scholarly journals PerfuPul—A Versatile Perfusable Platform to Assess Permeability and Barrier Function of Air Exposed Pulmonary Epithelia

2021 ◽  
Vol 9 ◽  
Author(s):  
Patrick Carius ◽  
Aurélie Dubois ◽  
Morvarid Ajdarirad ◽  
Arbel Artzy-Schnirman ◽  
Josué Sznitman ◽  
...  
Keyword(s):  
Aerosol Deposition ◽  
Cell Types ◽  
Specific Cell ◽  
Fluorescein Sodium ◽  
Drug Products ◽  
Orally Inhaled Drug Products ◽  
Specialized Equipment ◽  
Static Conditions ◽  
Cells Cultured

Complex in vitro models, especially those based on human cells and tissues, may successfully reduce or even replace animal models within pre-clinical development of orally inhaled drug products. Microfluidic lung-on-chips are regarded as especially promising models since they allow the culture of lung specific cell types under physiological stimuli including perfusion and air-liquid interface (ALI) conditions within a precisely controlled in vitro environment. Currently, though, such models are not available to a broad user community given their need for sophisticated microfabrication techniques. They further require systematic comparison to well-based filter supports, in analogy to traditional Transwells®. We here present a versatile perfusable platform that combines the advantages of well-based filter supports with the benefits of perfusion, to assess barrier permeability of and aerosol deposition on ALI cultured pulmonary epithelial cells. The platform as well as the required technical accessories can be reproduced via a detailed step-by-step protocol and implemented in typical bio-/pharmaceutical laboratories without specific expertise in microfabrication methods nor the need to buy costly specialized equipment. Calu-3 cells cultured under liquid covered conditions (LCC) inside the platform showed similar development of transepithelial electrical resistance (TEER) over a period of 14 days as cells cultured on a traditional Transwell®. By using a customized deposition chamber, fluorescein sodium was nebulized via a clinically relevant Aerogen® Solo nebulizer onto Calu-3 cells cultured under ALI conditions within the platform. This not only allowed to analyze the transport of fluorescein sodium after ALI deposition under perfusion, but also to compare it to transport under traditional static conditions.

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.

scholarly journals Rho GTPases as Key Molecular Players within Intestinal Mucosa and GI Diseases

Cells ◽  
2021 ◽  
Vol 10 (1) ◽  
pp. 66
Author(s):  
Rashmita Pradhan ◽  
Phuong A. Ngo ◽  
Luz d. C. Martínez-Sánchez ◽  
Markus F. Neurath ◽  
Rocío López-Posadas
Keyword(s):  
Intestinal Mucosa ◽  
Rho Gtpases ◽  
Current Knowledge ◽  
Cell Types ◽  
Effector Proteins ◽  
Special Focus ◽  
Specific Cell ◽  
Rho Proteins

Rho proteins operate as key regulators of the cytoskeleton, cell morphology and trafficking. Acting as molecular switches, the function of Rho GTPases is determined by guanosine triphosphate (GTP)/guanosine diphosphate (GDP) exchange and their lipidation via prenylation, allowing their binding to cellular membranes and the interaction with downstream effector proteins in close proximity to the membrane. A plethora of in vitro studies demonstrate the indispensable function of Rho proteins for cytoskeleton dynamics within different cell types. However, only in the last decades we have got access to genetically modified mouse models to decipher the intricate regulation between members of the Rho family within specific cell types in the complex in vivo situation. Translationally, alterations of the expression and/or function of Rho GTPases have been associated with several pathological conditions, such as inflammation and cancer. In the context of the GI tract, the continuous crosstalk between the host and the intestinal microbiota requires a tight regulation of the complex interaction between cellular components within the intestinal tissue. Recent studies demonstrate that Rho GTPases play important roles for the maintenance of tissue homeostasis in the gut. We will summarize the current knowledge on Rho protein function within individual cell types in the intestinal mucosa in vivo, with special focus on intestinal epithelial cells and T cells.

Differential susceptibilities of isolated hamster lung cell types to amiodarone toxicity

1998 ◽  
Vol 76 (7-8) ◽  
pp. 721-727 ◽  
Author(s):  
M W Bolt ◽  
W J Racz ◽  
J F Brien ◽  
T M Bray ◽  
T E Massey
Keyword(s):  
Alveolar Macrophages ◽  
Gradient Centrifugation ◽  
Cell Types ◽  
Clara Cell ◽  
Blue Dye ◽  
Alveolar Type ◽  
Specific Cell ◽  
Type Ii ◽  
Clara Cells

Treatment of cardiac dysrhythmias with the iodinated benzofuran derivative amiodarone (AM) is limited by pulmonary toxicity. The susceptibilities of different lung cell types of male Golden Syrian hamsters to AM-induced cytotoxicity were investigated in vitro. Bronchoalveolar lavage and protease digestion to release cells, followed by centrifugal elutriation and density gradient centrifugation, resulted in preparations enriched with alveolar macrophages (98%), alveolar type II cells (75-85%), and nonciliated bronchiolar epithelial (Clara) cells (35-50%). Alveolar type II cell and Clara cell preparations demonstrated decreased viability (by 0.5% trypan blue dye exclusion) when incubated with 50 µM AM for 36 h, and all AM-treated cell preparations demonstrated decreased viability when incubated with 100 or 200 µM AM. Based on a viability index ((viability of AM-treated cells ÷ viability of controls) × 100%), the Clara cell fraction was significantly (p < 0.05) more susceptible than all of the other cell types to 50 µM AM. However, AM cytotoxicity was greatest (p < 0.05) in alveolar macrophages following incubation with 100 or 200 µM AM. There was no difference between any of the enriched cell preparations in the amount of drug accumulated following 24 h of incubation with 50 µM AM, whereas alveolar macrophages accumulated the most drug during incubation with 100 µM AM. Thus, the most susceptible cell type was dependent on AM concentration. AM-induced cytotoxicity in specific cell types may initiate processes leading to inflammation and pulmonary fibrosis.Key words: amiodarone, susceptibility, alveolar macrophage, accumulation.

Temporal changes in the expression of the insulin-like growth factor II gene associated with tissue maturation in the human fetus

Development ◽  
1989 ◽  
Vol 106 (3) ◽  
pp. 543-554 ◽  
Author(s):  
A.L. Brice ◽  
J.E. Cheetham ◽  
V.N. Bolton ◽  
N.C. Hill ◽  
P.N. Schofield
Keyword(s):  
Growth Factor ◽  
Cell Types ◽  
Specific Cell ◽  
Insulin Like Growth Factor ◽  
Vitro Fertilization ◽  
Age Related ◽  
Factor Ii ◽  
Metanephric Blastema

The insulin-like growth factors are broadly distributed in the human conceptus and are thought to play a role in the growth and differentiation of tissues during development. Using in situ hybridization we have shown that a wide variety of specific cell types within tissues express the gene for insulin-like growth factor II at times of development from 18 days to 14 weeks of gestation. Examination of blastocysts produced by in vitro fertilization showed no expression, thus bracketing the time of first accumulation of IGF-II mRNA to between 5 and 18 days postfertilization. The pattern of IGF-II expression shows specific age-related differences in different tissues. In the kidney, for example, expression is found in the cells of the metanephric blastema which is dramatically reduced as the blastema differentiates. The reverse is also seen, and we have noted an increase in expression of IGF-II in the cytotrophoblast layer of the placenta with gestational age. The sites of expression do not correlate with areas of either high mitotic activity or specific types of differentiation, but the observed pattern of expression in the kidney, adrenal glands and liver suggests an explanation for the abnormally high IGF-II mRNA expression in developmental tumours such as Wilms' tumour.

Vertebrate retinal ganglion cells are selected from competent progenitors by the action of Notch

Development ◽  
1995 ◽  
Vol 121 (11) ◽  
pp. 3637-3650 ◽  
Author(s):  
C.P. Austin ◽  
D.E. Feldman ◽  
J.A. Ida ◽  
C.L. Cepko
Keyword(s):  
Cell Fate ◽  
Ganglion Cells ◽  
Notch Pathway ◽  
Cell Types ◽  
Projection Neurons ◽  
Specific Cell ◽  
Vitro Assay ◽  
Notch Activity

The first cells generated during development of the vertebrate retina are the ganglion cells, the projection neurons of the retina. Although they are one of the most intensively studied cell types within the central nervous system, little is known of the mechanisms that determine ganglion cell fate. We demonstrate that ganglion cells are selected from a large group of competent progenitors that comprise the majority of the early embryonic retina and that differentiation within this group is regulated by Notch. Notch activity in vivo was diminished using antisense oligonucleotides or augmented using a retrovirally transduced constitutively active allele of Notch. The number of ganglion cells produced was inversely related to the level of Notch activity. In addition, the Notch ligand Delta inhibited retinal progenitors from differentiating as ganglion cells to the same degree as did activated Notch in an in vitro assay. These results suggest a conserved strategy for neurogenesis in the retina and describe a versatile in vitro and in vivo system with which to examine the action of the Notch pathway in a specific cell fate decision in a vertebrate.

scholarly journals Gold-coated plant virus as computed tomography imaging contrast agent

2019 ◽  
Vol 10 ◽  
pp. 1983-1993 ◽  
Author(s):  
Alaa A A Aljabali ◽  
Mazhar S Al Zoubi ◽  
Khalid M Al-Batanyeh ◽  
Ali Al-Radaideh ◽  
Mohammad A Obeid ◽  
...  
Keyword(s):  
Computed Tomography ◽  
Contrast Agent ◽  
Low Cost ◽  
Cell Types ◽  
Specific Cell ◽  
Scan Time ◽  
Ct Contrast Agent ◽  
Potential Applications ◽  
Resolution Imaging

Chemical modification of the surface of viruses, both the interior and the exterior, imparts new functionalities, that have potential applications in nanomedicine. In this study, we developed novel virus-based nanomaterials as a contrast agent for computed tomography (CT) imaging in vitro. The gold-coated cowpea mosaic virus (Au-CPMV) particles were generated by the electrostatic adsorption of positively charged electrolyte on the virus capsid with the subsequent incubation and reduction of anionic gold complexes. Au-CPMV particles as a CT contrast agent offer a fast scan time (less than 2 min), low cost, and biocompatibility and allow for high-resolution imaging with ca. 150 Hounsfield units (HU). The Au-CPMV surface was further modified allowing for the incorporation of targeting molecules of specific cell types.

scholarly journals Fluorescent PSC-Derived Cardiomyocyte Reporter Lines: Generation Approaches and Their Applications in Cardiovascular Medicine

Biology ◽  
2020 ◽  
Vol 9 (11) ◽  
pp. 402
Author(s):  
Naeramit Sontayananon ◽  
Charles Redwood ◽  
Benjamin Davies ◽  
Katja Gehmlich
Keyword(s):  
Drug Testing ◽  
Cardiac Development ◽  
Cardiac Regeneration ◽  
Cell Types ◽  
Specific Cell ◽  
Clinical Safety ◽  
Fluorescent Reporter ◽  
Advantages And Disadvantages ◽  
Attractive Option

Recent advances have made pluripotent stem cell (PSC)-derived cardiomyocytes an attractive option to model both normal and diseased cardiac function at the single-cell level. However, in vitro differentiation yields heterogeneous populations of cardiomyocytes and other cell types, potentially confounding phenotypic analyses. Fluorescent PSC-derived cardiomyocyte reporter systems allow specific cell lineages to be labelled, facilitating cell isolation for downstream applications including drug testing, disease modelling and cardiac regeneration. In this review, the different genetic strategies used to generate such reporter lines are presented with an emphasis on their relative technical advantages and disadvantages. Next, we explore how the fluorescent reporter lines have provided insights into cardiac development and cardiomyocyte physiology. Finally, we discuss how exciting new approaches using PSC-derived cardiomyocyte reporter lines are contributing to progress in cardiac cell therapy with respect to both graft adaptation and clinical safety.

scholarly journals Effects of Aminoglycoside Antibiotics on Human Embryonic Stem Cell Viability during Differentiation In Vitro

2017 ◽  
Vol 2017 ◽  
pp. 1-18 ◽  
Author(s):  
Divya S. Varghese ◽  
Shama Parween ◽  
Mustafa T. Ardah ◽  
Bright Starling Emerald ◽  
Suraiya A. Ansari
Keyword(s):  
Cell Viability ◽  
Disease Modeling ◽  
Culture Media ◽  
Embryonic Stem ◽  
Toxicity Testing ◽  
Cell Types ◽  
Specific Cell ◽  
Directed Differentiation ◽  
Embryonic Neurogenesis

Human embryonic stem cells (hESCs) are being used extensively in array of studies to understand different mechanisms such as early human embryogenesis, drug toxicity testing, disease modeling, and cell replacement therapy. The protocols for the directed differentiation of hESCs towards specific cell types often require long-term cell cultures. To avoid bacterial contamination, these protocols include addition of antibiotics such as pen-strep and gentamicin. Although aminoglycosides, streptomycin, and gentamicin have been shown to cause cytotoxicity in various animal models, the effect of these antibiotics on hESCs is not clear. In this study, we found that antibiotics, pen-strep, and gentamicin did not affect hESC cell viability or expression of pluripotency markers. However, during directed differentiation towards neural and hepatic fate, significant cell death was noted through the activation of caspase cascade. Also, the expression of neural progenitor markers Pax6, Emx2, Otx2, and Pou3f2 was significantly reduced suggesting that gentamicin may adversely affect early embryonic neurogenesis whereas no effect was seen on the expression of endoderm or hepatic markers during differentiation. Our results suggest that the use of antibiotics in cell culture media for the maintenance and differentiation of hESCs needs thorough investigation before use to avoid erroneous results.

scholarly journals HDAC4 Promotes Growth of Colon Cancer Cells via Repression of p21

2008 ◽  
Vol 19 (10) ◽  
pp. 4062-4075 ◽  
Author(s):  
Andrew J. Wilson ◽  
Do-Sun Byun ◽  
Shannon Nasser ◽  
Lucas B. Murray ◽  
Kanyalakshmi Ayyanar ◽  
...  
Keyword(s):  
Growth Promotion ◽  
Cell Types ◽  
Specific Cell ◽  
Physical Interaction ◽  
Down Regulation ◽  
Proliferative Zone ◽  
P21 Promoter ◽  
Hct116 Cells

The class II Histone deacetylase (HDAC), HDAC4, is expressed in a tissue-specific manner, and it represses differentiation of specific cell types. We demonstrate here that HDAC4 is expressed in the proliferative zone in small intestine and colon and that its expression is down-regulated during intestinal differentiation in vivo and in vitro. Subcellular localization studies demonstrated HDAC4 expression was predominantly nuclear in proliferating HCT116 cells and relocalized to the cytoplasm after cell cycle arrest. Down-regulating HDAC4 expression by small interfering RNA (siRNA) in HCT116 cells induced growth inhibition and apoptosis in vitro, reduced xenograft tumor growth, and increased p21 transcription. Conversely, overexpression of HDAC4 repressed p21 promoter activity. p21 was likely a direct target of HDAC4, because HDAC4 down-regulation increased p21 mRNA when protein synthesis was inhibited by cycloheximide. The importance of p21 repression in HDAC4-mediated growth promotion was demonstrated by the failure of HDAC4 down-regulation to induce growth arrest in HCT116 p21-null cells. HDAC4 down-regulation failed to induce p21 when Sp1 was functionally inhibited by mithramycin or siRNA-mediated down-regulation. HDAC4 expression overlapped with that of Sp1, and a physical interaction was demonstrated by coimmunoprecipitation. Chromatin immunoprecipitation (ChIP) and sequential ChIP analyses demonstrated Sp1-dependent binding of HDAC4 to the proximal p21 promoter, likely directed through the HDAC4–HDAC3–N-CoR/SMRT corepressor complex. Consistent with increased transcription, HDAC4 or SMRT down-regulation resulted in increased histone H3 acetylation at the proximal p21 promoter locus. These studies identify HDAC4 as a novel regulator of colon cell proliferation through repression of p21.

scholarly journals Porcine colonoids and enteroids keep the memory of their origin during regeneration.

2021 ◽  
Author(s):  
Alicia M. Barnett ◽  
Jane A. Mullaney ◽  
Charlotte Hendriks ◽  
Lisa Le Borgne ◽  
Warren C. McNabb ◽  
...  
Keyword(s):  
Gene Expression ◽  
Glucose Transporter ◽  
Expression Profiles ◽  
Cell Types ◽  
Specific Cell ◽  
Stem Cell Markers ◽  
Culture Methods ◽  
Glucose Transporter 1 ◽  
Expression Levels

The development of alternative in vitro culture methods has increased in the last decade as three-dimensional organoids of various tissues, including those of the small and large intestines. Due to their multicellular composition, organoids offer advantages over traditionally used immortalized or primary cell lines. However, organoids must be accurate models of their tissues of origin. This study compared gene expression profiles with respect to markers of specific cell-types (stem-cells, enterocytes, goblet and enteroendocrine cells) and barrier maturation (tight junctions) of colonoid and enteroid cultures with their tissues of origin, and colonoids with enteroids. Colonoids derived from three healthy pigs formed multi-lobed structures with a monolayer of cells similar to the crypt structures in colonic tissue. Colonoid and enteroid gene expression signatures were more similar to those found for the tissues of their origin than to each other. However, relative to their derived tissues, organoids had increased gene expression levels of stem-cell markers Sox9 and Lgr5 encoding Sex determining region Y-box 9 and leucine-rich repeat-containing G-protein coupled rector 5, respectively. In contrast, expression levels of Occl and Zo1 encoding occludin and zonula occludens 1 respectively, were decreased. Expression levels of the cell lineage markers Atoh1, Cga and Muc2 encoding atonal homolog 1, chromogranin A and mucin 2 respectively, were decreased in colonoids, while Sglt1 and Apn encoding sodium-glucose transporter 1 and aminopeptidase A respectively, were decreased in enteroids. These results indicate colonoid and enteroid cultures were predominantly comprised of undifferentiated cell-types with decreased barrier maturation relative to their tissues of origin.

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