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

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.

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

10.1101/2020.03.10.975482 ◽

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.

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Mechanobiology of Epithelia From the Perspective of Extracellular Matrix Heterogeneity

Frontiers in Bioengineering and Biotechnology ◽

10.3389/fbioe.2020.596599 ◽

2020 ◽

Vol 8 ◽

Author(s):

Aleksandra N. Kozyrina ◽

Teodora Piskova ◽

Jacopo Di Russo

Keyword(s):

Extracellular Matrix ◽

Three Dimensional ◽

Cell Sheets ◽

Ecm Remodeling ◽

Cellular Behavior ◽

New Concepts ◽

Matrix Heterogeneity ◽

The Impact

Understanding the complexity of the extracellular matrix (ECM) and its variability is a necessary step on the way to engineering functional (bio)materials that serve their respective purposes while relying on cell adhesion. Upon adhesion, cells receive messages which contain both biochemical and mechanical information. The main focus of mechanobiology lies in investigating the role of this mechanical coordination in regulating cellular behavior. In recent years, this focus has been additionally shifted toward cell collectives and the understanding of their behavior as a whole mechanical continuum. Collective cell phenomena very much apply to epithelia which are either simple cell-sheets or more complex three-dimensional structures. Researchers have been mostly using the organization of monolayers to observe their collective behavior in well-defined experimental setups in vitro. Nevertheless, recent studies have also reported the impact of ECM remodeling on epithelial morphogenesis in vivo. These new concepts, combined with the knowledge of ECM biochemical complexity are of key importance for engineering new interactive materials to support both epithelial remodeling and homeostasis. In this review, we summarize the structure and heterogeneity of the ECM before discussing its impact on the epithelial mechanobiology.

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

10.1101/2021.09.01.458444 ◽

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.

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The Impact of the Extracellular Matrix Environment on Sost Expression by the MLO-Y4 Osteocyte Cell Line

Bioengineering ◽

10.3390/bioengineering9010035 ◽

2022 ◽

Vol 9 (1) ◽

pp. 35

Author(s):

Robert T. Brady ◽

Fergal J. O’Brien ◽

David A. Hoey

Keyword(s):

Extracellular Matrix ◽

Cell Line ◽

Three Dimensional ◽

3D Culture ◽

Specific Gene ◽

Collagen Scaffolds ◽

Sost Gene ◽

The Impact

Bone is a dynamic organ that can adapt its structure to meet the demands of its biochemical and biophysical environment. Osteocytes form a sensory network throughout the tissue and orchestrate tissue adaptation via the release of soluble factors such as a sclerostin. Osteocyte physiology has traditionally been challenging to investigate due to the uniquely mineralized extracellular matrix (ECM) of bone leading to the development of osteocyte cell lines. Importantly, the most widely researched and utilized osteocyte cell line: the MLO-Y4, is limited by its inability to express sclerostin (Sost gene) in typical in-vitro culture. We theorised that culture in an environment closer to the in vivo osteocyte environment could impact on Sost expression. Therefore, this study investigated the role of composition and dimensionality in directing Sost expression in MLO-Y4 cells using collagen-based ECM analogues. A significant outcome of this study is that MLO-Y4 cells, when cultured on a hydroxyapatite (HA)-containing two-dimensional (2D) film analogue, expressed Sost. Moreover, three-dimensional (3D) culture within HA-containing collagen scaffolds significantly enhanced Sost expression, demonstrating the impact of ECM composition and dimensionality on MLO-Y4 behaviour. Importantly, in this bone mimetic ECM environment, Sost expression was found to be comparable to physiological levels. Lastly, MLO-Y4 cells cultured in these novel conditions responded accordingly to fluid flow stimulation with a decrease in expression. This study therefore presents a novel culture system for the MLO-Y4 osteocyte cell line, ensuring the expression of an important osteocyte specific gene, Sost, overcoming a major limitation of this model.

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Multiscale modelling of drug transport and metabolism in liver spheroids

Interface Focus ◽

10.1098/rsfs.2019.0041 ◽

2020 ◽

Vol 10 (2) ◽

pp. 20190041 ◽

Cited By ~ 3

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.

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Culturing Keratinocytes on Biomimetic Substrates Facilitates Improved Epidermal Assembly In Vitro

Cells ◽

10.3390/cells10051177 ◽

2021 ◽

Vol 10 (5) ◽

pp. 1177

Author(s):

Eve Hunter-Featherstone ◽

Natalie Young ◽

Kathryn Chamberlain ◽

Pablo Cubillas ◽

Ben Hulette ◽

...

Keyword(s):

Cell Biology ◽

Three Dimensional ◽

Epidermal Keratinocytes ◽

Mechanical Stimuli ◽

Linc Complex ◽

Mechanical Conditioning ◽

Keratinocyte Cell ◽

The Impact

Mechanotransduction is defined as the ability of cells to sense mechanical stimuli from their surroundings and translate them into biochemical signals. Epidermal keratinocytes respond to mechanical cues by altering their proliferation, migration, and differentiation. In vitro cell culture, however, utilises tissue culture plastic, which is significantly stiffer than the in vivo environment. Current epidermal models fail to consider the effects of culturing keratinocytes on plastic prior to setting up three-dimensional cultures, so the impact of this non-physiological exposure on epidermal assembly is largely overlooked. In this study, primary keratinocytes cultured on plastic were compared with those grown on 4, 8, and 50 kPa stiff biomimetic hydrogels that have similar mechanical properties to skin. Our data show that keratinocytes cultured on biomimetic hydrogels exhibited major changes in cellular architecture, cell density, nuclear biomechanics, and mechanoprotein expression, such as specific Linker of Nucleoskeleton and Cytoskeleton (LINC) complex constituents. Mechanical conditioning of keratinocytes on 50 kPa biomimetic hydrogels improved the thickness and organisation of 3D epidermal models. In summary, the current study demonstrates that the effects of extracellular mechanics on keratinocyte cell biology are significant and therefore should be harnessed in skin research to ensure the successful production of physiologically relevant skin models.

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Folding of the td pre-RNA with the help of the RNA chaperone StpA

Biochemical Society Transactions ◽

10.1042/bst0301175 ◽

2002 ◽

Vol 30 (6) ◽

pp. 1175-1180 ◽

Cited By ~ 11

Author(s):

O. Mayer ◽

C. Waldsich ◽

R. Grossberger ◽

R. Schroeder

Keyword(s):

Conformational Changes ◽

Three Dimensional ◽

Group I Intron ◽

Dimensional Structure ◽

Rna Chaperone ◽

Three Dimensional Structure ◽

Group I ◽

The Impact

The td group I intron is inserted in the reading frame of the thymidylate synthase gene, which is mainly devoid of structural elements. In vivo, translation of the pre-mRNA is required for efficient folding of the intron into its splicing-competent structure. The ribosome probably resolves exon-intron interactions that interfere with splicing. Uncoupling splicing from translation, by introducing a non-sense codon into the upstream exon, reduces the splicing efficiency of the mutant pre-mRNA. Alternatively to the ribosome, co-expression of genes that encode proteins with RNA chaperone activity promote folding of the td pre-mRNA in vivo. These proteins also efficiently accelerate folding of the td pre-mRNA in vitro. In order to understand the mechanism of action of RNA chaperones, we probed the impact of the RNA chaperone StpA on the structure of the td intron in vivo and compared it with that of the well characterized Cyt-18 protein, which is a group-I-intron-specific splicing factor. We found that the two proteins have opposite effects on the structure of the td intron. While StpA loosens the three-dimensional structure, Cyt-18 tightens it up. Furthermore, mutations that destabilize the intron structure render the mutants sensitive to StpA, whereas splicing of these mutants is rescued by Cyt-18. Our results provide direct evidence for protein-induced conformational changes within a catalytic RNA in vivo. Whereas StpA resolves tertiary contacts enabling the RNA to refold, Cyt-18 contributes to the stabilization of the native three-dimensional structure.

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

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100083035 ◽

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.

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

Current Topics in Medicinal Chemistry ◽

10.2174/1568026618666180410125850 ◽

2018 ◽

Vol 18 (4) ◽

pp. 246-255 ◽

Cited By ~ 1

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.

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