scholarly journals Microvalve Bioprinting of MSC-Chondrocyte Co-Cultures

Cells ◽  
2021 ◽  
Vol 10 (12) ◽  
pp. 3329
Author(s):  
Joseph Dudman ◽  
Ana Marina Ferreira ◽  
Piergiorgio Gentile ◽  
Xiao Wang ◽  
Kenneth Dalgarno
Keyword(s):  
High Throughput Screening ◽  
Cell Types ◽  
Inhibitory Effect ◽  
Small Scale ◽  
Indirect Immunofluorescence Staining ◽  
Dense Tissue ◽  
Novel Method ◽  
Tissue Models ◽  
Transwell System

Recent improvements within the fields of high-throughput screening and 3D tissue culture have provided the possibility of developing in vitro micro-tissue models that can be used to study diseases and screen potential new therapies. This paper reports a proof-of-concept study on the use of microvalve-based bioprinting to create laminar MSC-chondrocyte co-cultures to investigate whether the use of MSCs in ACI procedures would stimulate enhanced ECM production by chondrocytes. Microvalve-based bioprinting uses small-scale solenoid valves (microvalves) to deposit cells suspended in media in a consistent and repeatable manner. In this case, MSCs and chondrocytes have been sequentially printed into an insert-based transwell system in order to create a laminar co-culture, with variations in the ratios of the cell types used to investigate the potential for MSCs to stimulate ECM production. Histological and indirect immunofluorescence staining revealed the formation of dense tissue structures within the chondrocyte and MSC-chondrocyte cell co-cultures, alongside the establishment of a proliferative region at the base of the tissue. No stimulatory or inhibitory effect in terms of ECM production was observed through the introduction of MSCs, although the potential for an immunomodulatory benefit remains. This study, therefore, provides a novel method to enable the scalable production of therapeutically relevant micro-tissue models that can be used for in vitro research to optimise ACI procedures.

scholarly journals Microvalve bioprinting of MSC-Chondrocyte Co-Cultures

2021 ◽  
Author(s):  
Joseph Dudman ◽  
Ana Marina Ferreira ◽  
Piergiorgio Gentile ◽  
Xiao Wang ◽  
Kenneth Dalgarno
Keyword(s):  
High Throughput Screening ◽  
Cellular Therapy ◽  
Cell Types ◽  
Inhibitory Effect ◽  
Proof Of Concept ◽  
Concept Study ◽  
Indirect Immunofluorescence Staining ◽  
Chondrocyte Implantation ◽  
Tissue Models

Recent improvements within the fields of high-throughput screening and 3D tissue culture have provided the possibility of developing in vitro micro-tissue models that can be used to study diseases and screen potential new therapies. This paper reports a proof of concept study on the use of microvalve-based bioprinting to create laminar MSC-chondrocyte co-cultures as an in vitro model of autologous chondrocyte implantation (ACI), an established cellular therapy for osteoarthritis. Microvalve-based bioprinting uses microvalves to deposit cells suspended in a liquid in a consistent and repeatable manner. In this case MSCs and chondrocytes have been sequentially deposited into an insert based transwell system in order to create a laminar co-culture, with variations in the ratios of the cell types used to investigate the potential for MSCs to stimulate improved repair. Histological and indirect immunofluorescence staining revealed the formation of dense tissue structures within the chondrocyte and MSC-chondrocyte cell co-cultures, alongside the establishment of a proliferative region at the base of the tissue. No stimulatory or inhibitory effect in terms of ECM production was observed through the introduction of MSCs, although the potential for an immunomodulatory benefit remains. This proof-of-concept study therefore provides a novel method to enable the scalable production of therapeutically relevant micro-tissue models that can be used for in vitro research to optimise ACI procedures.

scholarly journals Tenovin-6 impairs autophagy by inhibiting autophagic flux

2017 ◽  
Vol 8 (2) ◽  
pp. e2608-e2608 ◽  
Author(s):  
Hongfeng Yuan ◽  
Brandon Tan ◽  
Shou-Jiang Gao
Keyword(s):  
Cell Types ◽  
Inhibitory Effect ◽  
Lymphocytic Leukemia ◽  
Autophagic Flux ◽  
Dependent Manner ◽  
Autophagy Pathway ◽  
Sarcoma Cells ◽  
Regulatory Functions

Abstract Tenovin-6 has attracted significant interest because it activates p53 and inhibits sirtuins. It has anti-neoplastic effects on multiple hematopoietic malignancies and solid tumors in both in vitro and in vivo studies. Tenovin-6 was recently shown to impair the autophagy pathway in chronic lymphocytic leukemia cells and pediatric soft tissue sarcoma cells. However, whether tenovin-6 has a general inhibitory effect on autophagy and whether there is any involvement with SIRT1 and p53, both of which are regulators of the autophagy pathway, remain unclear. In this study, we have demonstrated that tenovin-6 increases microtubule-associated protein 1 light chain 3 (LC3-II) level in diverse cell types in a time- and dose-dependent manner. Mechanistically, the increase of LC3-II by tenovin-6 is caused by inhibition of the classical autophagy pathway via impairing lysosomal function without affecting the fusion between autophagosomes and lysosomes. Furthermore, we have revealed that tenovin-6 activation of p53 is cell type dependent, and tenovin-6 inhibition of autophagy is not dependent on its regulatory functions on p53 and SIRT1. Our results have shown that tenovin-6 is a potent autophagy inhibitor, and raised the precaution in interpreting results where tenovin-6 is used as an inhibitor of SIRT1.

scholarly journals Electrophysiological Analysis of Brain Organoids: Current Approaches and Advancements

2021 ◽  
Vol 14 ◽  
Author(s):  
Austin P. Passaro ◽  
Steven L. Stice
Keyword(s):  
High Throughput Screening ◽  
Disease Modeling ◽  
Cell Types ◽  
Full Potential ◽  
Two Dimensional ◽  
Physiological Relevance ◽  
Human Cell Cultures ◽  
Brain Organoid ◽  
Electrophysiological Methods

Brain organoids, or cerebral organoids, have become widely used to study the human brain in vitro. As pluripotent stem cell-derived structures capable of self-organization and recapitulation of physiological cell types and architecture, brain organoids bridge the gap between relatively simple two-dimensional human cell cultures and non-human animal models. This allows for high complexity and physiological relevance in a controlled in vitro setting, opening the door for a variety of applications including development and disease modeling and high-throughput screening. While technologies such as single cell sequencing have led to significant advances in brain organoid characterization and understanding, improved functional analysis (especially electrophysiology) is needed to realize the full potential of brain organoids. In this review, we highlight key technologies for brain organoid development and characterization, then discuss current electrophysiological methods for brain organoid analysis. While electrophysiological approaches have improved rapidly for two-dimensional cultures, only in the past several years have advances been made to overcome limitations posed by the three-dimensionality of brain organoids. Here, we review major advances in electrophysiological technologies and analytical methods with a focus on advances with applicability for brain organoid analysis.

scholarly journals CARPOOL: A library-based platform to rapidly identify next generation chimeric antigen receptors

2021 ◽  
Author(s):  
Taeyoon Kyung ◽  
Khloe S Gordon ◽  
Caleb R Perez ◽  
Patrick V Holec ◽  
Azucena Ramos ◽  
...  
Keyword(s):  
High Throughput Screening ◽  
Cell Types ◽  
Tumor Control ◽  
Antigen Receptors ◽  
Cell Therapies ◽  
Promising Tool ◽  
Signaling Domain ◽  
Screening Platform

CD19-targeted CAR therapies have successfully treated B cell leukemias and lymphomas, but many responders later relapse or experience toxicities. CAR intracellular domains (ICDs) are key to converting antigen recognition into anti-tumor effector functions. Despite the many possible immune signaling domain combinations that could be included in CARs, almost all CARs currently rely upon CD3𝛇, CD28, and/or 4-1BB signaling. To explore the signaling potential of CAR ICDs, we generated a library of 700,000 CD19 CAR molecules with diverse signaling domains and developed a high throughput screening platform to enable optimization of CAR signaling for anti-tumor functions. Our strategy identifies CARs with novel signaling domain combinations that elicit distinct T cell behaviors from a clinically available CAR, including enhanced proliferation and persistence, lower exhaustion, potent cytotoxicity in an in vitro tumor rechallenge condition, and comparable tumor control in vivo. This approach is readily adaptable to numerous disease models, cell types, and selection conditions, making it a promising tool for rapidly improving adoptive cell therapies and expanding their utility to new disease indications.

scholarly journals Systematic Identification of Antiprion Drugs by High-Throughput Screening Based on Scanning for Intensely Fluorescent Targets

2005 ◽  
Vol 79 (12) ◽  
pp. 7785-7791 ◽  
Author(s):  
Uwe Bertsch ◽  
Konstanze F. Winklhofer ◽  
Thomas Hirschberger ◽  
Jan Bieschke ◽  
Petra Weber ◽  
...  
Keyword(s):  
High Throughput ◽  
Conformational Changes ◽  
High Throughput Screening ◽  
Prion Diseases ◽  
Inhibitory Effect ◽  
Helical Conformation ◽  
Response Curves ◽  
Vitro Assay ◽  
Systematic Identification

ABSTRACT Conformational changes and aggregation of specific proteins are hallmarks of a number of diseases, like Alzheimer's disease, Parkinson's disease, and prion diseases. In the case of prion diseases, the prion protein (PrP), a neuronal glycoprotein, undergoes a conformational change from the normal, mainly alpha-helical conformation to a disease-associated, mainly beta-sheeted scrapie isoform (PrPSc), which forms amyloid aggregates. This conversion, which is crucial for disease progression, depends on direct PrPC/PrPSc interaction. We developed a high-throughput assay based on scanning for intensely fluorescent targets (SIFT) for the identification of drugs which interfere with this interaction at the molecular level. Screening of a library of 10,000 drug-like compounds yielded 256 primary hits, 80 of which were confirmed by dose response curves with half-maximal inhibitory effects ranging from 0.3 to 60 μM. Among these, six compounds displayed an inhibitory effect on PrPSc propagation in scrapie-infected N2a cells. Four of these candidate drugs share an N′-benzylidene-benzohydrazide core structure. Thus, the combination of high-throughput in vitro assay with the established cell culture system provides a rapid and efficient method to identify new antiprion drugs, which corroborates that interaction of PrPC and PrPSc is a crucial molecular step in the propagation of prions. Moreover, SIFT-based screening may facilitate the search for drugs against other diseases linked to protein aggregation.

scholarly journals Molecules incorporating a benzothiazole core scaffold inhibit the N-myristoyltransferase of Plasmodium falciparum

2007 ◽  
Vol 408 (2) ◽  
pp. 173-180 ◽  
Author(s):  
Paul W. Bowyer ◽  
Ruwani S. Gunaratne ◽  
Munira Grainger ◽  
Chrislaine Withers-Martinez ◽  
Sasala R. Wickramsinghe ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Plasmodium Falciparum ◽  
High Throughput ◽  
High Throughput Screening ◽  
Small Scale ◽  
Expression And Purification ◽  
Purification System ◽  
Pure Protein ◽  
Ic50 Values

Recombinant N-myristoyltransferase of Plasmodium falciparum (termed PfNMT) has been used in the development of a SPA (scintillation proximity assay) suitable for automation and high-throughput screening of inhibitors against this enzyme. The ability to use the SPA has been facilitated by development of an expression and purification system which yields considerably improved quantities of soluble active recombinant PfNMT compared with previous studies. Specifically, yields of pure protein have been increased from 12 μg·l−1 to >400 μg·l−1 by use of a synthetic gene with codon usage optimized for expression in an Escherichia coli host. Preliminary small-scale ‘piggyback’ inhibitor studies using the SPA have identified a family of related molecules containing a core benzothiazole scaffold with IC50 values <50 μM, which demonstrate selectivity over human NMT1. Two of these compounds, when tested against cultured parasites in vitro, reduced parasitaemia by >80% at a concentration of 10 μM.

scholarly journals High Throughput Screening Method for Identification of New Lipofection Reagents

2001 ◽  
Vol 6 (4) ◽  
pp. 245-254 ◽  
Author(s):  
Anne E. Regelin ◽  
Erhard Fernholz ◽  
Harald F. Krug ◽  
Ulrich Massing
Keyword(s):  
High Throughput Screening ◽  
Screening Method ◽  
Genetic Material ◽  
Cell Types ◽  
Cationic Lipids ◽  
Adherent Cell ◽  
Gene Assay

Lipofection, the transfer of genetic material into cells by means of cationic lipids, is of growing interest for in vitro and in vivo approaches. In order to identify ideal lipofection reagents in a HTS, we have developed an automated lipofection method for the transfer of reporter genes into cells and for determination of the lipofection results. The method has specifically been designed and optimized for 96-well microtiter plates and can successfully be carried out by a pipetting robot with accessory equipment. It consists of two separate parts: (1) pretransfection (preparation of liposomes, formation of lipoplexes, and lipoplex transfer to the cells) and (2) posttransfection (determination of the reporter enzyme activity and the protein content of the transfected cells). Individual steps of the lipofection method were specifically optimized—for example, lipoplex formation and incubation time as well as cell lysis, cell cultivating, and the reporter gene assay. The HTS method facilitates characterization of the transfection properties (efficiency and cytotoxicity) of large numbers of (cationic) lipids in various adherent cell types.

scholarly journals A high-throughput microfluidic bilayer co-culture platform to study endothelial-pericyte interactions

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Miles T. Rogers ◽  
Ashley L. Gard ◽  
Robert Gaibler ◽  
Thomas J. Mulhern ◽  
Rivka Strelnikov ◽  
...  
Keyword(s):  
High Throughput ◽  
Fluid Shear Stress ◽  
Cell Types ◽  
Fluid Shear ◽  
Multiple Cell ◽  
Cell Type Specific ◽  
On Chip ◽  
Tissue Models ◽  
Macromolecular Permeability

AbstractMicrophysiological organ-on-chip models offer the potential to improve the prediction of drug safety and efficacy through recapitulation of human physiological responses. The importance of including multiple cell types within tissue models has been well documented. However, the study of cell interactions in vitro can be limited by complexity of the tissue model and throughput of current culture systems. Here, we describe the development of a co-culture microvascular model and relevant assays in a high-throughput thermoplastic organ-on-chip platform, PREDICT96. The system consists of 96 arrayed bilayer microfluidic devices containing retinal microvascular endothelial cells and pericytes cultured on opposing sides of a microporous membrane. Compatibility of the PREDICT96 platform with a variety of quantifiable and scalable assays, including macromolecular permeability, image-based screening, Luminex, and qPCR, is demonstrated. In addition, the bilayer design of the devices allows for channel- or cell type-specific readouts, such as cytokine profiles and gene expression. The microvascular model was responsive to perturbations including barrier disruption, inflammatory stimulation, and fluid shear stress, and our results corroborated the improved robustness of co-culture over endothelial mono-cultures. We anticipate the PREDICT96 platform and adapted assays will be suitable for other complex tissues, including applications to disease models and drug discovery.

Extracellular nucleotide receptor inhibits AVP-stimulated water permeability in inner medullary collecting duct

1995 ◽  
Vol 269 (6) ◽  
pp. F863-F869 ◽  
Author(s):  
B. K. Kishore ◽  
C. L. Chou ◽  
M. A. Knepper
Keyword(s):  
Water Permeability ◽  
Collecting Duct ◽  
Cell Types ◽  
Inhibitory Effect ◽  
Nucleotide Receptors ◽  
Nucleotide Receptor ◽  
Camp Analogue ◽  
Osmotic Water ◽  
Camp Levels

The P2u class of nucleotide receptors is linked to mobilization of intracellular Ca2+ in many cell types, including the renal collecting duct cells. In the present studies, we examined the effects of nucleotides (ATP, UTP, and ADP; 10 microM each) on the arginine vasopressin (AVP, 0.1 nM)-stimulated osmotic water permeability (Pf) in in vitro perfused terminal inner medullary collecting ducts (IMCD) of rat. ATP or UTP, when added to the bath, decreased the AVP-stimulated Pf by approximately 40%. These effects were reversible upon withdrawal of the nucleotides. However, addition of ADP to the bath or sham exchange of the bath had no significant effect on the Pf. Furthermore, ATP did not have any significant effect on the Pf stimulated either by a membrane-permeant, nonhydrolyzable adenosine 3',5'-cyclic monophosphate (cAMP) analogue [8-(4-chlorophenylthio)-cAMP, 0.1 mM] o by forskolin (1 microM). In line with these findings, ATP decreased the AVP-stimulated cAMP levels in IMCD suspensions to approximately 68%. In addition, ATP did not exert an inhibitory effect on the AVP-stimulated Pf in the presence of calphostin C (150 nM), an inhibitor of protein kinase C. These results lead us to conclude the following: 1) agonist occupancy of the putative nucleotide receptor in the terminal IMCD causes an inhibition of AVP-stimulated Pf; and 2) this effect is due to a decrease in cellular cAMP levels, most likely resulting from activation of the phosphoinositide signaling pathway.

scholarly journals Ribavirin shows antiviral activity against SARS-CoV-2 and downregulates the activity of TMPRSS2 and the expression of ACE2 in vitro

2021 ◽  
pp. 1-12 ◽  
Author(s):  
Mehmet Altay Unal ◽  
Ceylan Verda Bitirim ◽  
Gokce Yagmur Summak ◽  
Sidar Bereketoglu ◽  
Inci Cevher Zeytin ◽  
...  
Keyword(s):  
Antiviral Activity ◽  
Broad Spectrum ◽  
In Silico ◽  
Antiviral Drug ◽  
In Silico Analysis ◽  
Cell Types ◽  
Suppressive Effect ◽  
Inhibitory Effect ◽  
Molecular Techniques

Ribavirin is a guanosine analog with broad-spectrum antiviral activity against RNA viruses. Based on this, we aimed to show the anti-SARS-CoV-2 activity of this drug molecule via in vitro, in silico, and molecular techniques. Ribavirin showed antiviral activity in Vero E6 cells following SARS-CoV-2 infection, whereas the drug itself did not show any toxic effect over the concentration range tested. In silico analysis suggested that ribavirin has a broad-spectrum impact on SARS-CoV-2, acting at different viral proteins. According to the detailed molecular techniques, ribavirin was shown to decrease the expression of TMPRSS2 at both mRNA and protein levels 48 h after treatment. The suppressive effect of ribavirin in ACE2 protein expression was shown to be dependent on cell types. Finally, proteolytic activity assays showed that ribavirin also showed an inhibitory effect on the TMPRSS2 enzyme. Based on these results, we hypothesized that ribavirin may inhibit the expression of TMPRSS2 by modulating the formation of inhibitory G-quadruplex structures at the TMPRSS2 promoter. As a conclusion, ribavirin is a potential antiviral drug for the treatment against SARS-CoV-2, and it interferes with the effects of TMPRSS2 and ACE2 expression.

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