scholarly journals Single-cell transcriptomics elucidates in vitro reprogramming of human intestinal epithelium cultured in a physiodynamic gut-on-a-chip

2021 ◽  
Author(s):  
Woojung Shin ◽  
Zhe Su ◽  
S. Stephen Yi ◽  
Hyun Jung Kim
Keyword(s):  
Single Cell ◽  
Intestinal Epithelium ◽  
Drug Transport ◽  
Three Dimensional ◽  
Cancer Cell Line ◽  
Static Condition ◽  
On Chip ◽  
Epithelial Layers

The microphysiological human gut-on-a-chip has demonstrated in vivo-relevant cellular fidelity of intestinal epithelium compared to its cultures in a static condition. Microfluidic control of morphogen gradients and mechanical cues robustly induced morphological histogenesis with villi-like three-dimensional (3D) microarchitecture, lineage-associated cytodifferentiation, and physiological functions of a human intestinal Caco-2 epithelium. However, transcriptomic dynamics that orchestrates morphological and functional reprogramming of the epithelium in a microphysiological culture remains elusive. Single-cell transcriptomic analysis revealed that a gut-on-a-chip culture that offers physiological motions and flow drives three distinctive subclusters that offer distinct gene expression and unique spatial representation in 3D epithelial layers. The pseudotemporal trajectory of individual cells visualized the evolutionary transition from ancestral genotypes in static cultures into more heterogeneous phenotypes in physiodynamic cultures on cell cycles, differentiation, and intestinal functions including digestion, absorption, drug transport, and metabolism of xenobiotics. Furthermore, the inversed transcriptomic signature of oncogenes and tumor-suppressor genes of Caco-2 cells verified that a gut-on-a-chip culture drives a postmitotic reprogramming of cancer-associated phenotypes. Thus, we discovered that a physiodynamic on-chip culture is necessary and sufficient for a cancer cell line to be reprogrammed to elicit in vivo-relevant heterogeneous cell populations with restored normal physiological signatures.

scholarly journals Engineered Heterochronic Parabiosis in 3D Microphysiological System for Identification of Muscle Rejuvenating Factors

2020 ◽  
Author(s):  
Yunki Lee ◽  
Jeongmoon J. Choi ◽  
Song Ih Ahn ◽  
Nan Hee Leea ◽  
Woojin M. Han ◽  
...  
Keyword(s):  
Stem Cell ◽  
Cell Function ◽  
Three Dimensional ◽  
Muscle Stem Cell ◽  
Organ Systems ◽  
Systemic Factors ◽  
Underlying Mechanisms ◽  
On Chip

AbstractExposure of aged mice to a young systemic milieu revealed remarkable rejuvenation effects on aged tissues, including skeletal muscle. Although some candidate factors have been identified, the exact identity and the underlying mechanisms of putative rejuvenating factors remain elusive, mainly due to the complexity of in vivo parabiosis. Here, we present an in vitro muscle parabiosis system that integrates young- and old-muscle stem cell vascular niche on a three-dimensional microfluidic platform designed to recapitulate key features of native muscle stem cell microenvironment. This innovative system enables mechanistic studies of cellular dynamics and molecular interactions within the muscle stem cell niche, especially in response to conditional extrinsic stimuli of local and systemic factors. We demonstrate that vascular endothelial growth factor (VEGF) signaling from endothelial cells and myotubes synergistically contribute to the rejuvenation of the aged muscle stem cell function. Moreover, with the adjustable on-chip system, we can mimic both blood transfusion and parabiosis and detect the time-varying effects of anti-geronic and pro-geronic factors in a single organ or multi-organ systems. Our unique approach presents a complementary in vitro model to supplement in vivo parabiosis for identifying potential anti-geronic factors responsible for revitalizing aging organs.

scholarly journals Non-Invasive Quantification of the Growth of Cancer Cell Colonies by a Portable Optical Coherence Tomography

Micromachines ◽  
2019 ◽  
Vol 10 (1) ◽  
pp. 35 ◽  
Author(s):  
Meng-Tsan Tsai ◽  
Bo-Huei Huang ◽  
Chun-Chih Yeh ◽  
Kin Fong Lei ◽  
Ngan-Ming Tsang
Keyword(s):  
Optical Coherence Tomography ◽  
Cancer Cell ◽  
Cellular Response ◽  
Tumor Development ◽  
Three Dimensional ◽  
Cancer Cell Line ◽  
Optical Coherence ◽  
Non Invasive

Investigation of tumor development is essential in cancer research. In the laboratory, living cell culture is a standard bio-technology for studying cellular response under tested conditions to predict in vivo cellular response. In particular, the colony formation assay has become a standard experiment for characterizing the tumor development in vitro. However, quantification of the growth of cell colonies under a microscope is difficult because they are suspended in a three-dimensional environment. Thus, optical coherence tomography (OCT) imaging was develop in this study to monitor the growth of cell colonies. Cancer cell line of Huh 7 was used and the cells were applied on a layer of agarose hydrogel, i.e., a non-adherent surface. Then, cell colonies were gradually formed on the surface. The OCT technique was used to scan the cell colonies every day to obtain quantitative data for describing their growth. The results revealed the average volume increased with time due to the formation of cell colonies day-by-day. Additionally, the distribution of cell colony volume was analyzed to show the detailed information of the growth of the cell colonies. In summary, the OCT provides a non-invasive quantification technique for monitoring the growth of the cell colonies. From the OCT images, objective and precise information is obtained for higher prediction of the in vivo tumor development.

scholarly journals Multiscale modelling of drug transport and metabolism in liver spheroids

2020 ◽  
Vol 10 (2) ◽  
pp. 20190041 ◽  
Author(s):  
Joseph A. Leedale ◽  
Jonathan A. Kyffin ◽  
Amy L. Harding ◽  
Helen E. Colley ◽  
Craig Murdoch ◽  
...  
Keyword(s):  
Drug Transport ◽  
Temporal Dynamics ◽  
Three Dimensional ◽  
Culture Conditions ◽  
Isolated Cells ◽  
Modelling Framework ◽  
The Impact ◽  
Cells Cultured

In early preclinical drug development, potential candidates are tested in the laboratory using isolated cells. These in vitro experiments traditionally involve cells cultured in a two-dimensional monolayer environment. However, cells cultured in three-dimensional spheroid systems have been shown to more closely resemble the functionality and morphology of cells in vivo . While the increasing usage of hepatic spheroid cultures allows for more relevant experimentation in a more realistic biological environment, the underlying physical processes of drug transport, uptake and metabolism contributing to the spatial distribution of drugs in these spheroids remain poorly understood. The development of a multiscale mathematical modelling framework describing the spatio-temporal dynamics of drugs in multicellular environments enables mechanistic insight into the behaviour of these systems. Here, our analysis of cell membrane permeation and porosity throughout the spheroid reveals the impact of these properties on drug penetration, with maximal disparity between zonal metabolism rates occurring for drugs of intermediate lipophilicity. Our research shows how mathematical models can be used to simulate the activity and transport of drugs in hepatic spheroids and in principle any organoid, with the ultimate aim of better informing experimentalists on how to regulate dosing and culture conditions to more effectively optimize drug delivery.

scholarly journals Bioengineering a Miniaturized In Vitro 3D Myotube Contraction Monitoring Chip For Modelization of Muscular Dystrophies

2021 ◽  
Author(s):  
Nicolas Rose ◽  
Surabhi Sonam ◽  
Thao Nguyen ◽  
Gianluca Grenci ◽  
Anne Bigot ◽  
...  
Keyword(s):  
Three Dimensional ◽  
Congenital Muscular Dystrophy ◽  
Muscular Disorders ◽  
Adsorption Technology ◽  
Therapeutic Procedures ◽  
On Chip ◽  
Physical And Chemical ◽  
The Impact

Quantification of skeletal muscle functional strength is essential to assess the outcomes of therapeutic procedures for muscular disorders. Several muscle three-dimensional Organ-on-chip models have been developed to measure the generated force. Yet, these technologies require a substantial amount of biological material, which is problematic in the context of limited patient sample. Here we developed a miniaturized 3D myotube culture chip with contraction monitoring capacity. Combination of light-induced molecular adsorption technology and optimized micropatterned substrate design enabled to obtain high culture yields in tightly controlled physical and chemical microenvironments. Spontaneous twitch contractions in 3D myotubes derived from primary human myoblasts were observed, the generated force was measured and the contraction pattern characterized. In addition, the impact of three-dimensional culture on nuclear morphology was analyzed, confirming the similarity in organization between the obtained 3D myotubes and in vivo myofibers. Our system enabled to model LMNA-related Congenital Muscular Dystrophy (L-CMD) with successful development of mutant 3D myotubes displaying contractile dysfunction. We anticipate that this technology shall be used to study contraction characteristics and evaluate how specific diseases affect muscle organization and force generation. Our downsized model system might allow to substantially improve drug screening capability for therapeutic oriented research.

scholarly journals Three-dimensional drug screen identifies HDAC inhibitors as therapeutic agents in mTORC1-driven lymphangioleiomyomatosis

2021 ◽  
Author(s):  
Adam Pietrobon ◽  
Julien Yockell-Lelièvre ◽  
Nicole Melong ◽  
Laura J. Smith ◽  
Sean P. Delaney ◽  
...  
Keyword(s):  
Single Cell ◽  
Hdac Inhibitors ◽  
Three Dimensional ◽  
Drug Screen ◽  
Single Cell Level ◽  
Loss Of Function ◽  
Cell Level ◽  
Mtorc1 Signaling

Lymphangioleiomyomatosis (LAM) is a rare disease involving cystic lung destruction by invasive LAM cells. These cells harbor loss-of-function mutations in TSC2, conferring constitutive mTORC1 signaling. Rapamycin is the only clinically approved disease-modifying treatment, but its action is cytostatic and disease progresses upon its withdrawal. There is a critical need to identify novel agents that prevent the invasive phenotype and/or eradicate the neoplastic LAM cells. Here, we employed novel cellular and extracellular models to screen for candidate therapeutics in a physiologically relevant setting. We observed that lung-mimetic hydrogel culture of pluripotent stem cell-derived diseased cells more faithfully recapitulates human LAM biology compared to conventional culture on two-dimensional tissue culture plastic. Leveraging our culture system, we conducted a three-dimensional drug screen using a custom 800-compound library, tracking cytotoxicity and invasion modulation phenotypes at the single cell level. We identified histone deacetylase (HDAC) inhibitors as a group of anti-invasive agents that are also selectively cytotoxic towards TSC2-/- cells. Unexpectedly, we observed that next generation ATP-competitive mTORC1/2 inhibitors potentiate invasion. We determined anti-invasive effects of HDAC inhibitors to be independent of genotype, while selective cell death is mTORC1-dependent and mediated by apoptosis. Drug performance was subsequently evaluated at the single cell level in zebrafish xenografts. We observed consistent therapeutic efficacy in vivo at equivalent concentrations to those used in vitro, substantiating HDAC inhibitors as potential therapeutic candidates for pursuit in patients with LAM.

In vitro and in vivo polysaccharides assembly: ultrastructural and cytochemical study of growing plant cell wall components.

1978 ◽  
Vol 36 (2) ◽  
pp. 434-435
Author(s):  
D. Reis ◽  
B. Vian ◽  
J. C. Roland
Keyword(s):  
Cell Wall ◽  
Self Assembly ◽  
Plant Cell Wall ◽  
Higher Plants ◽  
Three Dimensional ◽  
Cytochemical Study ◽  
Wall Components

Wall morphogenesis in higher plants is a problem still open to controversy. Until now the possibility of a transmembrane control and the involvement of microtubules were mostly envisaged. Self-assembly processes have been observed in the case of walls of Chlamydomonas and bacteria. Spontaneous gelling interactions between xanthan and galactomannan from Ceratonia have been analyzed very recently. The present work provides indications that some processes of spontaneous aggregation could occur in higher plants during the formation and expansion of cell wall.Observations were performed on hypocotyl of mung bean (Phaseolus aureus) for which growth characteristics and wall composition have been previously defined.In situ, the walls of actively growing cells (primary walls) show an ordered three-dimensional organization (fig. 1). The wall is typically polylamellate with multifibrillar layers alternately transverse and longitudinal. Between these layers intermediate strata exist in which the orientation of microfibrils progressively rotates. Thus a progressive change in the morphogenetic activity occurs.

Epithelial Organotypic Cultures: A Viable Model to Address Mechanisms of Carcinogenesis by Epitheliotropic Viruses

2018 ◽  
Vol 18 (4) ◽  
pp. 246-255 ◽  
Author(s):  
Lara Termini ◽  
Enrique Boccardo
Keyword(s):  
Three Dimensional ◽  
Cell Types ◽  
Experimental Models ◽  
Biological Research ◽  
Specific Gene ◽  
Specific Cell ◽  
Organotypic Cultures ◽  
Skin Physiology

In vitro culture of primary or established cell lines is one of the leading techniques in many areas of basic biological research. The use of pure or highly enriched cultures of specific cell types obtained from different tissues and genetics backgrounds has greatly contributed to our current understanding of normal and pathological cellular processes. Cells in culture are easily propagated generating an almost endless source of material for experimentation. Besides, they can be manipulated to achieve gene silencing, gene overexpression and genome editing turning possible the dissection of specific gene functions and signaling pathways. However, monolayer and suspension cultures of cells do not reproduce the cell type diversity, cell-cell contacts, cell-matrix interactions and differentiation pathways typical of the three-dimensional environment of tissues and organs from where they were originated. Therefore, different experimental animal models have been developed and applied to address these and other complex issues in vivo. However, these systems are costly and time consuming. Most importantly the use of animals in scientific research poses moral and ethical concerns facing a steadily increasing opposition from different sectors of the society. Therefore, there is an urgent need for the development of alternative in vitro experimental models that accurately reproduce the events observed in vivo to reduce the use of animals. Organotypic cultures combine the flexibility of traditional culture systems with the possibility of culturing different cell types in a 3D environment that reproduces both the structure and the physiology of the parental organ. Here we present a summarized description of the use of epithelial organotypic for the study of skin physiology, human papillomavirus biology and associated tumorigenesis.

A Novel Triazole Derivative Drug Presenting In Vitro and In Vivo Anticancer Properties

2018 ◽  
Vol 18 (17) ◽  
pp. 1483-1493
Author(s):  
Ricardo Imbroisi Filho ◽  
Daniel T.G. Gonzaga ◽  
Thainá M. Demaria ◽  
João G.B. Leandro ◽  
Dora C.S. Costa ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cancer Cells ◽  
Cell Line ◽  
Cancer Cell ◽  
Cancer Cell Line ◽  
Hepatic Function ◽  
Anticancer Properties ◽  
Mcf 7

Background: Cancer is a major cause of death worldwide, despite many different drugs available to treat the disease. This high mortality rate is largely due to the complexity of the disease, which results from several genetic and epigenetic changes. Therefore, researchers are constantly searching for novel drugs that can target different and multiple aspects of cancer. Experimental: After a screening, we selected one novel molecule, out of ninety-four triazole derivatives, that strongly affects the viability and proliferation of the human breast cancer cell line MCF-7, with minimal effects on non-cancer cells. The drug, named DAN94, induced a dose-dependent decrease in MCF-7 cells viability, with an IC50 of 3.2 ± 0.2 µM. Additionally, DAN94 interfered with mitochondria metabolism promoting reactive oxygen species production, triggering apoptosis and arresting the cancer cells on G1/G0 phase of cell cycle, inhibiting cell proliferation. These effects are not observed when the drug was tested in the non-cancer cell line MCF10A. Using a mouse model with xenograft tumor implants, the drug preventing tumor growth presented no toxicity for the animal and without altering biochemical markers of hepatic function. Results and Conclusion: The novel drug DAN94 is selective for cancer cells, targeting the mitochondrial metabolism, which culminates in the cancer cell death. In the end, DAN94 has been shown to be a promising drug for controlling breast cancer with minimal undesirable effects.

scholarly journals The Revolutionary Roads to Study Cell–Cell Interactions in 3D In Vitro Pancreatic Cancer Models

Cancers ◽  
2021 ◽  
Vol 13 (4) ◽  
pp. 930
Author(s):  
Donatella Delle Cave ◽  
Riccardo Rizzo ◽  
Bruno Sainz ◽  
Giuseppe Gigli ◽  
Loretta L. del Mercato ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
Therapeutic Response ◽  
Three Dimensional ◽  
Cellular Models ◽  
Common Cancer ◽  
Cancer Models ◽  
Anti Cancer ◽  
Tumor Environment

Pancreatic cancer, the fourth most common cancer worldwide, shows a highly unsuccessful therapeutic response. In the last 10 years, neither important advancements nor new therapeutic strategies have significantly impacted patient survival, highlighting the need to pursue new avenues for drug development discovery and design. Advanced cellular models, resembling as much as possible the original in vivo tumor environment, may be more successful in predicting the efficacy of future anti-cancer candidates in clinical trials. In this review, we discuss novel bioengineered platforms for anticancer drug discovery in pancreatic cancer, from traditional two-dimensional models to innovative three-dimensional ones.

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