCI treatment was observed to induce death principally by apoptosis in the 3D constructs compared to the mostly necrotic cell death caused by PDT. At low oxygen levels (1%) both PDT and PCI were significantly less effective in the constructs. Statement of significanceAssessment of new drugs or delivery systems for cancer therapy prior to conducting in vivo studies often relies on the use of conventional 2D cell culture, however 3D cancer constructs can provide more physiologically relevant information owing to their 3D architecture and the presence of an extracellular matrix. This study investigates the efficacy of Photochemical Internalisation mediated drug delivery in 3D constructs. In 3D cultures, both oxygen and drug delivery to the cells are limited by diffusion through the extracellular matrix unlike 2D models, and in our model we have used compressed collagen constructs where the density of collagen mimics physiological values. These 3D constructs are therefore well suited to studying drug delivery using PCI. Our study highlights the potential of these constructs for identifying differences in therapeutic response to PCI of two ovarian carcinoma lines.

Abstract 3861: 3D ex vivo PDX cell model screening to better predict in vivo outcome

Abstract Patient-derived xenograft (PDX) models have been highly demanded in preclinical drug discovery and development due to several advantages over conventional 2D cell culture system: 1) reflection of the heterogeneity, molecular and histopathologic signatures of the original tumor than cell lines or genetically engineered mouse models, and 2) certain degree of correlation of their drug-response profiles with clinical response. Certain limitations of using PDX models, however, do exist, including low throughput in candidate drug screening, lack of dose-response curves, high cost and time-consuming studies, and progressive loss of human-derived stromal elements over passages. To overcome these disadvantages, we sought to develop ex vivo 3D assay format on cells isolated from early passage PDX models, and provide genetic mutation information to better interpret results. Moreover, we attempted to incorporate immunotherapy strategy into the 3D system as well. We have recently built up an ex vivo cell bank containing more than 300 frozen ex vivo tumor cells dissociated from freshly isolated PDX tumor tissues. We have collected five panels of such frozen ex vivo cells, each panel representing more than 50 models in a specific tumor type. SOC compounds corresponding to each unique tumor panel were tested, and will be discussed. Moreover, we have established a 3D immune cell and ex vivo somatic tumor cell co-culture system to evaluate drug efficacy of immune checkpoint inhibitors aPD1, aPDL1 and CTLA4 antibodies either as single agent or in combination with small-molecule inhibitors. The data will be presented. In summary, we have developed a robust and reproducible 3D ex vivo assay platform for medium-throughput compound screening with bioinformatics information for data interpretation. Citation Format: Xiaoxi Xu, Zhongliang Li, Yan Liu, Fanxiu Meng, Yu Lu, Songling Zhang, Chunlan Dong, Frank Xing, Qian Shi. 3D ex vivo PDX cell model screening to better predict in vivo outcome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3861.

Recent Advances in Engineering the Stem Cell Microniche in 3D.

Conventional 2D cell culture techniques have provided fundamental insights into key biochemical and biophysical mechanisms responsible for various cellular behaviors, such as cell adhesion, spreading, division, proliferation, and differentiation. However, 2D culture in vitro does not fully capture the physical and chemical properties of the native microenvironment. There is a growing body of research that suggests that cells cultured on 2D substrates differ greatly from those grown in vivo. This article focuses on recent progress in using bioinspired 3D matrices that recapitulate as many aspects of the natural extracellular matrix as possible. A range of techniques for the engineering of 3D microenvironment with precisely controlled biophysical and chemical properties, and the impact of these environments on cellular behavior, is reviewed. Finally, an outlook on future challenges for engineering the 3D microenvironment and how such approaches would further our understanding of the influence of the microenvironment on cell function is provided.

A vacuum-actuated microtissue stretcher for long-term exposure to oscillatory strain within a 3D matrix.

Although our understanding of cellular behavior in response to extracellular biological and mechanical stimuli has greatly advanced using conventional 2D cell culture methods, these techniques lack physiological relevance. To a cell, the extracellular environment of a 2D plastic petri dish is artificially flat, extremely rigid, static and void of matrix protein. In contrast, we developed the microtissue vacuum-actuated stretcher (MVAS) to probe cellular behavior within a 3D multicellular environment composed of innate matrix protein, and in response to continuous uniaxial stretch. An array format, compatibility with live imaging and high-throughput fabrication techniques make the MVAS highly suited for biomedical research and pharmaceutical discovery. We validated our approach by characterizing the bulk microtissue strain, the microtissue strain field and single cell strain, and by assessing F-actin expression in response to chronic cyclic strain of 10%. The MVAS was shown to be capable of delivering reproducible dynamic bulk strain amplitudes up to 13%. The strain at the single cell level was found to be 10.4% less than the microtissue axial strain due to cellular rotation. Chronic cyclic strain produced a 35% increase in F-actin expression consistent with cytoskeletal reinforcement previously observed in 2D cell culture. The MVAS may further our understanding of the reciprocity shared between cells and their environment, which is critical to meaningful biomedical research and successful therapeutic approaches.

Diels-Alder Click-Cross-Linked Hydrogels with Increased Reactivity Enable 3D Cell Encapsulation.

Engineered hydrogels have been extensively used to direct cell function in 3D cell culture models, which are more representative of the native cellular microenvironment than conventional 2D cell culture. Previously, hyaluronan-furan and bis-maleimide polyethylene glycol hydrogels were synthesized via Diels-Alder chemistry at acidic pH, which did not allow encapsulation of viable cells. In order to enable gelation at physiological pH, the reaction kinetics were accelerated by replacing the hyaluronan-furan with the more electron-rich hyaluronan-methylfuran. These new click-cross-linked hydrogels gel faster and at physiological pH, enabling encapsulation of viable cells, as demonstrated with 3D culture of 5 different cancer cell lines. The methylfuran accelerates Diels-Alder cycloaddition yet also increases the retro Diels-Alder reaction. Using computational analysis, we gain insight into the mechanism of the increased Diels-Alder reactivity and uncover that transition state geometry and an unexpected hydrogen-bonding interaction are important contributors to the observed rate enhancement. This cross-linking strategy serves as a platform for bioconjugation and hydrogel synthesis for use in 3D cell culture and tissue engineering.

3D Hypoxia Microenvironment Created by a Single Cell Layer-by-Layer Assembly Spheroid Demonstrates the Role of miR-382 and Cell Autophagy in Renal Fibrosis

Compared with conventional 2D cell culture systems, 3D cell culture systems can better mimic the sophisticated internal environment. Hyaluronic acid (HA) is a fundamental element of the extracellular matrix and can be modified in many ways to alter its properties. Herein, in order to develop a 3D hypoxia culture system, we firstly synthesized HA-EDA with HA and ethylene-diamine (EDA) and then wrapped single renal mesangial cell layer-by-layer (LBL) with cationic HA-EDA and anionic HA. Unexpectedly, these wrapped cells formed a 3D hypoxia multi-cellular spheroid after being treated with trypsin. Based on the developed 3D hypoxia microenvironment, we found miR-382 might play a vital role in renal fibrosis. Amazingly, cell autophagy was detected both in 3D spheroid and 3DM cells (cells migrated from the 3D spheroid). However, different from the 3D spheroid, the 3DM cells displayed alleviated accumulation of TGF-β1 and Fn, which demonstrated the renoprotective role of autophagy in renal fibrosis. The high ratio of CD24+ cells in 3DM cells revealed that cells surviving through the autophagic process could acquire some characterization of progenitor cells via dedifferentiation. The developed 3D multi-cellular spheroid highlighted the role of hypoxia and cell autophagy in renal fibrosis.

Billion-scale production of hepatocyte-like cells from human induced pluripotent stem cells

Human induced pluripotent stem (iPS) cell-derived hepatocyte-like cells are expected to be utilized in drug screening and regenerative medicine. However, hepatocyte-like cells have not been fully used in such applications because it is difficult to produce such cells on a large scale. In this study, we tried to establish a method to mass produce hepatocyte-like cells using a three-dimensional (3D) cell culture bioreactor called the Rotary Cell Culture System (RCCS). RCCS enabled us to obtain homogenous hepatocyte-like cells on a billion scale (>109 cells). The gene expression levels of some hepatocyte markers (alpha-1 antitrypsin, cytochrome (CYP) 1A2, CYP2D6, and hepatocyte nuclear factor 4alpha) were higher in 3D-cultured hepatocyte-like cells than in 2D-cultured hepatocyte-like cells. This result suggests that RCCS could provide more suitable conditions for hepatocyte maturation than the conventional 2D cell culture conditions. In addition, more than 90% of hepatocyte-like cells were positive for albumin and could uptake low-density lipoprotein in the culture medium. We succeeded in the large-scale production of homogenous and functional hepatocyte-like cells from human iPS cells. This technology will be useful in drug screening and regenerative medicine, which require enormous numbers of hepatocyte-like cells.

Current advances in skin-on-a-chip models for drug testing.

Skin-on-a-chip models are highly desirable in drug testing compared to conventional 2D cell culture and animal models as they can replicate organ-specific 3D structural organization and physiological functions at a relatively low cost. To engineer a physiologically relevant skin model, human skin structures have been integrated onto microfluidic platforms to construct skin-on-a-chip systems that can mimic the complex in vivo situation. In this mini-review, we first briefly introduce some critical technologies employed to develop in vitro skin-on-a-chip models. We then review the applications of the state-of-the-art skin-on-a-chip models in drug testing, with a focus on using models of full-thickness skin equivalents (FTSEs), skin models with additional components such as vasculature, immune cells and hair follicles as well as multi-organ-on-a-chip models. Finally, we discuss some current challenges and future directions of development of complex, and in vivo-like skin-on-a-chip models.

In Vitro Porcine Colon Culture.

Models have been extensively used to investigate disease pathogenesis. Animal models are costly, require extensive logistics for animal care, and samples are not always suitable for different analytical techniques or to answer the research question. In vitro cell culture models are generally focused on recreating a specific characteristic of an organ, and are limited to a single cell population that does not display the characteristic tissue architecture of the source organ. In addition, such models do not account for the many interactions between pathogens and the diverse cell subsets that are normally present in a given organ. Conclusions based on conventional 2D cell culture methods are limited, requiring extrapolation from a reductionist model to understand in vivo events. In vitro organ culture (IVOC) offers a way to overcome some of these limitations. Explants conserve important in vivo characteristics, such as different cell types and complex tissue architecture. This in vitro (ex vivo) organ culture protocol of the swine large intestine aims at maintaining viable colonic mucosa for up to 5days. The protocol described herein applies a combination of methods used for immortalized cell culture and stem cell stimulation to support the physiological cellular flow inherent of the intestinal mucosa. Required equipment includes a hyperoxic chamber and culture at the air-liquid interface.

Carboxybetaine-modified succinylated chitosan-based beads encourage pancreatic \u03b2-cells (Min-6) to form islet-like spheroids under in vitro conditions

In vitro, pancreatic β-cells tend to reduce their ability to aggregate into islets and lose insulin-producing ability, likely due to insufficient cell–cell and cell–matrix interactions that are essential for β-cell retention, viability and functionality. In response to these needs, surfaces of succinylated chitosan-based beads (NSC) were modified with zwitterionic carboxy-betaine (CB) moieties, a compatible osmolyte known to regulate cellular hydration state, and used to promote the formation of β-cell spheroids using a conventional 2D cell culture technique. The NSC were synthesised by ionic gelation and surface-functionalised with CB using carbodiimide chemistry. Scanning electron microscopy (SEM), dynamic laser scattering (DLS) and Fourier transform infrared spectroscopy (FTIR) were employed as characterisation tools to confirm the successful modification of the succinylated chitosan material into spherical beads with rough surfaces and a diameter of 0.4 µm. NSC with and without CB were re-suspended at concentrations of 0.1, 0.3 and 0.6 mg/mL in saline medium and tested in vitro with MIN6 murine pancreatic β-cell line. Results showed that a concentration of 0.3 mg/mL, NSC-CB encouraged pancreatic MIN6 cells to proliferate and form spheroids via E-cadherin and Pdx-1 activation within 48 h in culture. These spheroids, with a size of approximately 80 µm, exhibited high cell viability and enhanced insulin protein expression and secretion when compared to cells organised by the non-modified beads.

Organ-on-a-Chip Technology for Reproducing Multiorgan Physiology.

In the drug development process, the accurate prediction of drug efficacy and toxicity is important in order to reduce the cost, labor, and effort involved. For this purpose, conventional 2D cell culture models are used in the early phase of drug development. However, the differences between the in vitro and the in vivo systems have caused the failure of drugs in the later phase of the drug-development process. Therefore, there is a need for a novel in vitro model system that can provide accurate information for evaluating the drug efficacy and toxicity through a closer recapitulation of the in vivo system. Recently, the idea of using microtechnology for mimicking the microscale tissue environment has become widespread, leading to the development of "organ-on-a-chip." Furthermore, the system is further developed for realizing a multiorgan model for mimicking interactions between multiple organs. These advancements are still ongoing and are aimed at ultimately developing "body-on-a-chip" or "human-on-a-chip" devices for predicting the response of the whole body. This review summarizes recently developed organ-on-a-chip technologies, and their applications for reproducing multiorgan functions.

Abstract 1923: Paired isolation and expansion of CSC and CTC from primary small cell lung cancer patient tissue and blood using the 3DKUBE bioreactor platform

Abstract Surgical resection is rarely an option for small cell lung cancer (SCLC) patients as the majority present with extensive disease at diagnosis. This scarcity of patient samples suitable for research presents a significant road block for the development of SCLC targeted-therapeutics. To address the problem of tissue scarcity, we have developed a method for the isolation and expansion of cancer stem cells (CSC) and circulating tumor cells (CTC) from primary tissues and blood of SCLC patients using the 3DKUBE™ perfusion microbioreactor. We have established a label-free, combined chemical and functional selection method for the isolation of CSCs from SCLC samples, solid tumor as well as blood, that does not rely upon the bias imposed by marker-based selection. Cells enriched in this manner were further purified and expanded under optimized conditions (growth factors, ECM, scaffolding and oxygen tension) within the 3DKUBE™ perfusion microbioreactor. These isolated and expanded CSCs have maintained resistance to cisplatin and etoposide, stabilized the expression of traditional CSC markers, and been validated in vitro through serial spheroid formation assays. These CSCs are currently being characterized and compared to parental tissue through correlative genomic and phenomic analysis and validated through in vivo tumorigenesis models. These cells will be utilized to generate 3D microtumors to accurately predict SCLC drug response in vitro, a determination that is not accurately performed in conventional 2D cell culture and is inhibited by both cost and time in patient-derived xenografts (PDX) Citation Format: Melissa Millard, Alina Lotstein, Lillia Holmes, David Schammel, Ki Chung, Jeff Edenfield, Hal E. Crosswell, Tessa DesRochers. Paired isolation and expansion of CSC and CTC from primary small cell lung cancer patient tissue and blood using the 3DKUBE bioreactor platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1923. doi:10.1158/1538-7445.AM2017-1923

A Novel Cellular Spheroid-Based Autophagy Screen Applying Live Fluorescence Microscopy Identifies Nonactin as a Strong Inducer of Autophagosomal Turnover

Dysregulation of the basal autophagic flux has been linked to several pathological conditions, including neurodegenerative diseases and cancer. In addition, autophagy has profound effects on the response of tumor cells to therapy. Hence, the search for pharmacological modulators of autophagy is of great clinical relevance. We established a drug screening assay in which the autophagic flux is measured by recording the fluorescence emission of the tandem fusion protein mRFP-GFP-LC3 by dynamic live-cell imaging. We optimized the assay for the identification of autophagy modulators in three dimensions with U343 glioma cell spheroids, which represent a more realistic cancer model than conventional 2D cell cultures. We validated the assay by screening a library of known autophagy modulators. As the first application, a small library of 94 natural compounds was screened for its impact on autophagy. We discovered the cyclic ionophore nonactin as a new and potent autophagy inducer. This novel autophagy screening assay based on 3D tumor spheroids is robust, reproducible, and scalable. It provides a valuable tool for both basic research and drug screening campaigns.

Creating Multiple Organotypic Models on a Single 3D Cell Culture Platform

BioTechniquesVol. 62, No. 3 Sponsored Paper - Application ForumOpen AccessCreating Multiple Organotypic Models on a Single 3D Cell Culture PlatformSei Hien Lim & Andrea PavesiSei Hien LimAIM Biotech Pte Lte, 51 Ayer Rajah Crescent #02-12/14, Singapore, 139948Search for more papers by this author & Andrea PavesiAIM Biotech Pte Lte, 51 Ayer Rajah Crescent #02-12/14, Singapore, 139948Search for more papers by this authorPublished Online:16 Mar 2018https://doi.org/10.2144/000114526AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInReddit IntroductionIn vitro 3D cell culture models have emerged as a bridge between conventional 2D cell culture models and the complex & expensive in vivo animal models. By observing, analyzing and comparing the biological behavior of tissues embedded in 3 dimensional hydrogels, results are significantly different from the classic 2D cell culture. In particular, literature shows differences in terms of proliferation, morphology, drug response and gene expression [1–4]. These differences have been attributed to the topographically complex 3D environment surrounding the cells, where cell adhesion, structure, effector transport and mechanotransduction are substantially altered [5]. A carefully designed 3D model can therefore provide more physiologically relevant information using experimental designs unachievable by conventional 2D assays, at a fraction of the cost of in vivo models.Current 3D cell culture assays like hanging drop culture often lack the capability to organize different co-cultured cell types in a meaningful way. The application of chemical gradients or flow is usually not possible.We are now able to address this issue in a microfluidic platform (Figure 1a). The platform consists of a hydrogel channel and two flanking media channels, where cells can be meaningfully compartmentalized to study their interactions. The miniature posts that border the hydrogel channel maintain a vertical gel interface. Given the multi-channel design, a transient gradient of chemical factors can be set up across the hydrogel to yield a more physiologically relevant microenvironment.Materials and methodsCulture Cells in Media ChannelsFill polymerizable hydrogel (e.g. type I collagen, fibrin gel, Matrigel) into the gel channel (the middle channel shaded in red in Figure 1b). Once the gel has polymerized, fill cell culture medium into the media channels (the flanking channels shaded in blue in Figure 1b). Seed cells into the media channels. Set up chemical gradient across the gel region or apply flow in the channels, depending on your applications. Allow the cells to grow and interact with the microenvironment in an incubator. Observe the biological processes with phase contrast, fluorescent or confocal microscopy. Perform downstream analysis (e.g. ELISA, RT-PCR) on media or extracts collected from the chips to further understand the biological processes.Figure 1. A schematic drawing to illustrate (a) the three-channel design of the chip and the workflow for (b) culturing cells in media channels; (c) culturing cells in hydrogel and media channels.Table 1. Biological models that have been successfully created in the platform. Additional references to related publications can be found at http://www.aimbiotech.com/publications.htmlCulture Cells in Hydrogel ChannelsMix cell suspension or cell aggregates with the hydrogel of choice. Fill the hydrogel with cells into the gel channel. Once it has polymerized, fill cell culture medium into the media channels. Seed cells, of the same cell type or of a second cell type, into the media channels. Observe and study the biological processes as described above.ResultsDifferent biological models can be devised depending on the cell types and the locations of the cells being cultured. Cells (e.g. cancer cells) can be seeded in the media channels and be positioned at the gel interface for cell migration assays. With appropriate chemical gradients, the cells can invade into the 3D hydrogel and this process can be monitored in real time. Endothelial cells tend to form a monolayer in the media channel which itself is useful for permeability tests. In addition, with the appropriate angiogenic signals and interstitial flow, an endothelial monolayer can be induced to form sprouts in the 3D hydrogel and serve as an angiogenesis model. The same monolayer can also be co-cultured with tumor cells and be used to study transendothelial migration, i.e. extravasation or intravasation, depending on the location of the target cells.Endothelial cells mixed with fibrin gel will undergo morphogenesis to form vascular networks in the gel through vasculogenesis. The vascular networks can then be used for more advanced assays including flowing cells through the networks.Solid tumor dissemination can be modelled by seeding tumor cell aggregates in the hydrogel. Under the right culture conditions (with co-culture or stimuli), tumor cells will disseminate from the aggregates and invade the surrounding matrix, marking the first step of metastasis.Please visit www.AIMBiotech.com for more information.

Bioluminescence Imaging of Spheroids for High-throughput Longitudinal Studies on 3D Cell Culture Models.

Bioluminescent (BL) cell-based assays based on two-dimensional (2D) monolayer cell cultures represent well-established bioanalytical tools for preclinical screening of drugs. However, cells in 2D cultures do not often reflect the morphology and functionality of living organisms, thus limiting the predictive value of 2D cell-based assays. Conversely, 3D cell models have the capability to generate the extracellular matrix and restore cell-to-cell communications; thus, they are the most suitable model to mimic invivo physiology. In this work, we developed a nondestructive real-time BL imaging assay of spheroids for longitudinal studies on 3D cell models. A high-throughput BL 3D cell-based assay in micropatterned 96-well plate format is reported. The assay performance was assessed using the transcriptional regulation of nuclear factor K beta response element in human embryonic kidney (HEK293) cells. We compared concentration-response curves for tumor necrosis factor-α with those obtained using conventional 2D cell cultures. One of the main advantages of this approach is the nonlysing nature of the assay, which allows for repetitive measurements on the same sample. The assay can be implemented in any laboratory equipped with basic cell culture facilities and paves the way to the development of new 3D bioluminescent cell-based assays.

Tumour-like druggable gene expression pattern of CaCo2 cells in microfluidic chip

The human-on-chip technology provides an efficient basis for preclinical studies and has potentially a greater predictive power for human drug response than classical 2D cell culture. Here we report the expression profile of druggable genes in the human colon cancer cells CaCo2 in static culture and within a microfluidic chip. We identified gene expression pattern under flow to be closer to the one of CaCo2 primary xenograft tumours as compared to those cells grown without circulation. The obtained results indicate that a microenvironment connected to a circulation within a chip brings the cells closer to in vivo situation. Hence the human-on-chip technology is a more powerful tool for drug development than conventional 2D cell culture.

A three-dimensional collagen scaffold cell culture system for screening anti-glioma therapeutics.

Three-dimensional (3D) culture, which can simulate in vivo microenvironments, has been increasingly used to study tumor cell biology. Since most preclinical anti-glioma drug tests still rely on conventional 2D cell culture, we established a collagen scaffold for 3D glioma cell culture. Glioma cells cultured on these 3D scaffolds showed greater degree of dedifferentiation and quiescence than cells in 2D culture. 3D-cultured cells also exhibited enhanced resistance to chemotherapeutic alkylating agents, with a much higher proportion of glioma stem cells and upregulation of O6-methylguanine DNA methyltransferase (MGMT). Importantly, tumor cells in 3D culture showed chemotherapy resistance patterns similar to those observed in glioma patients. Our results suggest that 3D collagen scaffolds are promising in vitro research platforms for screening new anti-glioma therapeutics.

Abstract B17: Traf2- and NCK-interacting kinase (TNIK) inhibitor down-regulates Wnt signaling pathway in cancer cells

Abstract Introduction: Aberrant activation of the Wnt signaling pathway has been implicated as the key driver of carcinogenesis, particularly in colorectal cancers. Because nearly 90% of colorectal cancers carry mutations either APC or β-catenin, downstream signaling molecules of the β-catenin destructive complex are considered as more attractive therapeutic targets in drug discovery research for the treatment of colorectal cancers. Recently, Traf2- and NCK-interacting kinase (TNIK) has been identified as one of components of the T-cell factor-4 (TCF4) transcriptional complex and essential for the expression of Wnt target genes. Knockdown of TNIK gene expression suppressed the transcriptional activity of the Wnt signaling pathway, and decreased in proliferation of colorectal cancer cells. Therefore inhibitors of TNIK may represent a promising therapeutic approach for treating Wnt-signal activated cancers. Previously we reported a thiazol-based TNIK inhibitor, N5355 have single digit nM IC50 value and the compound significantly down-regulated the Wnt target genes such as AXIN2 and cMYC in colorectal cancer cell line, HCT116. Further evaluations of N5355 have been implemented to characterize the effects of TNIK inhibition on cancer cell lines. Methods: To study the effects of N5355 on the Wnt signaling pathway, the expression levels of Wnt target proteins were analyzed by Western blotting using Wnt-signal activated cancer cell lines. The ability of N5355 for blocking of nuclear translocation of phosphorylated TNIK was also analyzed by Western blotting using HCT116 cells. Anti-proliferative activity profile of N5355 was studied using a panel of 44 cancer cell lines. The pharmacokinetic profile of TNIK inhibitor was also analyzed. Results: The Wnt signal inhibition was validated by significant suppression of Wnt target protein expressions by treating with N5355 in several cancer cells. In addition, Wnt co-receptor proteins LRP6 and LRP5 were found to be down-regulated by N5355 treatment. N5355 exhibited strong anti-proliferation activities against Wnt-driven cancer cell lines. Interestingly in DLD1 colorectal cancer cell lines, N5355 showed no anti-proliferative effect in conventional 2D cell culture assay, but displayed strong inhibitory activity in 3D cell culture assay using soft agar, which is biologically relevant assay system. The fact that N5355 inhibited the autophosphorylation of TNIK in the cytosol and subsequent nuclear translocation of TNIK in HCT116 cells suggested the depletion of TNIK proteins in nucleus resulted in the observed Wnt signal inhibition effect. Detailed results will be presented in the conference. Citation Format: Yuko Uno, Hideki Moriyama, Shigeki Kashimoto, Mari Masuda, Masaaki Sawa, Tesshi Yamada. Traf2- and NCK-interacting kinase (TNIK) inhibitor down-regulates Wnt signaling pathway in cancer cells. [abstract]. In: Proceedings of the AACR Special Conference: Developmental Biology and Cancer; Nov 30-Dec 3, 2015; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(4_Suppl):Abstract nr B17.

Experimental analysis of effect of electric field on mouse myoblast cells using high throughput microfluidic bioreactor

Event Abstract Back to Event Experimental analysis of effect of electric field on mouse myoblast cells using high throughput microfluidic bioreactor Sharmistha Naskar1, Bikramjit Basu1, 2 and V Kumaran1, 3 1 Indian Institute of Science, Centre for Biosystems Science and Engineering (BSSE), India 2 Indian Institute of Science, Materials Research Centre, India 3 Indian Institute of Science, Department of Chemical Engineering, India Application of electric field on excitable cells like myoblast[1] or neuroblast[2], causes cell phenotype changes which have already been investigated using conventional 2D cell culture. But this may not be a good representation of the in vivo conditions due to very high ratio of volume of culture media to the number of cells and also due to absence of the shear stress in comparison to in vivo conditions where cells continuously experience fluid flow around them. In this study, we had attempted to investigate the effect of electric field on the cells by introducing biomimetic devices that simulates the in vivo environment, using microfluidic technology. Microfluidics has already started to invade almost every corner of life sciences with the promise of more effective and efficient performance than the existing techniques with the requirement of very scanty amount of sample volume, reduction in the running cost of the experiments, high accuracy due to almost zero dead-volume and fast response time. In order to achieve an environment having close resemblance with the in vivo conditions, C2C12 (mouse myoblast) cells were cultured within the microfluidic bioreactor along with flow of culture media. Designing of this microfluidic bioreactor involved some crucial processes, including the microfluidic layout, selection of materials, fabrication process, and sterilization technique along with necessary requirements like the maintenance of sterility and biocompatibility. This microfluidic bioreactor was fabricated through photolithography by procuring a silicon based SU8 master. This SU8 master was used as a mold for device fabrication using Polydimethylsiloxane (PDMS). The C2C12 cells were then seeded inside the microfluidic channels (width of 20μm - 40μm) of the bioreactor device. By limiting both the space and nutrient for the cultured cells along with an exposure to shear stress due to flowing of fluid, this system provided a close resemblance with the in vivo scenarios. Prior exposure of mouse myoblast cells to an electric field of 12.5 V/cm for 12 hours resulted in morphological alterations with elongated nucleus, roughening of the cell suface topography, myogenesis, as revealed by immunofluorescence techniques, electron microscopy and PCR supported with biochemical assays. Overall, the results confirmed considerable morphological changes of the myoblasts, when stimulated electrically compared to the non-stimulated cells. Prof. Bikramjit Basu; Prof. V. Kumaran

A Microfluidic Bioreactor for Toxicity Testing of Stem Cell Derived 3D Cardiac Bodies.

Modeling tissues and organs using conventional 2D cell cultures is problematic as the cells rapidly lose their in vivo phenotype. In microfluidic bioreactors the cells reside in microstructures that are continuously perfused with cell culture medium to provide a dynamic environment mimicking the cells natural habitat. These micro scale bioreactors are sometimes referred to as organs-on-chips and are developed in order to improve and extend cell culture experiments. Here, we describe the two manufacturing techniques photolithography and soft lithography that are used in order to easily produce microfluidic bioreactors. The use of these bioreactors is exemplified by a toxicity assessment on 3D clustered human pluripotent stem cells (hPSC)-derived cardiomyocytes by beating frequency imaging.