scholarly journals Defining cell culture conditions to improve human norovirus infectivity assays

2013 ◽  
Vol 67 (4) ◽  
pp. 863-868 ◽  
Author(s):  
T. M. Straub ◽  
J. R. Hutchison ◽  
R. A. Bartholomew ◽  
C. O. Valdez ◽  
N. B. Valentine ◽  
...  
Keyword(s):  
Cell Line ◽  
Three Dimensional ◽  
Culture Conditions ◽  
Qrt Pcr ◽  
Infectivity Assay ◽  
Polymerase Chain ◽  
Single Laboratory ◽  
Cell Culture Conditions ◽  
Human Intestinal Cells

Significant difficulties remain for determining whether human noroviruses (hNoV) recovered from water, food, and environmental samples are infectious. Three-dimensional (3-D) tissue culture of human intestinal cells has shown promise in developing an infectivity assay, but reproducibility, even within a single laboratory, remains problematic. From the literature and our observations, we hypothesized that the common factors that lead to more reproducible hNoV infectivity in vitro requires that the cell line be (1) of human gastrointestinal origin, (2) expresses apical microvilli, and (3) be a positive secretor cell line. The C2BBe1 cell line, which is a brush-border producing clone of Caco-2, meets these three criteria. When challenged with Genogroup II viruses, we observed a 2 Log10 increase in viral RNA titer. A passage experiment with GII viruses showed evidence of the ability to propagate hNoV by both quantitative reverse transcription polymerase chain reaction (qRT-PCR) and microscopy. In our hands, using 3-D C2BBe1 cells improves reproducibility of the infectivity assay for hNoV, but the assay can still be variable. Two sources of variability include the cells themselves (mixed phenotypes of small and large intestine) and initial titer measurements using qRT-PCR that measures all RNA vs. plaque assays that measure infectious virus.

CHEMOTHERAPEUTIC POTENTIAL OF NOVEL XANTHONE SOURCED FROM SWERTIA CHIRATA AGAINST SKIN CARCINOGENESIS

2020 ◽  
pp. 84-88
Author(s):  
ATISH BARUA ◽  
PRITHA CHOUDHURY ◽  
CHINMAY KUMAR PANDA ◽  
PROSENJIT SAHA
Keyword(s):  
Skin Cancer ◽  
Antitumor Activity ◽  
Cell Line ◽  
Cell Lines ◽  
Cancer Cell ◽  
Cancer Cell Lines ◽  
Self Renewal ◽  
Qrt Pcr ◽  
Swertia Chirata

Objective: Swertia chirata forms a rich source of bio-active compounds, among which xanthones form an important part. Among the xanthones present in it, 1,5,8 Tri-hydroxy-3-methoxy xanthone (TMX) was found to be the most active. The present study aims to evaluate the chemotherapeutic potential of it against metastatic skin cancer cell lines. Methods: In this study, the antitumor activity of TMX (the active component of chirata plant) was evaluated in A431, SKMEL-5, and A375 cell line by using in-vitro assays such as cell viability assay, cell cycle analysis, caspase 3 activity assay, intracellular reactive oxygen species (ROS) level determination by dichlorofluorescein diacetate, and quantitative real-time polymerase chain reaction (qRT-PCR). Results: In vitro studies showed that TMX from S. chirata exhibited significant antitumor activity by inducing apoptosis and restricting proliferation in both melanoma and non-melanoma skin cancer cell lines, but no such activity was seen in normal skin cancer cell line WS1. The qRT-PCR analysis revealed that in both the melanoma ad non-melanoma cell lines, TMX could exert its antitumor activity by downregulating c-Myc, cyclin-D1, and β-catenin and up-regulating Wnt antagonist gsk-3β, thereby suppressing wnt self-renewal pathway, but such regulation was absent in normal cell line. Conclusions: TMX from chirata could effectively inhibit the proliferation of metastatic skin cancer (both melanoma and non-melanoma) cell lines while being non-toxic to normal cell lines. The chemotherapeutic potential of TMX against metastatic skin cancer cell lines was achieved by downregulating several key regulatory genes enabling the suppression of the self-renewal pathway, the chief reason behind the invasiveness of cancer cells.

Comparison of gene expression and activation of transcription factors in organoid-derived monolayer intestinal epithelial cells and organoids

2021 ◽  
Author(s):  
Yu Takahashi ◽  
Yu Inoue ◽  
Keitaro Kuze ◽  
Shintaro Sato ◽  
Makoto Shimizu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Epithelial Cells ◽  
Gene Expression Regulation ◽  
Intestinal Epithelial Cells ◽  
Three Dimensional ◽  
Culture Conditions ◽  
Dependent Manner ◽  
Intestinal Epithelial

Abstract Intestinal organoids better represent in vivo intestinal properties than conventionally used established cell lines in vitro. However, they are maintained in three-dimensional culture conditions that may be accompanied by handling complexities. We characterized the properties of human organoid-derived two-dimensionally cultured intestinal epithelial cells (IECs) compared with those of their parental organoids. We found that the expression of several intestinal markers and functional genes were indistinguishable between monolayer IECs and organoids. We further confirmed that their specific ligands equally activate intestinal ligand-activated transcriptional regulators in a dose-dependent manner. The results suggest that culture conditions do not significantly influence the fundamental properties of monolayer IECs originating from organoids, at least from the perspective of gene expression regulation. This will enable their use as novel biological tools to investigate the physiological functions of the human intestine.

scholarly journals Enhancing innate antiviral immune responses in rainbow trout by double stranded RNA delivered with cationic phytoglycogen nanoparticles

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Tamiru N. Alkie ◽  
Jondavid de Jong ◽  
Kristof Jenik ◽  
Karl M. Klinger ◽  
Stephanie J. DeWitte-Orr
Keyword(s):  
Rainbow Trout ◽  
Immune Responses ◽  
Cell Line ◽  
Culture Conditions ◽  
Type I Interferons ◽  
Poly I:C ◽  
Synthetic Analogue ◽  
Type I ◽  
Viral Dsrna

Abstract Innate immunity is induced when pathogen-associated molecular patterns (PAMPs) bind host pattern recognition receptors (PRRs). Polyinosinic:polycytidylic acid [poly(I:C)] is a synthetic analogue of viral dsRNA that acts as a PAMP, inducing type I interferons (IFNs) in vertebrates. In the present study, the immunostimulatory effects of high molecular weight (HMW) poly(I:C) in rainbow trout cells were measured when bound to a cationic phytoglycogen nanoparticle (Nano-HMW). The physical characteristics of the nanoparticle itself, when bound to different lengths of dsRNA and when cell associated was evaluated. Optimal concentration and timing for innate immune stimulation was measured using the RTG-P1 reporter cell line. The immunostimulatory effects of HMW poly (I:C) was compared to Nano-HMW in vitro using the RTgutGC cell line cultured in a conventional monolayer or a transwell culture system. The ability of an activated intestinal epithelium to transmit an antiviral signal to macrophages was evaluated using a co-culture of RTgutGC cells and RTSll (a monocyte/macrophage cell). In all culture conditions, Nano-HMW was a more effective inducer of IFN-related antiviral immune responses compared to HMW poly (I:C) alone. This study introduces the use of cationic phytoglycogen nanoparticles as a novel delivery system for immunomodulatory molecules to enhance immune responses in aquatic vertebrates.

scholarly journals Tissue Specific Differentiation of Human Chondrocytes Depends on Cell Microenvironment and Serum Selection

Cells ◽  
2019 ◽  
Vol 8 (8) ◽  
pp. 934 ◽  
Author(s):  
Ecke ◽  
Lutter ◽  
Scholka ◽  
Hansch ◽  
Becker ◽  
...  
Keyword(s):  
Articular Chondrocytes ◽  
Culture Conditions ◽  
Serum Supplement ◽  
3 Dimensional ◽  
The Matrix ◽  
Differentiation Degree ◽  
Overlay Technique ◽  
Fetal Bovine ◽  
Cell Culture Conditions

Therapeutic options to cure osteoarthritis (OA) are not yet available, although cell-based therapies for the treatment of traumatic defects of cartilage have already been developed using, e.g., articular chondrocytes. In order to adapt cell-based therapies to treat OA, appropriate cell culture conditions are necessary. Chondrocytes require a 3-dimensional (3D) environment for redifferentiation after 2-dimensional (2D) expansion. Fetal bovine serum (FBS) is commonly used as a medium supplement, although the usage of a xenogeneic serum could mask the intrinsic behavior of human cells in vitro. The aim of this study was to compare human articular chondrocytes cultivated as monolayers (2D) and the development of microtissues (3D) in the presence of FBS with those cultivated with human serum (HS). Evaluation of the expression of various markers via immunocytochemistry on monolayer cells revealed a higher dedifferentiation degree of chondrocytes cultivated with HS. Scaffold-free microtissues were generated using the agar overlay technique, and their differentiation level was evaluated via histochemistry and immunohistochemistry. Microtissues cultivated in the medium with FBS showed a higher redifferentiation level. This was evidenced by bigger microtissues and a more cartilage-like composition of the matrix with not any/less positivity for cartilage-specific markers in HS versus moderate-to-high positivity in FBS-cultured microtissues. The present study showed that the differentiation degree of chondrocytes depends both on the microenvironment of the cells and the serum type with FBS achieving the best results. However, HS should be preferred for the engineering of cartilage-like microtissues, as it rather enables a "human-based" situation in vitro. Hence, cultivation conditions might be further optimized to gain an even more adequate and donor-independent redifferentiation of chondrocytes in microtissues, e.g., designing a suitable chemically-defined serum supplement.

Hedgehog inhibition impaired platinum response in high-grade serous ovarian cancer harboring high hedgehog ligand expression and mTOR pathway activation.

2017 ◽  
Vol 35 (15_suppl) ◽  
pp. 5583-5583
Author(s):  
Gwo Yaw Ho ◽  
Elizabeth Lieschke ◽  
Elizabeth Kyran ◽  
Kristy Shield-Artin ◽  
Olga Kondrashova ◽  
...  
Keyword(s):  
Cell Line ◽  
Mtor Pathway ◽  
High Grade ◽  
Serous Ovarian Cancer ◽  
Qrt Pcr ◽  
Gli1 Expression ◽  
Pathway Activation ◽  
In Vivo Treatment

5583 Background: Elevated Glioma-associated Oncogene Homolog-1 (Gli1) protein expression is associated with Hedgehog (Hh) pathway activation in high-grade serous ovarian cancer (HGSOC). Inhibition of Hh signaling in Gli1-overexpressing HGSOC patient-derived xenograft (PDX) inhibited tumour growth, particularly in combination with chemotherapy. Early phase HGSOC clinical trials of vismodegib, a potent Hh inhibitor (SMO inhibitor), were disappointing. We identified a HGSOC PDX harboring both Indian Hh ligand-overexpression and bi-allelic deletion of TSC1, which latter event is reported to derepress the mTOR pathway, driving non-cannonical Gli1 expression. We explored the effect of vismodegib in combination with cisplatin or the mTOR inhibitor, everolimus, in this model. Methods: A cell-line was generated from the well-characterised PDX (identity confirmed by PDX-specific p53 mutation). In vitro response to vismodegib was assessed. qRT-PCR was performed to establish Hh-ligand and Gli1 expression with/without SMO inhibition. A PDX was generated from this cell-line and randomized to in vivo treatment with cisplatin, vismodegib, everolimus or vehicle alone, or vismodegib in combination with cisplatin or everolimus. Results: The HGSOC cell-line was sensitive to vismodegib in vitro (EC50 of 3.5µM) and qRT-PCR analysis revealed down-regulation of Hh-ligand and Gli1 expression following in vitro SMO inhibition, confirming on-target vismodegib activity. In vivo treatment with vismodegib or everolimus alone did not result in reproducible in vivo efficacy. The combination of vismodegib + everolimus caused short-lived responses in 3 of 6 mice. Strikingly, in vivo treatment with vismodegib in combination with cisplatin impaired median survival (19 days) when compared with cisplatin treatment alone (43 days; p = 0.039) due to rapid tumour progression. Conclusions: Combining chemotherapy with Hh inhibition in Hh ligand-overexpressing HGSOC PDX with mTOR pathway activation may be detrimental. These findings highlight the importance of an in-depth understanding of tumour biology in order to effectively combine therapeutic approaches.

scholarly journals Development of a Scalable, High-Throughput-Compatible Assay to Detect Tau Aggregates Using iPSC-Derived Cortical Neurons Maintained in a Three-Dimensional Culture Format

2016 ◽  
Vol 21 (8) ◽  
pp. 804-815 ◽  
Author(s):  
X. Medda ◽  
L. Mertens ◽  
S. Versweyveld ◽  
A. Diels ◽  
L. Barnham ◽  
...  
Keyword(s):  
High Throughput ◽  
Cortical Neurons ◽  
High Throughput Screening ◽  
Three Dimensional ◽  
Culture Conditions ◽  
Novel Treatments ◽  
Entry Points ◽  
Induced Pluripotent ◽  
Tau Aggregation

Tau aggregation is the pathological hallmark that best correlates with the progression of Alzheimer’s disease (AD). The presence of neurofibrillary tangles (NFTs), formed of hyperphosphorylated tau, leads to neuronal dysfunction and loss, and is directly associated with the cognitive decline observed in AD patients. The limited success in targeting β-amyloid pathologies has reinforced the hypothesis of blocking tau phosphorylation, aggregation, and/or spreading as alternative therapeutic entry points to treat AD. Identification of novel therapies requires disease-relevant and scalable assays capable of reproducing key features of the pathology in an in vitro setting. Here we use induced pluripotent stem cells (iPSCs) as a virtually unlimited source of human cortical neurons to develop a robust and scalable tau aggregation model compatible with high-throughput screening (HTS). We downscaled cell culture conditions to 384-well plate format and used Matrigel to introduce an extra physical protection against cell detachment that reduces shearing stress and better recapitulates pathological conditions. We complemented the assay with AlphaLISA technology for the detection of tau aggregates in a high-throughput-compatible format. The assay is reproducible across users and works with different commercially available iPSC lines, representing a highly translational tool for the identification of novel treatments against tauopathies, including AD.

Laminin-adherent versus suspension-non-adherent cell culture conditions for the isolation of cancer stem cells in the DAOY medulloblastoma cell line

Tumor Biology ◽  
2016 ◽  
Vol 37 (9) ◽  
pp. 12359-12370 ◽  
Author(s):  
Javier de la Rosa ◽  
Ander Sáenz Antoñanzas ◽  
Mehdi H. Shahi ◽  
Bárbara Meléndez ◽  
Juan A. Rey ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Culture ◽  
Cancer Stem Cells ◽  
Cell Line ◽  
Culture Conditions ◽  
Adherent Cell ◽  
Medulloblastoma Cell ◽  
Medulloblastoma Cell Line ◽  
Adherent Cell Culture ◽  
Cell Culture Conditions

Ins(1,4,5)P3 3-kinase-A overexpression induces cytoskeletal reorganization via a kinase-independent mechanism

2008 ◽  
Vol 414 (3) ◽  
pp. 407-417 ◽  
Author(s):  
Sabine Windhorst ◽  
Christine Blechner ◽  
Hong-Ying Lin ◽  
Christian Elling ◽  
Marcus Nalaskowski ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Calcium Release ◽  
Morphological Changes ◽  
Three Dimensional ◽  
Culture Conditions ◽  
Enzymatic Function ◽  
Marker Proteins ◽  
Independent Mechanism ◽  
Kinase A

In the present study, effects of increased IP3K-A [Ins(1,4,5)P3 3-kinase-A] expression were analysed. H1299 cells overexpressing IP3K-A formed branching protrusions, and under three-dimensional culture conditions, they exhibited a motile fibroblast-like morphology. They lost the ability to form actin stress fibres and showed increased invasive migration in vitro. Furthermore, expression levels of the mesenchymal marker proteins vimentin and N-cadherin were increased. The enzymatic function of IP3K-A is to phosphorylate the calcium-mobilizing second messenger Ins(1,4,5)P3 to (Ins(1,3,4,5)P4. Accordingly, cells overexpressing IP3K-A showed reduced calcium release and altered concentrations of InsPs, with decreasing concentrations of Ins(1,4,5)P3, InsP6 and Ins(1,2,3,4,5)P5, and increasing concentrations of Ins(1,3,4,5)P4. However, IP3K-A-induced effects on cell morphology do not seem to be dependent on enzyme activity, since a protein devoid of enzyme activity also induced the formation of branching protrusions. Therefore we propose that the morphological changes induced by IP3K-A are mediated by non-enzymatic activities of the protein.

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