scholarly journals Microfluidic chambers using fluid walls for cell biology

2018 ◽  
Vol 115 (26) ◽  
pp. E5926-E5933 ◽  
Author(s):  
Cristian Soitu ◽  
Alexander Feuerborn ◽  
Ann Na Tan ◽  
Henry Walker ◽  
Pat A. Walsh ◽  
...  
Keyword(s):  
Cell Culture ◽  
Cell Biology ◽  
Mammalian Cells ◽  
Soft Lithography ◽  
Petri Dish ◽  
Microtiter Plate ◽  
The Media ◽  
Downstream Analysis ◽  
Microfluidic Chambers ◽  
Made In

Many proofs of concept have demonstrated the potential of microfluidics in cell biology. However, the technology remains inaccessible to many biologists, as it often requires complex manufacturing facilities (such as soft lithography) and uses materials foreign to cell biology (such as polydimethylsiloxane). Here, we present a method for creating microfluidic environments by simply reshaping fluids on a substrate. For applications in cell biology, we use cell media on a virgin Petri dish overlaid with an immiscible fluorocarbon. A hydrophobic/fluorophilic stylus then reshapes the media into any pattern by creating liquid walls of fluorocarbon. Microfluidic arrangements suitable for cell culture are made in minutes using materials familiar to biologists. The versatility of the method is demonstrated by creating analogs of a common platform in cell biology, the microtiter plate. Using this vehicle, we demonstrate many manipulations required for cell culture and downstream analysis, including feeding, replating, cloning, cryopreservation, lysis plus RT-PCR, transfection plus genome editing, and fixation plus immunolabeling (when fluid walls are reconfigured during use). We also show that mammalian cells grow and respond to stimuli normally, and worm eggs develop into adults. This simple approach provides biologists with an entrée into microfluidics.

scholarly journals Enhancement of Virus Infection Using Dynamic Cell Culture in a Microchannel

Micromachines ◽  
2018 ◽  
Vol 9 (10) ◽  
pp. 482
Author(s):  
Jeong Kim ◽  
Hye Choi ◽  
Chul Kim ◽  
Hee Jin ◽  
Jae-sung Bae ◽  
...  
Keyword(s):  
Cell Culture ◽  
Virus Infection ◽  
Soft Lithography ◽  
Fluorescent Protein ◽  
Microfluidic Devices ◽  
Petri Dish ◽  
Culture Method ◽  
Dynamic Culture ◽  
Conventional Culture ◽  
Dynamic Cell

With increasing interest in induced pluripotent stem cells (iPSCs) in the field of stem cell research, highly efficient infection of somatic cells with virus factors is gaining importance. This paper presents a method of employing microfluidic devices for dynamic cell culture and virus infection in a microchannel. The closed space in the microchannel provided a better environment for viruses to diffuse and contact cell surfaces to infect cells. The microfluidic devices were fabricated by photolithography and soft lithography. NIH/3T3 fibroblast cells were cultured in the microfluidic device in static and dynamic conditions and compared with the conventional culture method of using Petri dishes. Virus infection was evaluated using an enhanced green fluorescent protein virus as a model. Dynamic culture in the microchannel showed similar growth of cells to that in Petri dish culture, but the virus infection efficiency was four-times higher. The proposed dynamic culture system could be useful in iPSC research by providing efficient virus infection tools.

Microengineering the Environment of Mammalian Cells in Culture

MRS Bulletin ◽  
2005 ◽  
Vol 30 (3) ◽  
pp. 194-201 ◽  
Author(s):  
Christopher S. Chen ◽  
Xingyu Jiang ◽  
George M. Whitesides
Keyword(s):  
Cell Biology ◽  
Mammalian Cells ◽  
Soft Lithography ◽  
Materials Science ◽  
Molecular Level ◽  
Extrinsic Factors ◽  
New Techniques ◽  
Biological Responses ◽  
Physical Environments ◽  
Attached Culture

AbstractAssays based on observations of the biological responses of individual cells to their environment have the potential to make enormous contributions to cell biology and biomedicine.To carry out well-defined experiments using cells, both the environments in which the cells live and the cells themselves must be well defined. Cell-based assays are now plagued by inconsistencies and irreproducibility, and a primary challenge in the development of informative assays is to understand the fundamental bases for these inconsistencies and to limit them. It now seems that multiple factors may contribute to the variability in the response of individual cells to stimuli; some of these factors may be extrinsic to the cells, some intrinsic. New techniques based on microengineering—especially using soft lithography to pattern surfaces at the molecular level and to fabricate microfluidic systems—have provided new capabilities to address the extrinsic factors. This review discusses recent advances in materials science that provide well-defined physical environments that can be used to study cells, both individually and in groups, in attached culture. It also reviews the challenges that must be addressed in order to make cell-based assays reproducible.

Separation of Extracellular Vesicles by DNA-Directed Immunocapturing Followed by Enzymatic Release

2020 ◽  
Author(s):  
Dario Brambilla ◽  
Laura Sola ◽  
Elisa Chiodi ◽  
Natasa Zarovni ◽  
Diogo Fortunato ◽  
...  
Keyword(s):  
Cell Culture ◽  
Extracellular Vesicles ◽  
Culture Media ◽  
Biological Fluids ◽  
Imaging Techniques ◽  
Cell Communication ◽  
Disease Diagnosis ◽  
Cell Culture Supernatant ◽  
Transmission Electron ◽  
Downstream Analysis

Extracellular vesicles (EVs) have attracted great interest among researchers due to their role in cell-cell communication, disease diagnosis, and drug delivery. In spite of their potential in the medical field, there is no consensus on the best method for separating microvesicles from cell culture supernatant and complex biological fluids. Obtaining a good recovery yield and preserving physical characteristics is critical for the diagnostic and therapeutic use of EVs. The separation is made complex by the fact that blood and cell culture media, contain a large number of nanoparticles in the same size range. Methods that exploit immunoaffinity capture provide high purity samples and overcome the issues of currently used separation methods. However, the release of captured nanovesicles requires harsh conditions that hinder their use in certain types of downstream analysis. Herein, a novel capture and release approach for small extracellular vesicles (sEVs), based on DNAdirected immobilization of antiCD63 antibody is presented. The flexible DNAlinker increases the capture efficiency and allows releasing of EVs by exploiting the endonucleasic activity of DNAse I. This separation protocol works under mild conditions, enabling the release of intact vesicles that can be successfully analyzed by imaging techniques. In this article sEVs recovered from plasma were characterized by established techniques for EVs analysis including nanoparticle tracking and transmission electron microscopy.<br>

Experimental Measurements in the Acquisition of Biosignals from a Neuronal Cell Culture for an Exoprosthesis Command

2018 ◽  
Vol 69 (10) ◽  
pp. 2948-2939 ◽  
Author(s):  
Carmen Moldovan ◽  
Lidia Dobrescu ◽  
Violeta Ristoiu ◽  
Bogdan Firtat ◽  
Silviu Dinulescu ◽  
...  
Keyword(s):  
Cell Culture ◽  
Glass Plate ◽  
Neuronal Cell ◽  
Cellular Biology ◽  
Experimental Measurements ◽  
Neuron Culture ◽  
Neuronal Cell Culture ◽  
Made In

This article presents experimental measurements performed in order to connect a neuronal cell culture to an exoprosthesis. The experiments focused on the biosignals� acquisition from the cell culture. A special gold-plated glass plate device was realized and several constructive variants were analyzed. A Olympus microscope with fluorescence and photo system was used. The acquisition of bio signals from the neuron culture is realized and described in the paper. The measurements were made in the sterile environment within the laboratory of Institute of Cellular Biology and Pathology. The measurements have been made for the pair of electrodes 1-1 at the edge of the glass plate.

scholarly journals CALINCA—A Novel Pipeline for the Identification of lncRNAs in Podocyte Disease

Cells ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 692
Author(s):  
Sweta Talyan ◽  
Samantha Filipów ◽  
Michael Ignarski ◽  
Magdalena Smieszek ◽  
He Chen ◽  
...  
Keyword(s):  
Cell Biology ◽  
Mammalian Cells ◽  
De Novo ◽  
Depth Information ◽  
Gene Products ◽  
Classical Analysis ◽  
Protein Coding ◽  
Bioinformatic Pipeline ◽  
Non Coding Rnas ◽  
Filtration Unit

Diseases of the renal filtration unit—the glomerulus—are the most common cause of chronic kidney disease. Podocytes are the pivotal cell type for the function of this filter and focal-segmental glomerulosclerosis (FSGS) is a classic example of a podocytopathy leading to proteinuria and glomerular scarring. Currently, no targeted treatment of FSGS is available. This lack of therapeutic strategies is explained by a limited understanding of the defects in podocyte cell biology leading to FSGS. To date, most studies in the field have focused on protein-coding genes and their gene products. However, more than 80% of all transcripts produced by mammalian cells are actually non-coding. Here, long non-coding RNAs (lncRNAs) are a relatively novel class of transcripts and have not been systematically studied in FSGS to date. The appropriate tools to facilitate lncRNA research for the renal scientific community are urgently required due to a row of challenges compared to classical analysis pipelines optimized for coding RNA expression analysis. Here, we present the bioinformatic pipeline CALINCA as a solution for this problem. CALINCA automatically analyzes datasets from murine FSGS models and quantifies both annotated and de novo assembled lncRNAs. In addition, the tool provides in-depth information on podocyte specificity of these lncRNAs, as well as evolutionary conservation and expression in human datasets making this pipeline a crucial basis to lncRNA studies in FSGS.

Preparation of Porous Organically-Modified Silicate Hybrids for Cell Culture Matrix

2007 ◽  
Vol 330-332 ◽  
pp. 1177-1180 ◽  
Author(s):  
Kanji Tsuru ◽  
Satoshi Hayakawa ◽  
Yuki Shirosaki ◽  
T. Okayama ◽  
K. Kataoka ◽  
...  
Keyword(s):  
Cell Culture ◽  
Mammalian Cells ◽  
Pore Diameter ◽  
Sol Gel ◽  
Three Dimensional ◽  
Culture Methods ◽  
Organically Modified ◽  
Dimensional Cell

Porous & rubbery organic-inorganic hybrids were synthesized from tetraethoxysilane (TEOS) and polydimethylsiloxane (PDMS) through a sol-gel route using sieved sucrose granules as a porogen. The porous hybrids with a high content of PDMS behaved like polymer sponge. The porosity was over 90% irrespective of the hybrid composition and the pore diameter ranged from 100 to 500 μm. In the three-dimensional cell culture, mammalian cells were well cultured in the porous hybrids. The present results indicate that the hybrids may be a promising scaffold for developing such functional culture methods.

scholarly journals Towards Modelling Genetic Kidney Diseases with Human Pluripotent Stem Cells

Nephron ◽  
2021 ◽  
pp. 1-12
Author(s):  
Kirsty M. Rooney ◽  
Adrian S. Woolf ◽  
Susan J. Kimber
Keyword(s):  
Stem Cell ◽  
Kidney Disease ◽  
Cell Biology ◽  
Kidney Diseases ◽  
Premature Mortality ◽  
Cystic Kidney Disease ◽  
Cystic Kidney ◽  
Metanephric Mesenchyme ◽  
Potential Therapeutic Application ◽  
Made In

<b><i>Background:</i></b> Kidney disease causes major suffering and premature mortality worldwide. With no cure for kidney failure currently available, and with limited options for treatment, there is an urgent need to develop effective pharmaceutical interventions to slow or prevent kidney disease progression. <b><i>Summary:</i></b> In this review, we consider the feasibility of using human pluripotent stem cell-derived kidney tissues, or organoids, to model genetic kidney disease. Notable successes have been made in modelling genetic tubular diseases (e.g., cystinosis), polycystic kidney disease, and medullary cystic kidney disease. Organoid models have also been used to test novel therapies that ameliorate aberrant cell biology. Some progress has been made in modelling congenital glomerular disease, even though glomeruli within organoids are developmentally immature. Less progress has been made in modelling structural kidney malformations, perhaps because sufficiently mature metanephric mesenchyme-derived nephrons, ureteric bud-derived branching collecting ducts, and a prominent stromal cell population are not generated together within a single protocol. <b><i>Key Messages:</i></b> We predict that the field will advance significantly if organoids can be generated with a full complement of cell lineages and with kidney components displaying key physiological functions, such as glomerular filtration. The future economic upscaling of reproducible organoid generation will facilitate more widespread research applications, including the potential therapeutic application of these stem cell-based technologies.

scholarly journals Macropinocytosis and Clathrin-Dependent Endocytosis Play Pivotal Roles for the Infectious Entry of Puumala Virus

2019 ◽  
Author(s):  
Sandy Bauherr ◽  
Filip Larsberg ◽  
Annett Petrich ◽  
Hannah Sabeth Sperber ◽  
Victoria Klose ◽  
...  
Keyword(s):  
Rna Interference ◽  
Cell Biology ◽  
Mammalian Cells ◽  
Case Fatality ◽  
Hemorrhagic Fever ◽  
Puumala Virus ◽  
Virus Species ◽  
Global Public Health ◽  
Emerging Pathogens ◽  
Cellular Mechanisms

AbstractViruses from the taxonomic familyHantaviridaeare encountered as emerging pathogens causing two life-threatening human zoonoses: hemorrhagic fever with renal syndrome (HFRS) and hantavirus cardiopulmonary syndrome (HCPS) with case fatalities of up to 50%. Here we comprehensively investigated entry of the Old-World Hantavirus, Puumala virus (PUUV), into mammalian cells, showing that upon treatment with pharmacological inhibitors of macropinocytosis and clathrin-mediated endocytosis, PUUV infections are significantly reduced. We demonstrated that the inhibitors did not interfere with viral replication and that RNA interference, targeting cellular mediators of macropinocytosis, is able to decrease PUUV infection levels significantly. Moreover, we established lipophilic tracer staining of PUUV virus particles and showed co-localization of stained virions and markers of macropinocytic uptake. Cells treated with lysosomotrophic agents were shown to exhibit an increased resistance to infection, confirming previous data suggesting that a low pH-dependent step is involved in PUUV infection. Finally, we observed a significant increase in the fluid-phase uptake of cell infected with PUUV, indicative of a virus-triggered promotion of macropinocytosis.Author SummaryTheHantaviridaefamily comprises a very diverse group of virus species and is considered an emerging global public health threat. Human pathogenic hantaviruses are primarily rodent-borne. Zoonosis is common with more than 150,000 annually registered cases and a case fatality index of up to 50%. Individual hantavirus species differ significantly in terms of their pathogenicity, but also their cell biology and host-pathogen interactions. In this study, we focused on the most prevalent pathogenic hantavirus in Europe, Puumala virus (PUUV), and investigated the entry and internalization of PUUV virions into mammalian cells. We showed that both, clathrin-mediated endocytosis and macropinocytosis, are cellular pathways exploited by the virus to establish productive infections and demonstrated that pharmacological inhibition of macropinocytosis or its targeted knockdown using RNA interference significantly reduced viral infections. We also found indications for an increase of macropinocytic uptake upon PUUV infections, suggesting that the virus triggers specific cellular mechanisms in order to promote its own internalization and facilitate infections.

scholarly journals The Road Less Traveled? Unconventional Protein Secretion at Parasite–Host Interfaces

2021 ◽  
Vol 9 ◽  
Author(s):  
Erina A. Balmer ◽  
Carmen Faso
Keyword(s):  
Protein Secretion ◽  
Cell Biology ◽  
Mammalian Cells ◽  
Cell Model ◽  
Signal Sequence ◽  
Model Organism ◽  
Secreted Proteins ◽  
Current Status ◽  
Secretory Proteins ◽  
Secretion Pathways

Protein secretion in eukaryotic cells is a well-studied process, which has been known for decades and is dealt with by any standard cell biology textbook. However, over the past 20 years, several studies led to the realization that protein secretion as a process might not be as uniform among different cargos as once thought. While in classic canonical secretion proteins carry a signal sequence, the secretory or surface proteome of several organisms demonstrated a lack of such signals in several secreted proteins. Other proteins were found to indeed carry a leader sequence, but simply circumvent the Golgi apparatus, which in canonical secretion is generally responsible for the modification and sorting of secretory proteins after their passage through the endoplasmic reticulum (ER). These alternative mechanisms of protein translocation to, or across, the plasma membrane were collectively termed “unconventional protein secretion” (UPS). To date, many research groups have studied UPS in their respective model organism of choice, with surprising reports on the proportion of unconventionally secreted proteins and their crucial roles for the cell and survival of the organism. Involved in processes such as immune responses and cell proliferation, and including far more different cargo proteins in different organisms than anyone had expected, unconventional secretion does not seem so unconventional after all. Alongside mammalian cells, much work on this topic has been done on protist parasites, including genera Leishmania, Trypanosoma, Plasmodium, Trichomonas, Giardia, and Entamoeba. Studies on protein secretion have mainly focused on parasite-derived virulence factors as a main source of pathogenicity for hosts. Given their need to secrete a variety of substrates, which may not be compatible with canonical secretion pathways, the study of mechanisms for alternative secretion pathways is particularly interesting in protist parasites. In this review, we provide an overview on the current status of knowledge on UPS in parasitic protists preceded by a brief overview of UPS in the mammalian cell model with a focus on IL-1β and FGF-2 as paradigmatic UPS substrates.

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