scholarly journals Study Cellular Hypoxic Response under Cyclic Oxygen Gradients Generated in Microfluidic Devices Using Real-Time Fluorescence Imaging

2021 ◽  
Author(s):  
Dao-Ming Chang ◽  
Yi-Chung Tung
Keyword(s):  
Cell Culture ◽  
Intracellular Calcium ◽  
Real Time ◽  
Oxygen Tension ◽  
Calcium Concentration ◽  
A549 Cells ◽  
Cell Types ◽  
Hypoxic Response ◽  
Oxygen Gradient ◽  
Oxygen Gradients

Abstract In this paper, an integrated approach to study cellular hypoxic response under cyclic oxygen gradients is developed. In the experiments, a cell culture system based on a microfluidic device is constructed to generate cyclic oxygen gradients with fast response. The oxygen gradient is generated by alternatively introducing gases with specific compositions by a computer-controlled gas supply system into the microfluidic channels next to the cell culture one separated by thin channel walls. Observation of cellular hypoxic responses is performed using real-time fluorescence imaging of dyes sensitive to extra-and intra-cellular oxygen tensions as well as intracellular calcium concentrations. The capability of consistent cyclic oxygen gradient generation is confirmed by characterizing spatiotemporal oxygen tension profiles within the device. In the cell experiments, cellular hypoxic responses of human aortic smooth muscle cells (AoSMCs) and lung carcinoma epithelium (A549) cells including intracellular oxygen and calcium levels are measured. The relationships between the oxygen probe intensity and extracellular oxygen tension for both cell types are highly linear, and difference between the intensity measured from the two cell types is observed between 4 to 8% O2 culture environment. In addition, both cell types show detectable intracellular calcium concentration variation when the environmental O2 varies between normoxia and lower than 4%. The AoSMCs and A549 cells show decrease and increase of the intracellular calcium concentration during hypoxic response when facing change from normoxia to hypoxia environments, respectively. With the capability of real-time cellular responses monitoring under cyclic oxygen gradients, the developed approach provides a useful scheme to investigate cellular hypoxic response in vitro under microenvironments mimicking various in vivo physiological and pathological conditions.

scholarly journals Numerical modeling of oxygen distributions in cortical and cancellous bone: oxygen availability governs osteonal and trabecular dimensions

2010 ◽  
Vol 299 (5) ◽  
pp. C922-C929 ◽  
Author(s):  
Adam M. Zahm ◽  
Michael A. Bucaro ◽  
Portonovo S. Ayyaswamy ◽  
Vickram Srinivas ◽  
Irving M. Shapiro ◽  
...  
Keyword(s):  
Numerical Modeling ◽  
Trabecular Bone ◽  
Oxygen Tension ◽  
Cancellous Bone ◽  
Bone Cells ◽  
Oxygen Availability ◽  
Oxygen Utilization ◽  
Oxygen Gradient ◽  
Stage Of Development ◽  
Oxygen Gradients

Whereas recent work has demonstrated the role of oxygen tension in the regulation of skeletal cell function and viability, the microenvironmental oxemic status of bone cells remains unknown. In this study, we have employed the Krogh cylinder model of oxygen diffusion to predict the oxygen distribution profiles in cortical and cancellous bone. Under the assumption of saturation-type Michaelis-Menten kinetics, our numerical modeling has indicated that, under steady-state conditions, there would be oxygen gradients across mature osteons and trabeculae. In Haversian bone, the calculated oxygen tension decrement ranges from 15 to 60%. For trabecular bone, a much shallower gradient is predicted. We note that, in Haversian bone, the gradient is largely dependent on osteocyte oxygen utilization and tissue oxygen diffusivity; in trabecular bone, the gradient is dependent on oxygen utilization by cells lining the bone surface. The Krogh model also predicts dramatic differences in oxygen availability during bone development. Thus, during osteon formation, the modeling equations predict a steep oxygen gradient at the initial stage of development, with the gradient becoming lesser as osteonal layers are added. In contrast, during trabeculum formation, the oxygen gradient is steepest when the diameter of the trabeculum is maximal. Based on these results, it is concluded that significant oxygen gradients exist within cortical and cancellous bone and that the oxygen tension may regulate the physical dimensions of both osteons and bone trabeculae.

scholarly journals Extracellular NAD+ regulates intracellular calcium levels and induces activation of human granulocytes

2006 ◽  
Vol 393 (3) ◽  
pp. 697-704 ◽  
Author(s):  
Santina Bruzzone ◽  
Iliana Moreschi ◽  
Lucrezia Guida ◽  
Cesare Usai ◽  
Elena Zocchi ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Intracellular Calcium ◽  
Blood Cells ◽  
Inflammatory Cytokine ◽  
Calcium Concentration ◽  
Cell Types ◽  
Adp Ribosyl Cyclase ◽  
Human Granulocytes ◽  
Kinase A ◽  
Stimulation Of

β-NAD+e (extracellular β-NAD+), present at nanomolar levels in human plasma, has been implicated in the regulation of [Ca2+]i (the intracellular calcium concentration) in various cell types, including blood cells, by means of different mechanisms. Here, we demonstrate that micromolar NAD+e (both the α and the β extracellular NAD+ forms) induces a sustained [Ca2+]i increase in human granulocytes by triggering the following cascade of causally related events: (i) activation of adenylate cyclase and overproduction of cAMP; (ii) activation of protein kinase A; (iii) stimulation of ADP-ribosyl cyclase activity and consequent overproduction of cADP-ribose, a universal Ca2+ mobilizer; and (iv) influx of extracellular Ca2+. The NAD+e-triggered [Ca2+]i elevation translates into granulocyte activation, i.e. superoxide and nitric oxide generation, and enhanced chemotaxis in response to 0.1–10 μM NAD+e. Thus extracellular β-NAD+e behaves as a novel pro-inflammatory cytokine, stimulating human granulocytes and potentially recruiting them at sites of inflammation.

scholarly journals Real-time monitoring of liver fibrosis through embedded sensors in a microphysiological system

2021 ◽  
Vol 8 (1) ◽  
Author(s):  
Hafiz Muhammad Umer Farooqi ◽  
Bohye Kang ◽  
Muhammad Asad Ullah Khalid ◽  
Abdul Rahim Chethikkattuveli Salih ◽  
Kinam Hyun ◽  
...  
Keyword(s):  
Cell Culture ◽  
Liver Fibrosis ◽  
Real Time ◽  
Hepatic Fibrosis ◽  
Transforming Growth Factor ◽  
Cell Monolayer ◽  
Embedded Sensors ◽  
Matrix Deposition ◽  
Tgf Β1

AbstractHepatic fibrosis is a foreshadowing of future adverse events like liver cirrhosis, liver failure, and cancer. Hepatic stellate cell activation is the main event of liver fibrosis, which results in excessive extracellular matrix deposition and hepatic parenchyma's disintegration. Several biochemical and molecular assays have been introduced for in vitro study of the hepatic fibrosis progression. However, they do not forecast real-time events happening to the in vitro models. Trans-epithelial electrical resistance (TEER) is used in cell culture science to measure cell monolayer barrier integrity. Herein, we explored TEER measurement's utility for monitoring fibrosis development in a dynamic cell culture microphysiological system. Immortal HepG2 cells and fibroblasts were co-cultured, and transforming growth factor β1 (TGF-β1) was used as a fibrosis stimulus to create a liver fibrosis-on-chip model. A glass chip-based embedded TEER and reactive oxygen species (ROS) sensors were employed to gauge the effect of TGF-β1 within the microphysiological system, which promotes a positive feedback response in fibrosis development. Furthermore, albumin, Urea, CYP450 measurements, and immunofluorescent microscopy were performed to correlate the following data with embedded sensors responses. We found that chip embedded electrochemical sensors could be used as a potential substitute for conventional end-point assays for studying fibrosis in microphysiological systems.

scholarly journals Human Immunodeficiency Viruses Pseudotyped with SARS-CoV-2 Spike Proteins Infect a Broad Spectrum of Human Cell Lines through Multiple Entry Mechanisms

Viruses ◽  
2021 ◽  
Vol 13 (6) ◽  
pp. 953
Author(s):  
Chuan Xu ◽  
Annie Wang ◽  
Ke Geng ◽  
William Honnen ◽  
Xuening Wang ◽  
...  
Keyword(s):  
Cell Lines ◽  
Viral Entry ◽  
Broad Spectrum ◽  
A549 Cells ◽  
Cell Types ◽  
Pseudotyped Virus ◽  
Angiotensin Converting Enzyme 2 ◽  
Human Immunodeficiency Viruses ◽  
Tyrosine Protein Kinase ◽  
Overexpressing Cell

Severe acute respiratory syndrome-related coronavirus (SARS-CoV-2), the causative agent of coronavirus disease 19 (COVID-19), enters cells through attachment to the human angiotensin converting enzyme 2 (hACE2) via the receptor-binding domain (RBD) in the surface/spike (S) protein. Several pseudotyped viruses expressing SARS-CoV-2 S proteins are available, but many of these can only infect hACE2-overexpressing cell lines. Here, we report the use of a simple, two-plasmid, pseudotyped virus system comprising a SARS-CoV-2 spike-expressing plasmid and an HIV vector with or without vpr to investigate the SARS-CoV-2 entry event in various cell lines. When an HIV vector without vpr was used, pseudotyped SARS-CoV-2 viruses produced in the presence of fetal bovine serum (FBS) were able to infect only engineered hACE2-overexpressing cell lines, whereas viruses produced under serum-free conditions were able to infect a broader range of cells, including cells without hACE2 overexpression. When an HIV vector containing vpr was used, pseudotyped viruses were able to infect a broad spectrum of cell types regardless of whether viruses were produced in the presence or absence of FBS. Infection sensitivities of various cell types did not correlate with mRNA abundance of hACE2, TMPRSS2, or TMPRSS4. Pseudotyped SARS-CoV-2 viruses and replication-competent SARS-CoV-2 virus were equally sensitive to neutralization by an anti-spike RBD antibody in cells with high abundance of hACE2. However, the anti-spike RBD antibody did not block pseudotyped viral entry into cell lines with low abundance of hACE2. We further found that CD147 was involved in viral entry in A549 cells with low abundance of hACE2. Thus, our assay is useful for drug and antibody screening as well as for investigating cellular receptors, including hACE2, CD147, and tyrosine-protein kinase receptor UFO (AXL), for the SARS-CoV-2 entry event in various cell lines.

scholarly journals Half-life modeling of basic fibroblast growth factor released from growth factor-eluting polyelectrolyte multilayers

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Ivan Ding ◽  
Amy M. Peterson
Keyword(s):  
Cell Culture ◽  
Growth Factor ◽  
Fibroblast Growth Factor ◽  
Basic Fibroblast Growth Factor ◽  
Half Life ◽  
Cell Types ◽  
Polyelectrolyte Multilayers ◽  
Enzyme Linked Immunosorbent Assay ◽  
Fibroblast Growth ◽  
Cell Culture Study

AbstractGrowth factor-eluting polymer systems have been widely reported to improve cell and tissue outcomes; however, measurements of actual growth factor concentration in cell culture conditions are limited. The problem is compounded by a lack of knowledge of growth factor half-lives, which impedes efforts to determine real-time growth factor concentrations. In this work, the half-life of basic fibroblast growth factor (FGF2) was determined using enzyme linked immunosorbent assay (ELISA). FGF2 release from polyelectrolyte multilayers (PEMs) was measured and the data was fit to a simple degradation model, allowing for the determination of FGF2 concentrations between 2 and 4 days of culture time. After the first hour, the FGF2 concentration for PEMs assembled at pH = 4 ranged from 2.67 ng/mL to 5.76 ng/mL, while for PEMs assembled at pH = 5, the concentration ranged from 0.62 ng/mL to 2.12 ng/mL. CRL-2352 fibroblasts were cultured on PEMs assembled at pH = 4 and pH = 5. After 2 days, the FGF2-eluting PEM conditions showed improved cell count and spreading. After 4 days, only the pH = 4 assembly condition had higher cells counts, while the PEM assembled at pH = 5 and PEM with no FGF2 showed increased spreading. Overall, the half-life model and cell culture study provide optimal concentration ranges for fibroblast proliferation and a framework for understanding how temporal FGF2 concentration may affect other cell types.

scholarly journals Identification of Novel FNIN2 and FNIN3 Fibronectin-Derived Peptides That Promote Cell Adhesion, Proliferation and Differentiation in Primary Cells and Stem Cells

2021 ◽  
Vol 22 (6) ◽  
pp. 3042
Author(s):  
Eun Ju Lee ◽  
Khurshid Ahmad ◽  
Shiva Pathak ◽  
SunJu Lee ◽  
Mohammad Hassan Baig ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Culture ◽  
Cell Adhesion ◽  
3D Culture ◽  
Human Mesenchymal Stem Cells ◽  
Cell Types ◽  
Primary Cells ◽  
Cell Culture Techniques ◽  
Culture Techniques ◽  
Promote Cell Adhesion

In recent years, a major rise in the demand for biotherapeutic drugs has centered on enhancing the quality and efficacy of cell culture and developing new cell culture techniques. Here, we report fibronectin (FN) derived, novel peptides fibronectin-based intergrin binding peptide (FNIN)2 (18-mer) and FNIN3 (20-mer) which promote cell adhesion proliferation, and the differentiation of primary cells and stem cells. FNIN2 and 3 were designed based on the in silico interaction studies between FN and its receptors (integrin α5β1, αvβ3, and αIIbβ3). Analysis of the proliferation of seventeen-cell types showed that the effects of FNINs depend on their concentration and the existence of expressed integrins. Significant rhodamine-labeled FNIN2 fluorescence on the membranes of HeLa, HepG2, A498, and Du145 cells confirmed physical binding. Double coating with FNIN2 or 3 after polymerized dopamine (pDa) or polymerized tannic acid (pTA) precoating increased HBEpIC cell proliferation by 30–40 percent, suggesting FNINs potently affect primary cells. Furthermore, the proliferation of C2C12 myoblasts and human mesenchymal stem cells (MSCs) treated with FNINs was significantly increased in 2D/3D culture. FNINs also promoted MSC differentiation into osteoblasts. The results of this study offer a new approach to the production of core materials (e.g., cell culture medium components, scaffolds) for cell culture.

scholarly journals Real-time imaging of cellular forces using optical interference

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Andrew T. Meek ◽  
Nils M. Kronenberg ◽  
Andrew Morton ◽  
Philipp Liehm ◽  
Jan Murawski ◽  
...  
Keyword(s):  
Cell Culture ◽  
Real Time ◽  
High Throughput ◽  
Mechanical Activity ◽  
Dynamic Processes ◽  
Imaging Method ◽  
Neonatal Cardiomyocytes ◽  
A Cell ◽  
Cellular Forces ◽  
Time Acquisition

AbstractImportant dynamic processes in mechanobiology remain elusive due to a lack of tools to image the small cellular forces at play with sufficient speed and throughput. Here, we introduce a fast, interference-based force imaging method that uses the illumination of an elastic deformable microcavity with two rapidly alternating wavelengths to map forces. We show real-time acquisition and processing of data, obtain images of mechanical activity while scanning across a cell culture, and investigate sub-second fluctuations of the piconewton forces exerted by macrophage podosomes. We also demonstrate force imaging of beating neonatal cardiomyocytes at 100 fps which reveals mechanical aspects of spontaneous oscillatory contraction waves in between the main contraction cycles. These examples illustrate the wider potential of our technique for monitoring cellular forces with high throughput and excellent temporal resolution.

scholarly journals Orientia tsutsugamushi Inhibits Apoptosis of Macrophages by Retarding Intracellular Calcium Release

2002 ◽  
Vol 70 (8) ◽  
pp. 4692-4696 ◽  
Author(s):  
Mee-Kyung Kim ◽  
Seung-Yong Seong ◽  
Ju-Young Seoh ◽  
Tae-Hee Han ◽  
Hyeon-Je Song ◽  
...  
Keyword(s):  
Intracellular Calcium ◽  
Calcium Concentration ◽  
Calcium Release ◽  
Cytosolic Calcium ◽  
Orientia Tsutsugamushi ◽  
Cytosolic Calcium Concentration ◽  
Infected Cells ◽  
Intracellular Calcium Release ◽  
Calcium Redistribution ◽  
In Cells

ABSTRACT Orientia tsutsugamushi shows both pro- and antiapoptotic activities in infected vertebrate cells. Apoptosis of THP-1 cells induced by beauvericin was inhibited by O. tsutsugamushi infection. Beauvericin-induced calcium redistribution was significantly reduced and retarded in cells infected with O. tsutsugamushi. Antiapoptotic activities of O. tsutsugamushi in infected cells are most probably due to inhibition of the increase in the cytosolic calcium concentration.

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