scholarly journals Culturing Keratinocytes on Biomimetic Substrates Facilitates Improved Epidermal Assembly In Vitro

Cells ◽  
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.

scholarly journals Current Advances in 3D Tissue and Organ Reconstruction

2021 ◽  
Vol 22 (2) ◽  
pp. 830
Author(s):  
Georgia Pennarossa ◽  
Sharon Arcuri ◽  
Teresina De Iorio ◽  
Fulvio Gandolfi ◽  
Tiziana A. L. Brevini
Keyword(s):  
Cell Biology ◽  
Drug Testing ◽  
Three Dimensional ◽  
3D Culture ◽  
In Vitro Models ◽  
The Body ◽  
Cellular Functions ◽  
Culture Systems

Bi-dimensional culture systems have represented the most used method to study cell biology outside the body for over a century. Although they convey useful information, such systems may lose tissue-specific architecture, biomechanical effectors, and biochemical cues deriving from the native extracellular matrix, with significant alterations in several cellular functions and processes. Notably, the introduction of three-dimensional (3D) platforms that are able to re-create in vitro the structures of the native tissue, have overcome some of these issues, since they better mimic the in vivo milieu and reduce the gap between the cell culture ambient and the tissue environment. 3D culture systems are currently used in a broad range of studies, from cancer and stem cell biology, to drug testing and discovery. Here, we describe the mechanisms used by cells to perceive and respond to biomechanical cues and the main signaling pathways involved. We provide an overall perspective of the most recent 3D technologies. Given the breadth of the subject, we concentrate on the use of hydrogels, bioreactors, 3D printing and bioprinting, nanofiber-based scaffolds, and preparation of a decellularized bio-matrix. In addition, we report the possibility to combine the use of 3D cultures with functionalized nanoparticles to obtain highly predictive in vitro models for use in the nanomedicine field.

scholarly journals Mechanobiology of Epithelia From the Perspective of Extracellular Matrix Heterogeneity

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.

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

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.

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 PI3Kδ Sustains Keratinocyte Hyperproliferation and Epithelial Inflammation: Implications for a Topically Druggable Target in Psoriasis

Cells ◽  
2021 ◽  
Vol 10 (10) ◽  
pp. 2636
Author(s):  
Laura Mercurio ◽  
Martina Morelli ◽  
Claudia Scarponi ◽  
Giovanni Luca Scaglione ◽  
Sabatino Pallotta ◽  
...  
Keyword(s):  
Skin Diseases ◽  
Inflammatory Responses ◽  
Basal Layer ◽  
Regulatory Subunit ◽  
Psoriatic Skin ◽  
Epidermal Keratinocytes ◽  
Epithelial Inflammation ◽  
The Impact

The phosphatidylinositol 3-kinase (PI3K)-dependent signaling pathway is aberrantly activated in psoriatic lesions and contributes to disease pathogenesis. Among PI3Ks enzymes, PI3Kα, β, and δ isoforms are known to bind the p85 regulatory subunit and mediate activation of AKT and other downstream effectors. In this study, we deepened our understanding of the expression and function of PI3Kδ in skin lesions of patients affected by psoriasis. For the first time, we found that PI3Kδ is overexpressed in psoriatic plaques, and its expression is not only confined to infiltrating immune cells but also accumulates in proliferating keratinocytes of the epidermal basal layer. We investigated the function of PI3Kδ in psoriatic skin by evaluating the impact of seletalisib, a newly developed selective PI3Kδ inhibitor, in both in vitro and in vivo experimental models of psoriasis. Of note, we found that PI3Kδ sustains keratinocyte hyperproliferation and impaired terminal differentiation induced by IL-22, as well as induces epithelial inflammation and resistance to apoptosis mediated by TNF-α in human keratinocytes. Mechanistically, PI3Kδ promotes PDK1 phosphorylation and signals through AKT-dependent or -independent pathways. It is worth mentioning that PI3Kδ inhibition by seletalisib attenuates the severity of psoriasiform phenotype induced in the Imiquimod-induced mouse model of psoriasis by restoring the physiological proliferation and differentiation programs in epidermal keratinocytes and contrasting the cutaneous inflammatory responses. Therefore, we suggest PI3Kδ as a potential topically druggable target in psoriasis and skin diseases characterized by epidermal hyperproliferation and skin inflammation.

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.

The impact of confocal microscopy in biomedical research

1992 ◽  
Vol 50 (2) ◽  
pp. 1158-1159
Author(s):  
William B. Amos
Keyword(s):  
Cell Biology ◽  
Three Dimensional ◽  
Optical Microscope ◽  
Ion Concentration ◽  
Reaction Products ◽  
Embryonic Life ◽  
The Many ◽  
Calcium Ion Concentration ◽  
The Impact

The confocal optical microscope, using laser illumination, has now gained widespread acceptance (see volume edited by Pawley) Its advantage in providing clear optical sections, particularly with fluorescent specimens, is well known. Of the many confocal instruments now in use in cell biology, the applications can be classified into five different categories.The chief use is to give three-dimensional information about conventionally prepared fluorescent specimens. A notable example is the in vivo mapping of an identified neurone through several days of embryonic life by O'Rourke, Scott Fraser and colleagues at Irvine, USA. There has also been much work on in situ hybridisation, morphometry of solid tumours, oncogene product localisation and many aspects of the cytoskeleton.The second use has been in reflection imaging of cell surface contacts, of isolated microtubules and microorganisms, of parts of the eye and of reaction products such as peroxidase.The third application is the measurement of intracellular parameters such as pH and calcium ion concentration within a defined volume.

scholarly journals The Impact of a Single Phosphorylation Site Mutation in the Glucocorticoid Receptor on the Molecular and Cellular Development of the Cerebral Cortex

2021 ◽  
Vol 5 (Supplement_1) ◽  
pp. A818-A819
Author(s):  
Herschel Raj Gupta ◽  
Prathi Pitchyaiah ◽  
Neerupma Silswal ◽  
Suban Burale ◽  
Joseph Bean ◽  
...  
Keyword(s):  
Glucocorticoid Receptor ◽  
Cell Biology ◽  
Cellular Proliferation ◽  
Phosphorylation Site ◽  
Psychiatric Disease ◽  
Site Mutation ◽  
The Impact

Abstract Premature birth leads to a significant increase in adverse clinical outcomes, including Respiratory Distress Syndrome, Bronchopulmonary Dysplasia, Necrotizing Enterocolitis and Intraventricular Hemorrhage. Synthetic Glucocorticoids (sGC) are administered prenatally to pregnant mothers at risk to reduce the chance of these complications. However, there is a correlation between long-term neurological defects in the infant and the clinical use of sGC prenatally. The use of the sGCs have been linked to the development of cerebral palsy and deficits in attention and concentration. To investigate the cellular basis of these abnormalities, we examined the consequences of sGC administration of the developing murine brain. Our studies demonstrated that premature exposure to sGC alters neural stem cell biology and has long term consequences for adult behavior in mice. In humans, site-specific phosphorylation of the Glucocorticoid Receptor (GR) on Serine 211 versus Serine 226 is associated with activated or repressed transcriptional states and clinical studies indicate that the ratio of S220/S226 phosphorylation is associated with increased predisposition to specific psychiatric disease states, including Major Depressive Disorder and Bipolar Disorder. To examine the role of these phosphorylation sites in the development of behavioral abnormalities, we utilized a knock-in mouse model where Serine 220 (equivalent to human Serine 211) was replaced with an alanine (S220A). In-vitro microarray analysis of neural stem cells and QPCR validation were performed to examine the expression changes in individual transcripts in critical pathways that may correlate with long-term neurologic disorders. Our results indicated that changing the phosphorylation status of GR alters the expression of 2570 genes. Ingenuity Pathway Analysis indicated that the major pathways altered include those involved in cellular proliferation, mitochondrial function, Valine degradation and G-coupled protein receptors involved in neurotransmission. Both in-vitro and in-vivo experiments indicated that the S220A mutation alters the cells response to sGC administration by impacting proliferation and differentiation. The long-term goal of these experiments was to demonstrate a role for S220 phosphorylation in the development of neuropsychiatric disorders.

scholarly journals Compartmentalized three-dimensional human neuromuscular tissue models fabricated on a well-plate-format microdevice

2021 ◽  
Author(s):  
Kazuki Yamamoto ◽  
Nao Yamaoka ◽  
Yu Imaizumi ◽  
Takunori Nagashima ◽  
Taiki Furutani ◽  
...  
Keyword(s):  
Motor Neurons ◽  
Neuromuscular Junctions ◽  
Neuromuscular Disorders ◽  
Three Dimensional ◽  
Induced Pluripotent Stem Cell ◽  
Motor Units ◽  
Mechanical Stimuli ◽  
Tissue Models

Engineered three-dimensional models of neuromuscular tissues are promising for use in mimicking their disorder states in vitro. Although several models have been developed, it is still challenging to mimic the physically separated structures of motor neurons (MNs) and skeletal muscle (SkM) fibers in the motor units in vivo. In this study, we aimed to develop microdevices for precisely compartmentalized coculturing of MNs and engineered SkM tissues. The developed microdevices, which fit a well of 24 well plates, had a chamber for MNs and chamber for SkM tissues. The two chambers were connected by microtunnels for axons, permissive to axons but not to cell bodies. The axons from human-induced-pluripotent-stem-cell-derived MN spheroids in the MN chamber elongated in microtunnels, reached the tissue-engineered human SkM in the SkM chamber, and formed functional neuromuscular junctions with the muscle fibers. The cocultured SkM tissues with MNs on the device contracted spontaneously in response to spontaneous firing of MNs. The addition of a neurotransmitter, glutamate, into the MN chamber induced contraction of the cocultured SkM tissues. Selective addition of tetrodotoxin or vecuronium bromide into either chamber induced SkM tissue relaxation, which could be explained by the inhibitory mechanisms. We also demonstrated the application of chemical or mechanical stimuli to the middle of the axons of cocultured tissues on the device. Thus, compartmentalized neuromuscular tissue models fabricated on the device could be used for phenotypic screening to evaluate the cellular type specific efficacy of drug candidates and would be a useful tool in fundamental research and drug development for neuromuscular disorders.

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

2002 ◽  
Vol 30 (6) ◽  
pp. 1175-1180 ◽  
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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