Conventional Cell Culture Models Research Articles

RNA-seq transcriptomic profiling of TGF-β2-exposed human trabecular meshwork explants: Advancing insights beyond conventional cell culture models

Primary open-angle glaucoma (POAG), a leading cause of irreversible vision loss, is closely linked to increased intraocular pressure (IOP), with the trabecular meshwork (TM) playing a critical role in its regulation. The TM, located at the iridocorneal angle, acts as a sieve, filtering the aqueous humor from the eye into the collecting ducts, thus maintaining proper IOP levels. The transforming growth factor-beta 2 (TGF-β2) signaling pathway has been implicated in the pathophysiology of primary open-angle glaucoma POAG particularly, in the dysfunction of the TM. This study utilizes human TM explants to closely mimic in vivo conditions, thereby minimizing transcriptional changes that could arise from cell culture enabling an exploration of the transcriptomic impacts of TGF-β2. Through bulk RNA sequencing and immunohistological analysis, we identified distinct gene expression patterns and morphological changes induced by TGF-β2 exposure (5 ng/ml for 48 h). Bulk RNA sequencing identified significant upregulation in genes linked to extracellular matrix (ECM) regulation and fibrotic signaling. Immunohistological analysis further elucidated the morphological alterations, including cytoskeletal rearrangements and ECM deposition, providing a visual confirmation of the transcriptomic data. Notably, the enrichment analysis unveils TGF-β2’s influence on both bone morphogenic protein (BMP) and Wnt signaling pathways, suggesting a complex interplay of molecular mechanisms contributing to TM dysfunction in glaucoma. This characterization of the transcriptomic modifications on an explant model of TM obtained under the effect of this profibrotic cytokine involved in glaucoma is crucial in order to develop and test new molecules that can block their signaling pathways.

BIOL-19. DIFFUSE MIDLINE GLIOMA CEREBRAL ORGANOID MODEL AND MULTIOMICS CHARACTERIZATION

Abstract Diffuse midline glioma (DMG) are highly aggressive malignancies of the central nervous system that primarily affect the pediatric population. These tumors are historically universally fatal with no curative treatment options available. There is a need to identify more targeted and optimal treatments for these patients. Current approaches to pre-clinical therapeutic testing have been limited by many obstacles to effectively translate theses to patients. It is known that the interactions between tumors and the other components of the tumor microenvironment (TME) can change the response to therapeutic interventions. This especially holds true for brain tumors and the complex neural network encompassed within their TME. Given this, it is crucial to develop more realistic DMG models that integrate this to conduct therapeutic testing rather than relying upon conventional cell culture models. The goal of our study was to develop a three-dimensional DMG cerebral organoid model derived from human induced pluripotent stem cells (iPSCs) co-cultured with three different DMG patient-derived xenograft (PDX) cell lines to better mimic the TME for therapeutic testing. We were able to successfully integrate our three cell lines into the cerebral organoids, capturing TME interactions along with performing multiomic profiling for better characterization. We next plan to perform therapeutic testing to further validate the model and improve preclinical drug screening for DMG.

Single-Cell Analysis of Unidirectional Migration of Glioblastoma Cells Using a Fiber-Based Scaffold.

Glioblastoma (GBM) is a malignant incurable brain tumor in which immature neoplastic cells infiltrate brain tissue by spreading along nerve fibers. The aim of the study was to compare the migration abilities of glioma cells with those of other cancer cells and elucidate the migratory profiles underlying the differential migration of glioma cells using a fiber-based quantitative migration assay. Here, wound healing and transwell assays were used to assess cell mobility in four cell lines: U87-MG glioblastoma cells, MDA-MB-231 breast cancer cells, HCT116 colorectal cancer cells, and MKN45 gastric cancer cells. We also assessed cell mobility using a fiber model that mimics nerve fibers. Time-lapse video microscopy was used to observe cell migration and morphology. The cytoskeleton arrangement was assessed in the fiber model and compared with that in the conventional cell culture model. The conventional evaluation of cell migration ability revealed that the migration ability of breast cancer and glioblastoma cell lines was higher than that of colon cancer and gastric cancer cell lines. The fiber model confirmed that the glioblastoma cell line had a significantly higher migration ability than other cell lines. Tubulin levels were significantly higher in the glioblastoma cells than in other cell lines. In conclusion, the developed fiber-based culture model revealed the specific migratory profile of GBM cells during invasion.

Immunohistochemistry (IHC) staining of in-vitro cancer cell-generated tumoroids

Targeting different pathways in combinational therapy may lead to synergistic effects with higher drug efficiency. Due to a large number of candidate drugs and the variability in the genomic landscape of the disease, conventional cell culture models have limited success. Three-dimensional (3D) cell culture platforms such as tumoroids not only provide a pathophysiological relevant condition but also allow for low-cost and high-throughput drug screening strategies. Immunostaining of targeted proteins within a tumoroid is challenging as the interior cells are difficult to access via a non-destructive method. Immunohistochemistry (IHC) is an important technique in clinical research to explore the expression of various biomarkers. IHC staining of tumoroids allows non-destructive detection of unstable proteins by direct fixation of cells at the state of tumor microenvironment (TME) context, providing two main advantages. First, the target protein can be fixed without dissociating cells and disintegration of tumoroids into a single-cell suspension. Second, staining the preserved structure of tumoroids helps identify the location of the target proteins as well as the spatial distribution throughout the tumoroid geometry. In this protocol, we describe the detailed methodology of a non-destructive IHC staining of cancer biomarkers which minimizes the manipulation of tumoroids prior to fixation by eliminating multiple centrifugations and shaking steps typically required for removing excess hydrogel and collecting tumoroids. The protocol can be used in studies involving prognostic and predictive biomarker investigations in new anti-tumor drug development strategies.

3D organ-on-a-chip: The convergence of microphysiological systems and organoids.

Medicine today faces the combined challenge of an increasing number of untreatable diseases and fewer drugs reaching the clinic. While pharmaceutical companies have increased the number of drugs in early development and entering phase I of clinical trials, fewer actually successfully pass phase III and launch into the market. In fact, only 1 out of every 9 drugs entering phase I will launch. In vitro preclinical tests are used to predict earlier and better the potential of new drugs and thus avoid expensive clinical trial phases. The most recent developments favor 3D cell culture and human stem cell biology. These 3D humanized models known as organoids better mimic the 3D tissue architecture and physiological cell behavior of healthy and disease models, but face critical issues in production such as small-scale batches, greater costs (when compared to monolayer cultures) and reproducibility. To become the gold standard and most relevant biological model for drug discovery and development, organoid technology needs to integrate biological culture processes with advanced microtechnologies, such as microphysiological systems based on microfluidics technology. Microphysiological systems, known as organ-on-a-chip, mimic physiological conditions better than conventional cell culture models since they can emulate perfusion, mechanical and other parameters crucial for tissue and organ physiology. In addition, they reduce labor cost and human error by supporting automated operation and reduce reagent use in miniaturized culture systems. There is thus a clear advantage in combining organoid culture with microsystems for drug development. The main objective of this review is to address the recent advances in organoids and microphysiological systems highlighting crucial technologies for reaching a synergistic strategy, including bioprinting.

Construction of a Human Aorta Smooth Muscle Cell Organ-On-A-Chip Model for Recapitulating Biomechanical Strain in the Aortic Wall.

Conventional two-dimensional cell culture techniques and animal models have been used in the study of human thoracic aortic aneurysm and dissection (TAAD). However, human TAAD sometimes cannot be characterized by animal models. There is an apparent species gap between clinical human studies and animal experiments that may hinder the discovery of therapeutic drugs. In contrast, the conventional cell culture model is unable to simulate in vivo biomechanical stimuli. To this end, microfabrication and microfluidic techniques have developed greatly in recent years, providing novel techniques for establishing organoids-on-a-chip models that replicate the biomechanical microenvironment. In this study, a human aorta smooth muscle cell organ-on-a-chip (HASMC-OOC) model was developed to simulate the pathophysiological parameters of aortic biomechanics, including the amplitude and frequency of cyclic strain experienced by human aortic smooth muscle cells (HASMCs) that play a vital role in TAAD. In this model, the morphology of HASMCs became elongated in shape, aligned perpendicularly to the strain direction, and presented a more contractile phenotype under strain conditions than under static conventional conditions. This was consistent with the cell orientation and phenotype in native human aortic walls. Additionally, using bicuspid aortic valve-related TAAD (BAV-TAAD) and tricuspid aortic valve-related TAAD (TAV-TAAD) patient-derived primary HASMCs, we established BAV-TAAD and TAV-TAAD disease models, which replicate HASMC characteristics in TAAD. The HASMC-OOC model provides a novel in vitro platform that is complementary to animal models for further exploring the pathogenesis of TAAD and discovering therapeutic targets.

DIPG-39. New preclinical models for Diffuse Midline Glioma

Malignant brain tumors are the leading cause of childhood death in Germany, with Diffuse Midline Glioma (DMG) being the most lethal of all paediatric brain tumors. Current treatment strategies are limited to irradiation which prolongs survival only by a few months. Preclinical studies have identified effective drug candidates, but translation into the clinic remains a major obstacle. It is known that interactions between tumor cells and components of the TME (tumor microenvironment), such as cell to cell contacts between malignant and non-malignant cells or secreted factors, can increase therapy resistance and progression of brain tumors. However, these important factors are not present in most conventional cell culture models for drug testing. Consequently, there is a need for more realistic DMG models to improve the relevance and translational potential of current drug screening. Therefore, the goal of this study was to develop a new DMG model for drug testing, consisting of induced pluripotent stem cell (iPSC) derived human brain cells and patient derived DMG cells to better mimic the complex tumor microenvironment. We co-cultured three-dimensional cerebral organoids with DMG tumor spheres resulting in the formation of DMG-Brain-Organoids (DBO). Preliminary results show that co-culture induces distinct tumor cell subpopulations corresponding to those detected in DMG tumors by single cell RNA sequencing (Filbin et al., 2018). These subpopulations mainly differ in their proliferative capacity and their differential response to clinical interventions may be critical for therapeutic success. DBOs subjected to drug treatments (single or combination) were sectioned and individual therapy effects on tumor cell subpopulations and proliferative capacity were monitored using multiplexed immunofluorescence imaging. By observing drug effects in a realistic setup, we hope to improve the predictive power of our preclinical drug screens and to find new combination therapies for DMG.

Establishment of a Simple and Versatile Evaporation Compensation Model for in vitro Chronic Ethanol Treatment: Impact on Neuronal Viability.

Alcohol overconsumption is a major cause of preventable mental disorders and death in the United States and around the world. The pathogenesis of alcohol dependence, abuse, and toxicity to the central nervous system remains incompletely understood. In vitro and cell culture-based models have been highly valuable in studying the molecular and cellular mechanisms underlying the contribution of individual CNS cell types to ethanol's effects on the brain. However, conventional cell culture model systems carry the inherent disadvantage of rapid loss of ethanol due to evaporation following a bolus addition at the start of the treatment. We have established a multi-well cell culture plate-based ethanol evaporation compensation model that utilizes the inter-well space as a reservoir to compensate for the evaporative loss of ethanol in the cell treatment wells. Following a single bolus addition at the start of the treatment, ethanol concentration rapidly decreased over time. Through compensation using the multi-well plate platform, maintenance of ethanol concentrations ranging from 10-100 mM was achieved for up to 72 hours in a cell-free system. Greater effects in ethanol-induced decrease in neuronal cell viability were observed with than without compensation. Our method effectively compensates for the evaporative loss of ethanol typically observed in the traditional method. This method provides an economic, simple and effective in vitro model system for ethanol treatment over an extended timeframe where maintenance of a relatively constant concentration of ethanol is desired.

Longitudinal Monitoring of Intra-Tumoural Heterogeneity Using Optical Barcoding of Patient-Derived Colorectal Tumour Models.

Simple SummaryColorectal cancer (CRC) is the second most common cancer worldwide. Despite improvements in the clinical management of CRC, outcomes for those with metastatic disease remain extremely poor. One reason for this is tumour heterogeneity, which refers to the observation that each cell within complex tumour cell populations displays different genetic features and biological behaviours. Such tumour heterogeneity is known to impact treatment efficacy and promote tumour recurrence. Here, we present a multi-colour barcoding methodology that allows for different lineages of colorectal cancer cells to be identified and monitored, thus allowing for tumour heterogeneity to be quantified in real-time. We show that discrete cell lineages can be quantified by both fluorescence microscopy and flow cytometry. Using this approach, we show that the cell culture models that are traditionally used in cancer research display limited heterogeneity, whereas patient-derived organoids—which are generated from fresh tumour resections—more faithfully represent the heterogeneity observed in cancer patients.Geno- and phenotypic heterogeneity amongst cancer cell subpopulations are established drivers of treatment resistance and tumour recurrence. However, due to the technical difficulty associated with studying such intra-tumoural heterogeneity, this phenomenon is seldom interrogated in conventional cell culture models. Here, we employ a fluorescent lineage technique termed “optical barcoding” (OBC) to perform simultaneous longitudinal tracking of spatio-temporal fate in 64 patient-derived colorectal cancer subclones. To do so, patient-derived cancer cell lines and organoids were labelled with discrete combinations of reporter constructs, stably integrated into the genome and thus passed on from the founder cell to all its clonal descendants. This strategy enables the longitudinal monitoring of individual cell lineages based upon their unique optical barcodes. By designing a novel panel of six fluorescent proteins, the maximum theoretical subpopulation resolution of 64 discriminable subpopulations was achieved, greatly improving throughput compared with previous studies. We demonstrate that all subpopulations can be purified from complex clonal mixtures via flow cytometry, permitting the downstream isolation and analysis of any lineages of interest. Moreover, we outline an optimized imaging protocol that can be used to image optical barcodes in real-time, allowing for clonal dynamics to be resolved in live cells. In contrast with the limited intra-tumour heterogeneity observed in conventional 2D cell lines, the OBC technique was successfully used to quantify dynamic clonal expansions and contractions in 3D patient-derived organoids, which were previously demonstrated to better recapitulate the heterogeneity of their parental tumour material. In summary, we present OBC as a user-friendly, inexpensive, and high-throughput technique for monitoring intra-tumoural heterogeneity in in vitro cell culture models.

A narrative review of the migration and invasion features of non-small cell lung cancer cells upon xenobiotic exposure: insights from in vitro studies.

Lung cancer (LC) is the leading cause of cancer deaths worldwide, being non-small lung cancer (NSCLC) sub-types the most prevalent. Since most LC cases are only detected during the last stage of the disease the high mortality rate is strongly associated with metastases. For this reason, the migratory and invasive capacity of these cancer cells as well as the mechanisms involved have long been studied to uncover novel strategies to prevent metastases and improve the patients’ prognosis. This narrative review provides an overview of the main in vitro migration and invasion assays employed in NSCLC research. While several methods have been developed, experiments using conventional cell culture models prevailed, specifically the wound-healing and the transwell migration and invasion assays. Moreover, it is provided herewith a summary of the available information concerning chemical contaminants that may promote the migratory/invasive properties of NSCLC cells in vitro, shedding some light on possible LC risk factors. Most of the reported agents with pro-migration/invasion effects derive from cigarette smoking [e.g., Benzo(a)pyrene and cadmium] and air pollution. This review further presents several studies in which different dietary/plant-derived compounds demonstrated to impair migration/invasion processes in NSCLC cells in vitro. These chemicals that have been proposed as anti-migratory consisted mainly of natural bioactive substances, including polyphenols non-flavonoids, flavonoids, bibenzyls, terpenes, alkaloids, and steroids. Some of these compounds may eventually represent novel therapeutic strategies to be considered in the future to prevent metastasis formation in LC, which highlights the need for additional in vitro methodologies that more closely resemble the in vivo tumor microenvironment and cancer cell interactions. These studies along with adequate in vivo models should be further explored as proof of concept for the most promising compounds.

Bioengineered in vitro 3D model of myotonic dystrophy type 1 human skeletal muscle

Myotonic dystrophy type 1 (DM1) is the most common hereditary myopathy in the adult population. The disease is characterized by progressive skeletal muscle degeneration that produces severe disability. At present, there is still no effective treatment for DM1 patients, but the breakthroughs in understanding the molecular pathogenic mechanisms in DM1 have allowed the testing of new therapeutic strategies. Animal models and in vitro two-dimensional cell cultures have been essential for these advances. However, serious concerns exist regarding how faithfully these models reproduce the biological complexity of the disease. Biofabrication tools can be applied to engineer human three-dimensional (3D) culture systems that complement current preclinical research models. Here, we describe the development of the first in vitro 3D model of DM1 human skeletal muscle. Transdifferentiated myoblasts from patient-derived fibroblasts were encapsulated in micromolded gelatin methacryloyl-carboxymethyl cellulose methacrylate hydrogels through photomold patterning on functionalized glass coverslips. These hydrogels present a microstructured topography that promotes myoblasts alignment and differentiation resulting in highly aligned myotubes from both healthy and DM1 cells in a long-lasting cell culture. The DM1 3D microtissues recapitulate the molecular alterations detected in patient biopsies. Importantly, fusion index analyses demonstrate that 3D micropatterning significantly improved DM1 cell differentiation into multinucleated myotubes compared to standard cell cultures. Moreover, the characterization of the 3D cultures of DM1 myotubes detects phenotypes as the reduced thickness of myotubes that can be used for drug testing. Finally, we evaluated the therapeutic effect of antagomiR-23b administration on bioengineered DM1 skeletal muscle microtissues. AntagomiR-23b treatment rescues both molecular DM1 hallmarks and structural phenotype, restoring myotube diameter to healthy control sizes. Overall, these new microtissues represent an improvement over conventional cell culture models and can be used as biomimetic platforms to establish preclinical studies for myotonic dystrophy.

Human intestinal models to study interactions between intestine and microbes.

Implementations of suitable in vitro cell culture systems of the human intestine have been essential tools in the study of the interaction among organs, commensal microbiota, pathogens and parasites. Due to the great complexity exhibited by the intestinal tissue, researchers have been developing in vitro/ex vivo systems to diminish the gap between conventional cell culture models and the human intestine. These models are able to reproduce different structures and functional aspects of the tissue. In the present review, information is recapitulated on the most used models, such as cell culture, intestinal organoids, scaffold-based three-dimensional models, and organ-on-a-chip and their use in studying the interaction between human intestine and microbes, and their advantages and limitations are also discussed.

Cell Culture Model for Examining Fat-Soluble Nutrient Absorption <i>In Vitro</i>

Permeable support systems (PS) are employed in <i>in vitro </i>nutrient absorption studies but data are absent on their efficacy compared to conventional cell culture models (CONV). The<i> in vivo </i>absorption of fat soluble nutrients is influenced by its delivery vehicle, yet a fundamental understanding of the influence of the vehicle on cells in culture is lacking. We compared the efficacy of lutein absorption in Caco-2 cells cultured with CONV and PS, and examined the role of micelles, the physiological vehicle within the small intestine. After plating for 2 and 21 d to attain confluence and differentiation in CONV and PS, respectively, cells were treated with lutein in micelles or ethanol. After incubation, lutein in cell lysate, as well as apical and basolateral mediums, were quantified by HPLC-UV. After 24 h, cellular lutein in CONV was ≥460 and 8% greater in ethanol and micelle, respectively, than in PS. However, the intracellular AUC over time was only different for ethanol (<i>P</i> ≤ 0.05). In PS, 0.15% of micellized lutein was secreted into the basolateral medium in contrast to 0.016% of lutein in ethanol. The absorption of lutein (uptake + secretion), independent of the vehicle, in CONV increased in a linear manner with dose (0.35 to 4 or to 14.6 µg/mL for ethanol or micelle, respectively), while that in PS peaked at 1.18 μg/mL. Caco-2 cells cultured in PS grow to display the phenotype and function of small intestine enterocytes and suggest this <i>in vitro </i>platform generates information closest to the natural physiology of the absorptive process. However, although the CONV has the physiology of colonic tissue, it appears to display a greater efficacy for lutein uptake by Caco-2 cells and so can provide a more rapid, preliminary method for nutrient absorption studies.

PI3K-Akt-mTOR axis sustains rotavirus infection via the 4E-BP1 mediated autophagy pathway and represents an antiviral target

ABSTRACT Rotavirus infection is a major cause of severe dehydrating diarrhea in infants younger than 5 y old and in particular cases of immunocompromised patients irrespective to the age of the patients. Although vaccines have been developed, antiviral therapy is an important complement that cannot be substituted. Because of the lack of specific approved treatment, it is urgent to facilitate the cascade of further understanding of the infection biology, identification of druggable targets and the final development of effective antiviral therapies. PI3K-Akt-mTOR signaling pathway plays a vital role in regulating the infection course of many viruses. In this study, we have dissected the effects of PI3K-Akt-mTOR signaling pathway on rotavirus infection using both conventional cell culture models and a 3D model of human primary intestinal organoids. We found that PI3K-Akt-mTOR signaling is essential in sustaining rotavirus infection. Thus, blocking the key elements of this pathway, including PI3K, mTOR and 4E-BP1, has resulted in potent anti-rotavirus activity. Importantly, a clinically used mTOR inhibitor, rapamycin, potently inhibited both experimental and patient-derived rotavirus strains. This effect involves 4E-BP1 mediated induction of autophagy, which in turn exerts anti-rotavirus effects. These results revealed new insights on rotavirus-host interactions and provided new avenues for antiviral drug development.

Time- and cell-resolved dynamics of redox-sensitive Nrf2, HIF and NF-κB activities in 3D spheroids enriched for cancer stem cells

Cancer cells have an altered redox status, with changes in intracellular signaling pathways. The knowledge of how such processes are regulated in 3D spheroids, being well-established tumor models, is limited. To approach this question we stably transfected HCT116 cells with a pTRAF reporter that enabled time- and cell-resolved activity monitoring of three redox-regulated transcription factors Nrf2, HIF and NF-κB in spheroids enriched for cancer stem cells. At the first day of spheroid formation, these transcription factors were activated and thereafter became repressed. After about a week, both HIF and Nrf2 were reactivated within the spheroid cores. Further amplifying HIF activation in spheroids by treatment with DMOG resulted in a dominant quiescent stem-cell-like phenotype, with high resistance to stress-inducing treatments. Auranofin, triggering oxidative stress and Nrf2 activation, had opposite effects with increased differentiation and proliferation. These novel high-resolution insights into spatiotemporal activation patterns demonstrate a striking coordination of redox regulated transcription factors within spheroids not occurring in conventional cell culture models.

Development of a pneumatically driven active cover lid for multi-well microplates for use in perfusion three-dimensional cell culture.

Before microfluidic-based cell culture models can be practically utilized for bioassays, there is a need for a transitional cell culture technique that can improve conventional cell culture models. To address this, a hybrid cell culture system integrating an active cover lid and a multi-well microplate was proposed to achieve perfusion 3-D cell culture. In this system, a microfluidic-based pneumatically-driven liquid transport mechanism was integrated into the active cover lid to realize 6-unit culture medium perfusion. Experimental results revealed that the flow of culture medium could be pneumatically driven in a flow-rate uniform manner. We used the system to successfully perform a perfusion 3-D cell culture of mesenchymal stem cells (MSCs) for up to 16 days. Moreover, we investigated the effects of various cell culture models on the physiology of MSCs. The physiological nature of MSCs can vary with respect to the cell culture model used. Using the perfusion 3-D cell culture format might affect the proliferation and osteogenic differentiation of MSCs. Overall, we have developed a cell culture system that can achieve multi-well microplate-based perfusion 3-D cell culture in an efficient, cost-effective, and user-friendly manner. These features could facilitate the widespread application of perfusion cell culture models for cell-based assays.

An integrated microfluidic cell culture system for high-throughput perfusion three-dimensional cell culture-based assays: effect of cell culture model on the results of chemosensitivity assays

Although microfluidic cell culture systems are versatile tools for cellular assays, their use has yet to set in motion an evolutionary shift away from conventional cell culture methods. This situation is mainly due to technical hurdles: the operational barriers to the end-users, the lack of compatible detection schemes capable of reading out the results of a microfluidic-based cellular assay, and the lack of fundamental data to bridge the gap between microfluidic and conventional cell culture models. To address these issues, we propose a high-throughput, perfusion, three-dimensional (3-D) microfluidic cell culture system encompassing 30 microbioreactors. This integrated system not only aims to provide a user-friendly cell culture tool for biologists to perform assays but also to enable them to obtain precise data. Its technical features include (i) integration of a heater chip based on transparent indium tin oxide glass, providing stable thermal conditions for cell culturing; (ii) a microscale 3-D culture sample loading scheme that is both efficient and precise; (iii) a non-mechanical pneumatically driven multiplex medium perfusion mechanism; and (iv) a microplate reader-compatible waste medium collector array for the subsequent high throughput bioassays. In this study, we found that the 3-D culture sample loading method provided uniform sample loading [coefficient of variation (CV): 3.2%]. In addition, the multiplex medium perfusion mechanism led to reasonably uniform (CV: 3.6-6.9%) medium pumping rates in the 30 microchannels. Moreover, we used the proposed system to perform a successful cell culture-based chemosensitivity assay. To determine the effects of cell culture models on the cellular proliferation, and the results of chemosensitivity assays, we compared our data with that obtained using three conventional cell culture models. We found that the nature of the cell culture format could lead to different evaluation outcomes. Consequently, when establishing a cell culture model for in vitro cell-based assays, it might be necessary to investigate the fundamental physiological variations of the cultured cells in different culture systems to avoid any misinterpretation of data. As a whole, we have developed an integrated microfluidic cell culture system that overcomes several technical hurdles commonly encountered in the practical application of microfluidic cell culture systems, and we have obtained fundamental information to reconcile differences found with data acquired using conventional methods.

Cardiogenic Regulation of Stem-Cell Electrical Properties in a Laser-Patterned Biochip

Normal cardiomyocytes are highly dependent on the functional expression of ion channels to form action potentials and electrical coupling with other cells. To fully determine the scientific and therapeutic potential of stem cells for cardiovascular-disease treatment, it is necessary to assess comprehensively the regulation of stem-cell electrical properties during stem cell-cardiomyocyte interaction. It has been reported in the literature that contact with native cardiomyocytes induced and regulated stem-cell cardiogenic differentiation. However, in conventional cell-culture models, the importance of cell-cell contact for stem-cell functional coupling with cardiomyocytes has not been elucidated due to insufficient control of the cell-contact mode of individual cells. Using microfabrication and laser-guided cell micropatterning techniques, we created two biochips with contact-promotive and -preventive microenvironments to systematically study the effect of contact on cardiogenic regulation of stem-cell electrical properties. In contact-promotive biochips, connexin 43 expression was upregulated and relocated to the junction area between one stem cell and one cardiomyocyte. Only stem cells in contact with cardiomyocytes were induced by adjacent cardiomyocytes to acquire electrophysiological properties for action-potential formation similar to that of a cardiomyocyte.

Microengineered physiological biomimicry: Organs-on-Chips

Microscale engineering technologies provide unprecedented opportunities to create cell culture microenvironments that go beyond current three-dimensional in vitro models by recapitulating the critical tissue-tissue interfaces, spatiotemporal chemical gradients, and dynamic mechanical microenvironments of living organs. Here we review recent advances in this field made over the past two years that are focused on the development of 'Organs-on-Chips' in which living cells are cultured within microfluidic devices that have been microengineered to reconstitute tissue arrangements observed in living organs in order to study physiology in an organ-specific context and to develop specialized in vitro disease models. We discuss the potential of organs-on-chips as alternatives to conventional cell culture models and animal testing for pharmaceutical and toxicology applications. We also explore challenges that lie ahead if this field is to fulfil its promise to transform the future of drug development and chemical safety testing.

Developmental cell biology of human villous trophoblast: current research problems

The common gestational pathologies are placental and developmental in origin. However, much remains to be learned about the key events of morphogenesis and growth in the placenta. Metabolic settings established early in both the fetus and placenta define their capacity to respond to later challenges, as well as the quality of the response. Placental growth is exponential in the first trimester and involves coordinated events in trophoblast and mesenchyme, with early cell segregation events having a strong influence on growth potential. One of these is the differentiation of progenitor cytotrophoblasts into villous intermediate cells programmed to fuse with the syncytium, or, alternatively, into extravillous migratory cells that transform the maternal vascular supply. In the latter case, contact with decidual extracellular matrix stimulates differentiation, and therefore this decision is influenced by the number of contact sites at the placental periphery, which in turn is a function of branching in the villous tree. The villous trophoblast bilayer is the primary barrier between maternal and fetal tissues. The maternal-facing layer is syncytial and post-mitotic: this limits traffic of pathogens to the fetus and chimaerism arising by shedding of (non-proliferative) syncytial elements into maternal circulation. Conventional cell culture models fail to replicate this critical vectorial relationship. Tissue explants can overcome the problem to some extent, and have been used to show that turnover of trophoblast in the villous environment is regulated by signals from both fetal and maternal tissues. Maternally delivered insulin-like growth factors stimulate growth and might be therapeutically useful when endogenous growth pathways falter.