Fluid Lipid Multilayer Stabilization by Tetraethyl Orthosilicate for Underwater AFM Characterization and Cell Culture Applications

MRS Advances ◽  
2017 ◽  
Vol 2 (57) ◽  
pp. 3553-3558
Author(s):  
Aubrey E. Kusi-Appiah ◽  
Troy W. Lowry ◽  
Nicholas Vafai ◽  
David H. Van Winkle ◽  
Steven Lenhert
Keyword(s):  
Cell Culture ◽  
High Throughput ◽  
High Throughput Screening ◽  
Cell Types ◽  
Tetraethyl Orthosilicate ◽  
Force Microscopy ◽  
Essential Step ◽  
Atomic Force ◽  
Lipid Films ◽  
Cell Assays

ABSTRACTStabilization of surface supported fluid lipid multilayers for underwater characterization is an essential step in making them useful for scalable cell culture applications such as high throughput screening. To this end, we used tetraethyl orthosilicate (TEOS), recently shown to stabilize fluid lipid films while maintaining their fluidity and functionality under water, to stabilize lipid multilayer micropatterns of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). The treated multilayers were immersed under water and successfully imaged by atomic force microscopy (AFM), a difficult feat to perform on fluid lipid multilayers without TEOS treatment. The treated lipid multilayer showed an average swelling of approximately 18% in water but remained stable during the imaging process. The TEOS-treated lipid multilayers also proved compatible with cell culture as HeLa, MDCK, and HEK cell types all adhered and grew in high numbers over the multilayers. The results obtained here open the door to the use of fluid lipid multilayers in biotechnology applications such as microarray based high throughput cell assays.

scholarly journals Development of NS3/4A Protease-Based Reporter Assay Suitable for Efficiently Assessing Hepatitis C Virus Infection

2009 ◽  
Vol 53 (11) ◽  
pp. 4825-4834 ◽  
Author(s):  
Kao-Lu Pan ◽  
Jin-Ching Lee ◽  
Hsing-Wen Sung ◽  
Teng-Yuang Chang ◽  
John T.-A. Hsu
Keyword(s):  
Cell Culture ◽  
Life Cycle ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Virus Infection ◽  
High Throughput ◽  
High Throughput Screening ◽  
Reporter System ◽  
Viral Protease ◽  
Peptide Cleavage

ABSTRACT A cell culture system for the production of hepatitis C virus (HCV) whole virions has greatly accelerated studies of the virus life cycle and the discovery of anti-HCV agents. However, the quantification of the HCV titers in a whole-virus infection/replication system currently relies mostly on reverse transcription-PCR or immunofluorescence assay, which would be cumbersome for high-throughput drug screening. To overcome this problem, this study has generated a novel cell line, Huh7.5-EG(Δ4B5A)SEAP, that carries a dual reporter, EG(Δ4B5A)SEAP. The EG(Δ4B5A)SEAP reporter is a viral protease-cleavable fusion protein in which the enhanced green fluorescence protein is linked to secreted alkaline phosphatase (SEAP) in frame via Δ4B5A, a short peptide cleavage substrate for NS3/4A viral protease. This study demonstrates that virus replication/infection in the Huh7.5-EG(Δ4B5A)SEAP cells can be quantitatively indicated by measuring the SEAP activity in cell culture medium. The levels of SEAP released from HCV-infected Huh7.5-EG(Δ4B5A)SEAP cells correlated closely with the amounts of HCV in the inocula. The Huh7.5-EG(Δ4B5A)SEAP cells were also shown to be a suitable host for the discovery of anti-HCV inhibitors by using known compounds that target multiple stages of the HCV life cycle. The Z′-factor of this assay ranged from 0.64 to 0.74 in 96-well plates, indicating that this reporter system is suitable for high-throughput screening of prospective anti-HCV agents.

scholarly journals Complex Tumor Spheroids, a Tissue-Mimicking Tumor Model, for Drug Discovery and Precision Medicine

2021 ◽  
pp. 247255522110383
Author(s):  
Gurmeet Kaur ◽  
David M. Evans ◽  
Beverly A. Teicher ◽  
Nathan P. Coussens
Keyword(s):  
Drug Discovery ◽  
Precision Medicine ◽  
Cell Lines ◽  
High Throughput ◽  
High Throughput Screening ◽  
Cell Types ◽  
Response To Therapy ◽  
Cancer Drug ◽  
Malignant Cells ◽  
Pancreatic Cell

Malignant tumors are complex tissues composed of malignant cells, vascular cells, structural mesenchymal cells including pericytes and carcinoma-associated fibroblasts, infiltrating immune cells, and others, collectively called the tumor stroma. The number of stromal cells in a tumor is often much greater than the number of malignant cells. The physical associations among all these cell types are critical to tumor growth, survival, and response to therapy. Most cell-based screens for cancer drug discovery and precision medicine validation use malignant cells in isolation as monolayers, embedded in a matrix, or as spheroids in suspension. Medium- and high-throughput screening with multiple cell lines requires a scalable, reproducible, robust cell-based assay. Complex spheroids include malignant cells and two normal cell types, human umbilical vein endothelial cells and highly plastic mesenchymal stem cells, which rapidly adapt to the malignant cell microenvironment. The patient-derived pancreatic adenocarcinoma cell line, K24384-001-R, was used to explore complex spheroid structure and response to anticancer agents in a 96-well format. We describe the development of the complex spheroid assay as well as the growth and structure of complex spheroids over time. Subsequently, we demonstrate successful assay miniaturization to a 384-well format and robust performance in a high-throughput screen. Implementation of the complex spheroid assay was further demonstrated with 10 well-established pancreatic cell lines. By incorporating both human stromal and tumor components, complex spheroids might provide an improved model for tumor response in vivo.

Patterned superhydrophobic surfaces to process and characterize biomaterials and 3D cell culture

2018 ◽  
Vol 5 (3) ◽  
pp. 379-393 ◽  
Author(s):  
A. I. Neto ◽  
P. A. Levkin ◽  
J. F. Mano
Keyword(s):  
Cell Culture ◽  
High Throughput ◽  
High Throughput Screening ◽  
3D Cell Culture ◽  
Superhydrophobic Surfaces ◽  
Technological Breakthrough ◽  
Large Numbers

Microarrays are a technological breakthrough for high-throughput screening of large numbers of assays.

Atomic force microscopy: seeing molecules of lipid and immunoglobulin

1991 ◽  
Vol 37 (9) ◽  
pp. 1497-1501 ◽  
Author(s):  
H G Hansma ◽  
A L Weisenhorn ◽  
A B Edmundson ◽  
H E Gaub ◽  
P K Hansma
Keyword(s):  
Immunoglobulin M ◽  
Biological Molecules ◽  
Ammonium Bromide ◽  
Force Microscopy ◽  
Atomic Force ◽  
Head Groups ◽  
Afm Imaging ◽  
Wide Range ◽  
Lipid Films ◽  
Active Imaging

Abstract The atomic force microscope (AFM) can image individual molecules by raster-scanning a sharp tip over a surface. In this paper we present molecular-resolution images of immunoglobulin M (IgM) and of ultraviolet light-polymerized films of the lipid dimethyl-bis(pentacosadiynoyloxyethyl) ammonium bromide ("BRONCO"). The polar head groups of individual lipid molecules can be resolved on the surface of this and other lipid films. These lipid films also provide a good substrate for AFM imaging of DNA and of other molecules such as antibodies. Because the AFM scans surfaces, it is most often successful at imaging either molecules that can form an array on a surface or molecules that are quite firmly attached to a surface. The ability of the AFM to operate under water, buffers, and other liquids makes it possible to study biological molecules under conditions in which they are physiologically active. Imaging of the actual molecular process of fibrin polymerization shows the potential of the AFM for studying biological processes. In the six years since its invention, the AFM has excited much interest and has imaged molecules in a wide range of systems.

scholarly journals Actin-binding compounds, previously discovered by FRET-based high-throughput screening, differentially affect skeletal and cardiac muscle

2020 ◽  
Vol 295 (41) ◽  
pp. 14100-14110 ◽  
Author(s):  
Piyali Guhathakurta ◽  
Lien A. Phung ◽  
Ewa Prochniewicz ◽  
Sarah Lichtenberger ◽  
Anna Wilson ◽  
...  
Keyword(s):  
Cardiac Muscle ◽  
High Throughput ◽  
High Throughput Screening ◽  
Cell Types ◽  
Actin Binding ◽  
Time Resolved ◽  
Biochemical Assays ◽  
Numerous Cell ◽  
Cardiac Muscles ◽  
And Function

Actin's interactions with myosin and other actin-binding proteins are essential for cellular viability in numerous cell types, including muscle. In a previous high-throughput time-resolved FRET (TR-FRET) screen, we identified a class of compounds that bind to actin and affect actomyosin structure and function. For clinical utility, it is highly desirable to identify compounds that affect skeletal and cardiac muscle differently. Because actin is more highly conserved than myosin and most other muscle proteins, most such efforts have not targeted actin. Nevertheless, in the current study, we tested the specificity of the previously discovered actin-binding compounds for effects on skeletal and cardiac α-actins as well as on skeletal and cardiac myofibrils. We found that a majority of these compounds affected the transition of monomeric G-actin to filamentous F-actin, and that several of these effects were different for skeletal and cardiac actin isoforms. We also found that several of these compounds affected ATPase activity differently in skeletal and cardiac myofibrils. We conclude that these structural and biochemical assays can be used to identify actin-binding compounds that differentially affect skeletal and cardiac muscles. The results of this study set the stage for screening of large chemical libraries for discovery of novel compounds that act therapeutically and specifically on cardiac or skeletal muscle.

scholarly journals Dose-Response Modeling of High-Throughput Screening Data

2009 ◽  
Vol 14 (10) ◽  
pp. 1216-1227 ◽  
Author(s):  
Fred Parham ◽  
Chris Austin ◽  
Noel Southall ◽  
Ruili Huang ◽  
Raymond Tice ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Statistical Approach ◽  
Statistical Tests ◽  
Cell Types ◽  
Large Numbers ◽  
Rodent Cell ◽  
Response Modeling ◽  
Significant Concentration

The National Toxicology Program is developing a high-throughput screening (HTS) program to set testing priorities for compounds of interest, to identify mechanisms of action, and potentially to develop predictive models for human toxicity. This program will generate extensive data on the activity of large numbers of chemicals in a wide variety of biochemical- and cell-based assays. The first step in relating patterns of response among batteries of HTS assays to in vivo toxicity is to distinguish between positive and negative compounds in individual assays. Here, the authors report on a statistical approach developed to identify compounds positive or negative in an HTS cytotoxicity assay based on data collected from screening 1353 compounds for concentration-response effects in 9 human and 4 rodent cell types. In this approach, the authors develop methods to normalize the data (removing bias due to the location of the compound on the 1536-well plates used in the assay) and to analyze for concentration-response relationships. Various statistical tests for identifying significant concentration-response relationships and for addressing reproducibility are developed and presented.

scholarly journals Chemotherapy exposure increases leukemia cell stiffness

Blood ◽  
2006 ◽  
Vol 109 (8) ◽  
pp. 3505-3508 ◽  
Author(s):  
Wilbur A. Lam ◽  
Michael J. Rosenbluth ◽  
Daniel A. Fletcher
Keyword(s):  
Cell Death ◽  
Leukemia Cell ◽  
Vascular Complications ◽  
Cell Types ◽  
Cell Stiffness ◽  
Microfluidic Channels ◽  
Cell Deformability ◽  
Force Microscopy ◽  
Atomic Force ◽  
Acute Myeloid Leukemia Cells

Abstract Deformability of blood cells is known to influence vascular flow and contribute to vascular complications. Medications for hematologic diseases have the potential to modulate these complications if they alter blood cell deformability. Here we report the effect of chemotherapy on leukemia cell mechanical properties. Acute lymphoblastic and acute myeloid leukemia cells were incubated with standard induction chemotherapy, and individual cell stiffness was tracked with atomic force microscopy. When exposed to dexamethasone or daunorubicin, leukemia cell stiffness increased by nearly 2 orders of magnitude, which decreased their passage through microfluidic channels. This stiffness increase occurred before caspase activation and peaked after completion of cell death, and the rate of stiffness increase depended on chemotherapy type. Stiffening with cell death occurred for all cell types investigated and may be due to dynamic changes in the actin cytoskeleton. These observations suggest that chemotherapy itself may increase the risk of vascular complications in acute leukemia.

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