scholarly journals Quantitative Image-Based Cell Viability (QuantICV) Assay for Microfluidic 3D Tissue Culture Applications

Micromachines ◽  
2020 ◽  
Vol 11 (7) ◽  
pp. 669
Author(s):  
Louis Jun Ye Ong ◽  
Liang Zhu ◽  
Gabriel Jenn Sern Tan ◽  
Yi-Chin Toh
Keyword(s):  
Tissue Culture ◽  
Cell Viability ◽  
Cell Cultures ◽  
Drug Testing ◽  
3D Culture ◽  
Liquid Volume ◽  
Small Scale ◽  
Tissue Cultures ◽  
Cell Viability Assay ◽  
Quantitative Image

Microfluidic 3D tissue culture systems are attractive for in vitro drug testing applications due to the ability of these platforms to generate 3D tissue models and perform drug testing at a very small scale. However, the minute cell number and liquid volume impose significant technical challenges to perform quantitative cell viability measurements using conventional colorimetric or fluorometric assays, such as MTS or Alamar Blue. Similarly, live-dead staining approaches often utilize metabolic dyes that typically label the cytoplasm of live cells, which makes it difficult to segment and count individual cells in compact 3D tissue cultures. In this paper, we present a quantitative image-based cell viability (QuantICV) assay technique that circumvents current challenges of performing the quantitative cell viability assay in microfluidic 3D tissue cultures. A pair of cell-impermeant nuclear dyes (EthD-1 and DAPI) were used to sequentially label the nuclei of necrotic and total cell populations, respectively. Confocal microscopy and image processing algorithms were employed to visualize and quantify the cell nuclei in the 3D tissue volume. The QuantICV assay was validated and showed good concordance with the conventional bulk MTS assay in static 2D and 3D tumor cell cultures. Finally, the QuantICV assay was employed as an on-chip readout to determine the differential dose responses of parental and metastatic 3D oral squamous cell carcinoma (OSCC) to Gefitinib in a microfluidic 3D culture device. This proposed technique can be useful in microfluidic cell cultures as well as in a situation where conventional cell viability assays are not available.

scholarly journals Decellularized Human Gut as a Natural 3D Platform for Research in Intestinal Fibrosis

2019 ◽  
Vol 25 (11) ◽  
pp. 1740-1750 ◽  
Author(s):  
Paolo Giuffrida ◽  
Marco Curti ◽  
Walid Al-Akkad ◽  
Carin Biel ◽  
Claire Crowley ◽  
...  
Keyword(s):  
Cell Cultures ◽  
Drug Testing ◽  
Biomarker Discovery ◽  
Disease Modeling ◽  
3D Culture ◽  
Protein Composition ◽  
Intestinal Fibrosis ◽  
3D Cell Cultures ◽  
3D Architecture ◽  
Inflammatory Bowel

Abstract Background The current methodologies for the identification of therapeutic targets for inflammatory bowel disease (IBD) are limited to conventional 2-dimensional (2D) cell cultures and animal models. The use of 3D decellularized human intestinal scaffolds obtained from surgically resected intestine and engineered with human intestinal cells may provide a major advancement in the development of innovative intestinal disease models. The aim of the present study was to design and validate a decellularization protocol for the production of acellular 3D extracellular matrix (ECM) scaffolds from the human duodenum. Methods Scaffolds were characterized by verifying the preservation of the ECM protein composition and 3D architecture of the native intestine and were employed for tissue engineering with primary human intestinal myofibroblasts for up to 14 days. Results Engrafted cells showed the ability to grow and remodel the surrounding ECM. mRNA expression of key genes involved in ECM turnover was significantly different when comparing primary human intestinal myofibroblasts cultured in 3D scaffolds with those cultured in standard 2D cultures on plastic dishes. Moreover, incubation with key profibrogenic growth factors such as TGFβ1 and PDGF-BB resulted in markedly different effects in standard 2D vs 3D cultures, further emphasizing the importance of using 3D cell cultures. Conclusions These results confirm the feasibility of 3D culture of human intestinal myofibroblasts in intestinal ECM scaffolds as an innovative platform for disease modeling, biomarker discovery, and drug testing in intestinal fibrosis.

scholarly journals Comparative Analysis of Salmon Cell Lines and Zebrafish Primary Cell Cultures Infection with the Fish Pathogen Piscirickettsia salmonis

2021 ◽  
Vol 9 (12) ◽  
pp. 2516
Author(s):  
Javiera Ortiz-Severín ◽  
Julia I. Tandberg ◽  
Hanne C. Winther-Larsen ◽  
Francisco P. Chávez ◽  
Verónica Cambiazo
Keyword(s):  
Cell Culture ◽  
Cell Viability ◽  
Cell Lines ◽  
Cell Cultures ◽  
High Throughput ◽  
Primary Cell ◽  
Infection Cycle ◽  
Cell Viability Assay ◽  
Viability Assay ◽  
Piscirickettsia Salmonis

Piscirickettsia salmonis is the etiologic agent of piscirickettsiosis, a disease that causes significant losses in the salmon farming industry. In order to unveil the pathogenic mechanisms of P. salmonis, appropriate molecular and cellular studies in multiple cell lines with different origins need to be conducted. Toward that end, we established a cell viability assay that is suitable for high-throughput analysis using the alamarBlue reagent to follow the distinct stages of the bacterial infection cycle. Changes in host cell viability can be easily detected using either an absorbance- or fluorescence-based plate reader. Our method accurately tracked the infection cycle across two different Atlantic salmon-derived cell lines, with macrophage and epithelial cell properties, and zebrafish primary cell cultures. Analyses were also carried out to quantify intracellular bacterial replication in combination with fluorescence microscopy to visualize P. salmonis and cellular structures in fixed cells. In addition, dual gene expression analysis showed that the pro-inflammatory cytokines IL-6, IL-12, and TNFα were upregulated, while the cytokines IL1b and IFNγ were downregulated in the three cell culture types. The expression of the P. salmonis metal uptake and heme acquisition genes, together with the toxin and effector genes ospD3, ymt, pipB2 and pepO, were upregulated at the early and late stages of infection regardless of the cell culture type. On the other hand, Dot/Icm secretion system genes as well as stationary state and nutrient scarcity-related genes were upregulated only at the late stage of P. salmonis intracellular infection. We propose that these genes encoding putative P. salmonis virulence factors and immune-related proteins could be suitable biomarkers of P. salmonis infection. The infection protocol and cell viability assay described here provide a reliable method to compare the molecular and cellular changes induced by P. salmonis in other cell lines and has the potential to be used for high-throughput screenings of novel antimicrobials targeting this important fish intracellular pathogen.

A tissue culture technique for the assay of antibacterial immune sera

1971 ◽  
Vol 17 (1) ◽  
pp. 13-15 ◽  
Author(s):  
B. B. Diena ◽  
R. Wallace ◽  
C. P. Kenny ◽  
L. Greenberg
Keyword(s):  
Tissue Culture ◽  
Cell Cultures ◽  
Salmonella Typhi ◽  
Immune Serum ◽  
Culture Technique ◽  
Assay Method ◽  
Tissue Cultures ◽  
Significant Protection ◽  
Bacterial Challenge ◽  
Monkey Kidney Cell

A tissue culture assay method is described in which monkey kidney cell cultures were protected by antibacterial immune serum against infection by Salmonella typhi, Neisseria meningitidis, or Neisseria gonorrhoeae. The neutralization of the bacterial challenge was specific since neither heterologous nor normal sera gave significant protection to the tissue cultures.

Do We have a Satisfactory Cell Viability Assay? Review of the Currently Commercially-Available Assays

2020 ◽  
Vol 17 (1) ◽  
pp. 2-22 ◽  
Author(s):  
Abdel-Baset Halim
Keyword(s):  
Cell Viability ◽  
Data Interpretation ◽  
Positive Control ◽  
Live Cells ◽  
Proof Of Concept ◽  
Cell Viability Assay ◽  
Viability Assay ◽  
Current Cell ◽  
Dying Cells ◽  
Differential Permeability

:Cell-based assays are an important part of the drug discovery process and clinical research. One of the main hurdles is to design sufficiently robust assays with adequate signal to noise parameters while maintaining the inherent physiology of the cells and not interfering with the pharmacology of target being investigated.:A plethora of assays that assess cell viability (or cell heath in general) are commercially available and can be classified under different categories according to their concepts and principle of reactions. The assays are valuable tools, however, suffer from a large number of limitations. Some of these limitations can be procedural or operational, but others can be critical as those related to a poor concept or the lack of proof of concept of an assay, e.g. those relying on differential permeability of dyes in-and-out of viable versus compromised cell membranes. While the assays can differentiate between dead and live cells, most, if not all, of them can just assess the relative performance of cells rather than providing a clear distinction between healthy and dying cells. The possible impact of relatively high molecular weight dyes, used in most of the assay, on cell viability has not been addressed. More innovative assays are needed, and until better alternatives are developed, setup of current cell-based studies and data interpretation should be made with the limitations in mind. Negative and positive control should be considered whenever feasible. Also, researchers should use more than one orthogonal method for better assessment of cell health.

scholarly journals Marine Fish Primary Hepatocyte Isolation and Culture: New Insights to Enzymatic Dissociation Pancreatin Digestion

2021 ◽  
Vol 18 (4) ◽  
pp. 1380
Author(s):  
Neusa Figueiredo ◽  
Beatriz Matos ◽  
Mário Diniz ◽  
Vasco Branco ◽  
Marta Martins
Keyword(s):  
Cell Viability ◽  
Cell Cultures ◽  
Marine Fish ◽  
Sparus Aurata ◽  
Cost Effective ◽  
Primary Hepatocyte ◽  
Digestion Method ◽  
Trypan Blue Exclusion ◽  
And Function

Primary cell cultures from wild organisms have been gaining relevance in ecotoxicology as they are considered more sensitive than immortalized cell lines and retain the biochemical pathways found in vivo. In this study, the efficacy of two methods for primary hepatocyte cell isolation was compared using liver from two marine fish (Sparus aurata and Psetta maxima): (i) two-step collagenase perfusion and (ii) pancreatin digestion with modifications. Cell cultures were incubated in L-15 medium at 17 ± 1 °C and monitored for up to six days for cell viability and function using the trypan blue exclusion test, MTT test, lactate dehydrogenase (LDH) activity, and ethoxyresorufin O-deethylase (EROD) activity after Benzo[a]Pyrene exposure. The results showed significant differences between the number of viable cells (p < 0.05), the highest number being obtained for the pancreatin digestion method (average = 4.5 ± 1.9 × 107 cells). Moreover, the hepatocytes showed solid adherence to the culture plate and the rounded shape, changing into a triangular/polygonal shape. The cell viability and function obtained by pancreatin digestion were maintained for five days, and the EROD induction after exposure to the B[a]P showed that cells were metabolically active. This study shows that the optimized pancreatin digestion method is a valid, cost-effective, and simple alternative to the standard perfusion method for the isolation of primary hepatocytes from fish and is suitable for ecotoxicological studies involving marine pollutants, such as PAHs.

NUTRITION OF ANIMAL CELLS IN TISSUE CULTURE: VII. USE OF REPLICATE CELL CULTURES IN THE EVALUATION OF SYNTHETIC MEDIA

1954 ◽  
Vol 32 (1) ◽  
pp. 306-318
Author(s):  
Raymond C. Parker ◽  
George M. Healy ◽  
Dorothy C. Fisher
Keyword(s):  
Tissue Culture ◽  
Cell Cultures ◽  
Subsequent Treatment ◽  
Relative Toxicity ◽  
Animal Cells ◽  
Washed Cell ◽  
Mouse Cells ◽  
Synthetic Media ◽  
Simple Screening ◽  
Natural Media

The replicate culture assay procedures of Earle and his associates have been adapted for use in evaluating the effectiveness of synthetic media. For this purpose, use has also been made of Earle's L strain mouse cells. Washed and continuously stirred suspensions of these or similar strains of cells may be dispensed, with reasonable assurance of uniformity, into a series of replicate cultures, the number depending on the volume of the suspension and the capacity and effectiveness of the stirring and dispensing unit. For use with synthetic media, the original procedures for the preparation and care of the replicate cultures and for their subsequent treatment for the counting of isolated, stained nuclei have been modified considerably. This paper describes the procedures that were finally adopted and also describes a relatively simple screening procedure in which washed cell suspensions may be used to advantage in making preliminary assays of synthetic media and in testing the relative toxicity or growth stimulating effects of substances added to, or derived from, natural media.

NUTRITION OF ANIMAL CELLS IN TISSUE CULTURE: VII. USE OF REPLICATE CELL CULTURES IN THE EVALUATION OF SYNTHETIC MEDIA

1954 ◽  
Vol 32 (3) ◽  
pp. 306-318 ◽  
Author(s):  
Raymond C. Parker ◽  
George M. Healy ◽  
Dorothy C. Fisher
Keyword(s):  
Tissue Culture ◽  
Cell Cultures ◽  
Subsequent Treatment ◽  
Relative Toxicity ◽  
Animal Cells ◽  
Washed Cell ◽  
Mouse Cells ◽  
Synthetic Media ◽  
Simple Screening ◽  
Natural Media

The replicate culture assay procedures of Earle and his associates have been adapted for use in evaluating the effectiveness of synthetic media. For this purpose, use has also been made of Earle's L strain mouse cells. Washed and continuously stirred suspensions of these or similar strains of cells may be dispensed, with reasonable assurance of uniformity, into a series of replicate cultures, the number depending on the volume of the suspension and the capacity and effectiveness of the stirring and dispensing unit. For use with synthetic media, the original procedures for the preparation and care of the replicate cultures and for their subsequent treatment for the counting of isolated, stained nuclei have been modified considerably. This paper describes the procedures that were finally adopted and also describes a relatively simple screening procedure in which washed cell suspensions may be used to advantage in making preliminary assays of synthetic media and in testing the relative toxicity or growth stimulating effects of substances added to, or derived from, natural media.

Isolation of Arboviruses From Cattle and Insects at 2 Sentinel Sites in Queensland, Australia, 1979-85

1990 ◽  
Vol 38 (1) ◽  
pp. 25 ◽  
Author(s):  
DH Cybinski ◽  
MJ Muller
Keyword(s):  
Cell Cultures ◽  
Virus Isolation ◽  
Hemorrhagic Disease ◽  
Tissue Cultures ◽  
Akabane Virus ◽  
Biting Midges ◽  
Bovine Ephemeral Fever ◽  
Serotype 1 ◽  
Intrathoracic Inoculation ◽  
First Time

Blood samples were collected regularly from two sentinel herds of cattle in northern and southern Queensland between 1979 and 1985. From 2660 samples, virus isolation attempts using baby hamster kidney (BHK21) and Aedes albopictus (AA) tissue cultures and suckling mice produced 308 viruses of which 243 (79%) were in the Palyam subgroup of orbiviruses. Mosquitoes and biting midges were collected at the southern sentinel herd site in January-February 1984 and processed for virus isolation in BHK2l and AA tissue cultures and by intrathoracic inoculation of Ae. aegypti mosquitoes. Totals of 14 338 midges of four species in 156 pools, and 9030 mosquitoes of 27 species in 232 pools, were processed and yielded 59 isolations. Of the 35 viruses isolated from Culicoides brevitarsis, 17 were members of the Palyam subgroup. Bovine ephemeral fever (BEF) virus was isolated once from Anopheles bancroftii, once from C. brevitarsis and 17 times from cattle. Akabane virus was isolated for the first time from C. wadai, as well as a further 10 times from C. brevitarsis and 20 times from cattle. Other viruses isolated from cattle included bluetongue serotype 1, and serotypes 5, 6 and 7 of epizootic hemorrhagic disease of deer (EHD). A new BEF group virus, tentatively called Oak Vale, was isolated nine times from Culex edwardsi mosquitoes. Of the orbiviruses, those in the Palyam subgroup were isolated almost exclusively in BHK2l tissue cultures but those in the bluetongue and EHD subgroups were isolated almost exclusively in AA cell cultures or after passage through Ae. aegypti. Of 22 rhabdovirus isolations from blood and insects (BEF, Kimberley and Tibrogargan), 16 were made only in AA cell cultures or after passage through Ae. aegypti.

Monoisopropylglutathione ester protects A549 cells from the cytotoxic effects of sulphur mustard

1997 ◽  
Vol 16 (11) ◽  
pp. 636-644 ◽  
Author(s):  
Christopher D Lindsay ◽  
Joy L Hambrook ◽  
Alison F Lailey
Keyword(s):  
Cell Viability ◽  
Cell Cultures ◽  
A549 Cell ◽  
High Performance ◽  
Gentian Violet ◽  
A549 Cells ◽  
Rapid Onset ◽  
Neutral Red ◽  
Biochemical Basis ◽  
Sulphur Mustard

1 The A549 cell line was used to assess the toxicity of sulphur mustard (HD), using gentian violet (GV) and neutral red (NR) dyes as indicators of cell viability. It was found that exposure to concentrations in excess of 40 ?M HD resulted in a rapid onset of toxicity. 2 The ability of monoisopropylglutathione ester (MIPE) to protect A549 cells against the effects of a 100 ?M challenge dose ofHD was determined using the NR and GV assays. It was found that MIPE (8 mM) could protect cells against the effects ofHD though MIPE had to be present at the time of HD challenge. Cultures protected with MIPE were two times more viable than HD exposed cells 48 h after HD challenge when using the GV and NR assays to assess viability. Observations by phase contrast microscopy of NR and GV stained cultures confirmed these findings. Addition of MIPE after previously exposing the A549 cultures to HD (for up to 5 min) maintained cell viability at 72% compared to 37% for unprotected cultures, after which time viability fell significantly so that at 10 min there was no difference in viability between the MIPE treated and untreated cultures. 3 Pretreating A549 cultures with MIPE for 1 h followed by its removal prior to HD challenge did not maintain cell viability. Treatment of cultures with HD for 1 h followed by addition of MIPE did not maintain the viability of the cultures, thus the window within which it was possible for MIPE to rescue cell cultures from the effects of HD was of short duration. 4 High performance liquid chromatography was used to determine the biochemical basis of the actions of MIPE. It was found that whilst intracellular levels of cysteine were increased up to 40-fold following treatment of A549 cell cultures with MIPE, levels of reduced glutathione did not rise. The lack of protection seen in cultures pretreated with MIPE for 1 h prior to HD exposure suggests that raising intracellular cysteine levels was not an effective strategy for protecting cells from the effects of HD. The protection observed is probably due to extra cellular inactivation of HD by MIPE.

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