scholarly journals Picosecond Laser Processing of Photosensitive Glass for Generation of Biologically Relevant Microenvironments

2020 ◽  
Vol 10 (24) ◽  
pp. 8947
Author(s):  
Florin Jipa ◽  
Stefana Orobeti ◽  
Cristian Butnaru ◽  
Marian Zamfirescu ◽  
Emanuel Axente ◽  
...  
Keyword(s):  
Single Cell ◽  
Picosecond Laser ◽  
High Repetition Rate ◽  
Cellular Adhesion ◽  
Microfluidic Channels ◽  
Biologically Relevant ◽  
Photosensitive Glass ◽  
Smart Surfaces ◽  
Organ Models

Various material processing techniques have been proposed for fabrication of smart surfaces that can modulate cellular behavior and address specific clinical issues. Among them, laser-based technologies have attracted growing interest due to processing versatility. Latest development of ultrashort pulse lasers with pulse widths from several tens of femtoseconds (fs) to several picoseconds (ps) allows clean microfabrication of a variety of materials at micro- and nanoscale both at surface and in volume. In this study, we addressed the possibility of 3D microfabrication of photosensitive glass (PG) by high repetition rate ps laser-assisted etching (PLAE) to improve the fabrication efficiency for the development of useful tools to be used for specific biological applications. Microfluidic structures fabricated by PLAE should provide the flow aspects, 3D characteristics, and possibility of producing functional structures to achieve the biologically relevant microenvironments. Specifically, the microfluidic structures could induce cellular chemotaxis over extended periods in diffusion-based gradient media. More importantly, the 3D characteristics could reproduce capillaries for in vitro testing of relevant organ models. Single cell trapping and analysis by using the fabricated microfluidic structures are also essential for understanding individual cell behavior within the same population. To this end, this paper demonstrates: (1) generation of 3D structures in glass volume or on surface for fabrication of microfluidic channels, (2) subtractive 3D surface patterning to create patterned molds in a controlled manor for casting polydimethylsiloxane (PDMS) structures and developing single cell microchambers, and (3) designing glass photo-masks to be used for sequel additive patterning of biocompatible nanomaterials with controlled shapes, sizes, and periodicity. Mesenchymal stem cells grown on laser-processed glass surfaces revealed no sign of cytotoxicity, while a collagen thin coating improved cellular adhesion.

scholarly journals High Repetition Rate UV versus VIS Picosecond Laser Fabrication of 3D Microfluidic Channels Embedded in Photosensitive Glass

Nanomaterials ◽  
2018 ◽  
Vol 8 (8) ◽  
pp. 583 ◽  
Author(s):  
Florin Jipa ◽  
Stefana Iosub ◽  
Bogdan Calin ◽  
Emanuel Axente ◽  
Felix Sima ◽  
...  
Keyword(s):  
Repetition Rate ◽  
Three Dimensional ◽  
Picosecond Laser ◽  
Basic Structure ◽  
High Repetition Rate ◽  
Microfluidic Channels ◽  
Hollow Structures ◽  
High Repetition ◽  
Glass Processing ◽  
Fs Laser

Glass is an alternative solution to polymer for the fabrication of three-dimensional (3D) microfluidic biochips. Femtosecond (fs) lasers are nowadays the most promising tools for transparent glass processing. Specifically, the multiphoton process induced by fs pulses enables fabrication of embedded 3D channels with high precision. The subtractive fabrication process creating 3D hollow structures in glass, known as fs laser-assisted etching (FLAE), is based on selective removal of the laser-modified regions by successive chemical etching in diluted hydrofluoric acid solutions. In this work we demonstrate the possibility to generate embedded hollow channels in photosensitive Foturan glass volume by high repetition rate picosecond (ps) laser-assisted etching (PLAE). In particular, the influence of the critical irradiation doses and etching rates are discussed in comparison of two different wavelengths of ultraviolet (355 nm) and visible (532 nm) ranges. Fast and controlled fabrication of a basic structure composed of an embedded micro-channel connected with two open reservoirs, commonly used in the biochip design, are achieved inside glass. Distinct advantages such as good aspect-ratio, reduced processing time for large areas, and lower fabrication cost are evidenced.

scholarly journals Enhancing biological signals and detection rates in single-cell RNA-seq experiments with cDNA library equalization

2020 ◽  
Author(s):  
Rhonda Bacher ◽  
Li-Fang Chu ◽  
Cara Argus ◽  
Jennifer M. Bolin ◽  
Parker Knight ◽  
...  
Keyword(s):  
Single Cell ◽  
Cdna Libraries ◽  
Sequencing Data ◽  
Specific Expression ◽  
Detection Rates ◽  
Biologically Relevant ◽  
Expression Variability ◽  
Biological Signals ◽  
Experimental Protocols

AbstractConsiderable effort has been devoted to refining experimental protocols having reduced levels of technical variability and artifacts in single-cell RNA-sequencing data (scRNA-seq). We here present evidence that equalizing the concentration of cDNA libraries prior to pooling, a step not consistently performed in single-cell experiments, improves gene detection rates, enhances biological signals, and reduces technical artifacts in scRNA-seq data. To evaluate the effect of equalization on various protocols, we developed Scaffold, a simulation framework that models each step of an scRNA-seq experiment. Numerical experiments demonstrate that equalization reduces variation in sequencing depth and gene-specific expression variability. We then performed a set of experiments in vitro with and without the equalization step and found that equalization increases the number of genes that are detected in every cell by 17-31%, improves discovery of biologically relevant genes, and reduces nuisance signals associated with cell cycle. Further support is provided in an analysis of publicly available data.

Immunocartography: Charting vaccine-driven immunity by applying single cell proteomics to an in vitro human model

2021 ◽  
pp. 113083
Author(s):  
Jessica S. Duprez ◽  
Michael Cohen ◽  
Stephen Li ◽  
Derek Wilson ◽  
Roger H. Brookes ◽  
...  
Keyword(s):  
Single Cell ◽  
Human Model

scholarly journals Single-cell RNA sequencing reveals ex vivo signatures of SARS-CoV-2-reactive T cells through ‘reverse phenotyping’

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
David S. Fischer ◽  
Meshal Ansari ◽  
Karolin I. Wagner ◽  
Sebastian Jarosch ◽  
Yiqi Huang ◽  
...  
Keyword(s):  
T Cells ◽  
Single Cell ◽  
Rna Sequencing ◽  
Ex Vivo ◽  
Severe Disease ◽  
Human Leukocyte ◽  
Cell Receptor ◽  
Single Cell Rna Sequencing

AbstractThe in vivo phenotypic profile of T cells reactive to severe acute respiratory syndrome (SARS)-CoV-2 antigens remains poorly understood. Conventional methods to detect antigen-reactive T cells require in vitro antigenic re-stimulation or highly individualized peptide-human leukocyte antigen (pHLA) multimers. Here, we use single-cell RNA sequencing to identify and profile SARS-CoV-2-reactive T cells from Coronavirus Disease 2019 (COVID-19) patients. To do so, we induce transcriptional shifts by antigenic stimulation in vitro and take advantage of natural T cell receptor (TCR) sequences of clonally expanded T cells as barcodes for ‘reverse phenotyping’. This allows identification of SARS-CoV-2-reactive TCRs and reveals phenotypic effects introduced by antigen-specific stimulation. We characterize transcriptional signatures of currently and previously activated SARS-CoV-2-reactive T cells, and show correspondence with phenotypes of T cells from the respiratory tract of patients with severe disease in the presence or absence of virus in independent cohorts. Reverse phenotyping is a powerful tool to provide an integrated insight into cellular states of SARS-CoV-2-reactive T cells across tissues and activation states.

scholarly journals Investigation of the Wilson gene ATP7B transcriptional start site and the effect of core promoter alterations

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Clemens Höflich ◽  
Angela Brieger ◽  
Stefan Zeuzem ◽  
Guido Plotz
Keyword(s):  
Wilson Disease ◽  
Transcription Initiation ◽  
Transcriptional Activity ◽  
Core Promoter ◽  
Transcriptional Start Site ◽  
Copper Metabolism ◽  
Biologically Relevant ◽  
The Core ◽  
Translational Start

AbstractPathogenic genetic variants in the ATP7B gene cause Wilson disease, a recessive disorder of copper metabolism showing a significant variability in clinical phenotype. Promoter mutations have been rarely reported, and controversial data exist on the site of transcription initiation (the core promoter). We quantitatively investigated transcription initiation and found it to be located in immediate proximity of the translational start. The effects human single-nucleotide alterations of conserved bases in the core promoter on transcriptional activity were moderate, explaining why clearly pathogenic mutations within the core promoter have not been reported. Furthermore, the core promoter contains two frequent polymorphisms (rs148013251 and rs2277448) that could contribute to phenotypical variability in Wilson disease patients with incompletely inactivating mutations. However, neither polymorphism significantly modulated ATP7B expression in vitro, nor were copper household parameters in healthy probands affected. In summary, the investigations allowed to determine the biologically relevant site of ATP7B transcription initiation and demonstrated that genetic variations in this site, although being the focus of transcriptional activity, do not contribute significantly to Wilson disease pathogenesis.

scholarly journals Uncovering transcriptional dark matter via gene annotation independent single-cell RNA sequencing analysis

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Michael F. Z. Wang ◽  
Madhav Mantri ◽  
Shao-Pei Chou ◽  
Gaetano J. Scuderi ◽  
David W. McKellar ◽  
...  
Keyword(s):  
Single Cell ◽  
Genome Annotation ◽  
Gene Annotation ◽  
Active Regions ◽  
Sequencing Analysis ◽  
Biologically Relevant ◽  
Mole Rat ◽  
Genome Annotations ◽  
Cell Expression ◽  
High Quality Genome

AbstractConventional scRNA-seq expression analyses rely on the availability of a high quality genome annotation. Yet, as we show here with scRNA-seq experiments and analyses spanning human, mouse, chicken, mole rat, lemur and sea urchin, genome annotations are often incomplete, in particular for organisms that are not routinely studied. To overcome this hurdle, we created a scRNA-seq analysis routine that recovers biologically relevant transcriptional activity beyond the scope of the best available genome annotation by performing scRNA-seq analysis on any region in the genome for which transcriptional products are detected. Our tool generates a single-cell expression matrix for all transcriptionally active regions (TARs), performs single-cell TAR expression analysis to identify biologically significant TARs, and then annotates TARs using gene homology analysis. This procedure uses single-cell expression analyses as a filter to direct annotation efforts to biologically significant transcripts and thereby uncovers biology to which scRNA-seq would otherwise be in the dark.

Generation of three-dimensional pannus-like tissues in vitro from single cell suspensions of synovial fluid cells from arthritis patients

2004 ◽  
Vol 24 (2) ◽  
pp. 71-76 ◽  
Author(s):  
Samuel Solomon ◽  
Madhan Masilamani ◽  
Subhasis Mohanty ◽  
J�rg E. Schwab ◽  
Eva-Maria Boneberg ◽  
...  
Keyword(s):  
Synovial Fluid ◽  
Single Cell ◽  
Three Dimensional ◽  
Cell Suspensions ◽  
Synovial Fluid Cells

scholarly journals Impact of cigarette versus electronic cigarette aerosol conditioned media on aortic endothelial cells in a microfluidic cardiovascular model

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Om Makwana ◽  
Gina A. Smith ◽  
Hannah E. Flockton ◽  
Gary P. Watters ◽  
Frazer Lowe ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Human Monocyte ◽  
Conditioned Media ◽  
Electronic Cigarette ◽  
Microfluidic Channels ◽  
Adhesion Assay ◽  
Monocyte Adhesion ◽  
Aortic Endothelial Cells ◽  
Aortic Endothelial

AbstractAtherosclerosis is a complex process involving progressive pathological events, including monocyte adhesion to the luminal endothelial surface. We have developed a functional in vitro adhesion assay using BioFlux microfluidic technology to investigate THP-1 (human acute monocytic leukaemia cell) monocyte adhesion to human aortic endothelial cells (HAECs). The effect of whole smoke conditioned media (WSCM) generated from University of Kentucky reference cigarette 3R4F, electronic cigarette vapour conditioned media (eVCM) from an electronic nicotine delivery system (ENDS) product (Vype ePen) and nicotine on monocyte adhesion to HAECs was evaluated. Endothelial monolayers were grown in microfluidic channels and exposed to 0–1500 ng/mL nicotine or nicotine equivalence of WSCM or eVCM for 24 h. Activated THP-1 cells were perfused through the channels and a perfusion, adhesion period and wash cycle performed four times with increasing adhesion period lengths (10, 20, 30 and 40 min). THP-1 cell adhesion was quantified by counting adherent cells. WSCM induced dose-dependent increases in monocyte adhesion compared to vehicle control. No such increases were observed for eVCM or nicotine. Adhesion regulation was linked to increased ICAM-1 protein expression. Staining of ICAM-1 in HAECs and CD11b (MAC-1) in THP-1 cells demonstrated adhesion molecule co-localisation in BioFlux plates. The ICAM-1 adhesion response to WSCM was downregulated by transfecting HAECs with ICAM-1 siRNA. We conclude that the BioFlux system is able to model human monocyte adhesion to primary human endothelial cells in vitro and WSCM drives the greatest increase in monocyte adhesion via a mechanism involving endothelial ICAM-1 expression.

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