scholarly journals Human Co-culture Model of Neurons and Astrocytes to Test Acute Cytotoxicity of Neurotoxic Compounds

2017 ◽  
Vol 36 (6) ◽  
pp. 463-477 ◽  
Author(s):  
Uliana De Simone ◽  
Francesca Caloni ◽  
Laura Gribaldo ◽  
Teresa Coccini
Keyword(s):  
Cell Line ◽  
Cell Morphology ◽  
Cell Model ◽  
Culture Conditions ◽  
Test Method ◽  
Alternative Methods ◽  
Mitochondrial Activity ◽  
Neuroprotective Effects ◽  
Toxicological Studies

Alternative methods and their use in planning and conducting toxicology experiments have become essential for modern toxicologists, thus reducing or replacing living animals. Although in vitro human co-culture models allow the establishment of biologically relevant cell–cell interactions that recapitulate the tissue microenvironment and better mimic its physiology, the number of publications is limited specifically addressing this scientific area and utilizing this test method which could provide an additional valuable model in toxicological studies. In the present study, an in vitro model based on central nervous system (CNS) cell co-cultures was implemented using a transwell system combining human neuronal cells (SH-SY5Y cell line) and glial cells, namely astrocytes (D384 cell line), to investigate neuroprotection of D384 on SH-SY5Y and vice versa. The model was applied to test acute (24-48 hours) cytotoxicity of 3 different neurotoxicants: (1) methyl mercury (1-2.5 μM), (2) Fe3O4 nanoparticles (1-100 μg/mL), and (3) methylglyoxal (0.5-1 mM). Data were compared to mono-cultures evaluating the mitochondrial function and cell morphology. The results clearly showed that all compounds tested affected the mitochondrial activity and cell morphology in both mono-culture and co-culture conditions. However, astrocytes, when cultured together with neurons, diminish the neurotoxicant-induced cytotoxic effects that occurred in neurons cultured alone, and astrocytes become more resistant in the presence of neurons. This human CNS co-culture system seems a suitable cell model to feed high-throughput acute screening platforms and to evaluate both human neuronal and astrocytic toxicity and neuroprotective effects of new and emerging materials (eg, nanomaterials) and new products with improved sensitivity due to the functional neuron–astrocyte metabolic interactions.

scholarly journals The effect of strontium ranelate on titanium particle-induced periprosthetic osteolysis regulated by WNT/β-catenin signaling in vivo and in vitro

2021 ◽  
Vol 41 (1) ◽  
Author(s):  
Bolun Wang ◽  
Haohui Guo ◽  
Tianxiang Geng ◽  
Kening Sun ◽  
Liang Zhang ◽  
...  
Keyword(s):  
Bone Resorption ◽  
Aseptic Loosening ◽  
Cell Line ◽  
Conditioned Medium ◽  
Strontium Ranelate ◽  
Cell Model ◽  
Periprosthetic Osteolysis ◽  
A Cell

Abstract Aseptic loosening following periprosthetic osteolysis is the primary complication that limits the lifetime of total joint arthroplasty (TJA). The wear particles trigger a chronic inflammation response in the periprosthetic tissue and turn over the bone balance to bone resorption. The present study aimed to investigate the possible effect and mechanism of strontium ranelate (SR), a clinically safe drug for osteoporosis, on particle-induced periprosthetic osteolysis. Thirty-six female C57BL/6j mice underwent tibial Ti-nail implantation to establish an animal model of aseptic loosening. After 12 weeks, micro-CT results showed that strontium ranelate could inhibit periprosthetic bone resorption. In vitro, Ti particles were used to stimulate RAW264.7 cell line to collect conditioned medium, and co-culture MC3T3-E1 cell line with conditioned medium to establish a cell model of aseptic loosening. The results of alkaline phosphatase (ALP) detection, immunofluorescence, and flow cytometry demonstrated that strontium ranelate could regulate the expression of OPG/RANKL, promote differentiation and mineralization, and inhibit apoptosis in osteoblasts. Moreover, we revealed that SR’s exerted its therapeutic effect by down-regulating sclerostin, thereby activating the Wnt/β-catenin signal pathway. Therefore, this research suggests that strontium ranelate could be a potential drug for the prevention and treatment of particle-induced aseptic loosening post-TJA.

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.

Expression of Liver Functions in Imrnortalised Rot Hepotocyte Cell Lines

1994 ◽  
Vol 13 (6) ◽  
pp. 439-444 ◽  
Author(s):  
Caroline MacDonald ◽  
Maureen Vass ◽  
Brian Willett ◽  
Alexander Scott ◽  
Helen Grant
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Primary Cultures ◽  
Rat Hepatocytes ◽  
Hepatic Function ◽  
Glutathione S Transferase ◽  
Early Passage ◽  
Toxicological Studies ◽  
Well Differentiated

The differentiated hepatic function of two rat liver cell lines, P9 and SV40RH1, immortalised by transfection with SV40 DNA has been investigated in terms of the glutathione synthesis, and the activities of γ-glutamyltransferase, glutathione-S-transferase and UDP-glucuronosyltransferase. SV40RH1 is a highly differentiated cell line at early passage, but the expression of some aspects of its differentiated phenotype is unstable and some functions are lost by passage 12-13. P9 is a less-well differentiated cell line, with relatively stable expression of functions between passages 4 and 13. In terms of differentiated function both cell lines represent a marked improvement over primary cultures of rat hepatocytes which de-differentiate rapidly within 24-48 h in culture. This retention of liver function in proliferating cell lines offers the opportunity to use such cells in in vitro toxicological studies.

scholarly journals Characterization of a murine renal distal convoluted tubule cell line for the study of transcellular calcium transport

2004 ◽  
Vol 286 (3) ◽  
pp. F483-F489 ◽  
Author(s):  
Robin J. W. Diepens ◽  
Els den Dekker ◽  
Marcelle Bens ◽  
A. Freek Weidema ◽  
Alain Vandewalle ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Model ◽  
Collecting Duct ◽  
Primary Cultures ◽  
Ruthenium Red ◽  
Distal Convoluted Tubule ◽  
Molecular Regulation ◽  
Hek 293 ◽  
Calbindin D

To unravel the molecular regulation of renal transcellular Ca2+ transport, a murine distal convoluted tubule (mpkDCT) cell line derived from distal convoluted tubules (DCT) microdissected from a SV-PK/Tag transgenic mouse was characterized. This cell line originated from DCT only, as mRNA encoding for the DCT marker thiazide-sensitive Na+/Cl- cotransporter was expressed, whereas mRNA encoding for the connecting tubule and collecting duct marker aquaporin-2 was not detected, as determined by reverse-transcriptase PCR. mpkDCT cells expressed mRNA encoding the Ca2+ channels TRPV5 and TRPV6 and other key players necessary for transcellular Ca2+ transport, i.e., calbindin-D9k, calbindin-D28k, plasma membrane Ca2+-ATPase isoform 1b, and Na+/Ca2+ exchanger 1. Primary cultures of DCT cells exhibited net transcellular Ca2+ transport of 0.4 ± 0.1 nmol·h-1·cm-2, whereas net transcellular Ca2+ transport across mpkDCT cells was significantly higher at 2.4 ± 0.4 nmol·h-1·cm-2. Transcellular Ca2+ transport across mpkDCT cells was completely inhibited by ruthenium red, an inhibitor of TRPV5 and TRPV6, but not by the voltage-operated Ca2+ channel inhibitors felodipine and verapamil. With the use of patch-clamp analysis, the IC50 of ruthenium red on Na+ currents was between the values measured for TRPV5- and TRPV6-expressing HEK 293 cells, suggesting that TRPV5 and/or TRPV6 is possibly active in mpkDCT cells. Forskolin in combination with IBMX, 1,25-dihydroxyvitamin D3, and 1-deamino-8-d-arginine vasopressin increased transcellular Ca2+ transport, whereas PMA and parathyroid hormone had no significant effect. In conclusion, the murine mpkDCT cell line provides a unique cell model in which to study the molecular regulation of transcellular Ca2+ transport in the kidney in vitro.

scholarly journals Neuroprotective Effects of the Herbal Formula B401 in Both Cell and Mouse Models of Alzheimer’s Disease

2016 ◽  
Vol 2016 ◽  
pp. 1-17 ◽  
Author(s):  
Chih-Hsiang Hsu ◽  
Sheue-Er Wang ◽  
Ching-Lung Lin ◽  
Chun-Jen Hsiao ◽  
Shuenn-Jyi Sheu ◽  
...  
Keyword(s):  
Cell Model ◽  
Acetylcholinesterase Activity ◽  
Herbal Formula ◽  
Protective Effects ◽  
Neuroprotective Effects ◽  
Neurocognitive Dysfunctions ◽  
Subcutaneous Blood Flow ◽  
Triple Transgenic Mouse

In this study, we have reported the herbal formula B401 that has neuroprotective effects via multifunction, multitarget characteristics. It is possible that the herbal formula B401 may also provide new insights for AD. Here, we studied protective effects in the Tet-On Aβ42-GFP SH-SY5Y cell model and the APP/PS1/Tau triple transgenic mouse model by the herbal formula B401. Inin vitroexperiments, we showed that the herbal formula B401 treatment effectively reduces glutamate-induced excitotoxicity and acetylcholinesterase activity in Tet-On Aβ42-GFP SH-SY5Y cells. Inin vivoexperiments, we found that oral B401 treatment effectively ameliorates neurocognitive dysfunctions of 3× Tg-AD mice via motor and cognitive behavior tests. By using magnetic resonance imaging, moorFLPI instruments, and chemiluminescence methods, we reported that oral B401 treatment effectively alleviates brain atrophy, improves subcutaneous blood flow, and reduces blood ROS in 3× Tg-AD mice. As observed from results of immunohistochemistry staining and western blotting, we found that oral B401 treatment significantly enhances expressions of neuroprotective proteins, while reducing expressions of AD derived proteins such as amyloid beta, phosphorylated Tau, neurofibrillary tangles, and 3-nitrotyrosine in the brain of 3× Tg-AD mice. Thus, the herbal formula B401 may have the potential to be developed into optimum TCM for AD patients.

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.

scholarly journals Camptothecin regulates microglia polarization and exerts neuroprotective effects via the AKT/Nrf2/HO-1-NF-κB signal axis in vivo and in vitro

2020 ◽  
Author(s):  
dewei he ◽  
dianfeng liu ◽  
ang zhou ◽  
xiyu gao ◽  
yufei zhang ◽  
...  
Keyword(s):  
Parkinson’S Disease ◽  
Parkinson's Disease ◽  
Substantia Nigra ◽  
Cell Line ◽  
Dopaminergic Neuron ◽  
Inflammatory Responses ◽  
Neuroprotective Effects ◽  
Microglia Polarization

Abstract Background Parkinson's disease (PD), the second largest neurodegenerative disease seriously affects human health. Microglia, the main immune cells in the brain participate in the innate immune response in the central nervous system (CNS). Studies have shown that microglia can be polarized into pro-inflammatory M1 and anti-inflammatory M2 phenotypes. Accumulated evidences suggest that over-activated M1 microglia release pro-inflammatory mediators that damage neurons and lead to Parkinson's disease (PD). In contrast, M2 microglia release neuroprotective factors and exert the effects of neuroprotection. Camptothecin (CPT), an extract of the plant Camptotheca acuminate, has been reported to have anti-inflammation and antitumor effects. However the effect of CPT on microglia polarization and microglia-mediated inflammation responses has not been reported. Therefore, we aim to explore the effect of CPT on microglia polarization and its underlying mechanism on neuroinflammation. Methods C57BL/6 mice (25–30 g) were injected LPS or PBS into the substantia nigra (SN). Open-Field Test and Immunohistochemistry were performed to test the dyskinesia of mice and the loss of neurons in the substantia nigra (SN). Microglia cell line BV-2, the neuroblastoma SH-SY5Y and dopaminergic neuron MN9D cell were cultured. Cytotoxicity assay, reverse transcription quantitative real-time polymerase chain reaction (RT-PCR), Western blot, ELISA and Immunofluorescence staining were performed. All results were presented with mean ± SD. Results In vivo, CPT improved dyskinesia of mice, reduced the loss of neurons in the substantia nigra (SN) and inhibited neuro-inflammatory responses in LPS-injected mice. In vitro, CPT inhibited M1 polarization of microglia and promotes M2 polarization via the AKT/Nrf2/HO-1-NF-κB signal axis. Furthermore, CPT protected the neuroblastoma cell line SH-SY5Y and dopaminergic neuron cell line MN9D from neurotoxicity of mediated by microglia activation. Conclusion CPT regulates the microglia polarization phenotype via the AKT/Nrf2/HO-1-NF-κB signal axis, inhibits neuro-inflammatory responses and exerts neuroprotective effects in vivo and in vitro.

scholarly journals Morphometric analysis of a triple negative breast cancer cell line in hydrogel and monolayer culture environments

PeerJ ◽  
2018 ◽  
Vol 6 ◽  
pp. e4340 ◽  
Author(s):  
Manasi P. Jogalekar ◽  
Elba E. Serrano
Keyword(s):  
Breast Cancer ◽  
Cell Line ◽  
Triple Negative Breast Cancer ◽  
Monolayer Culture ◽  
Triple Negative ◽  
Treatment Strategies ◽  
Cancer Cell Line ◽  
Intercellular Junctions ◽  
Culture Conditions

Triple negative breast cancer (TNBC) is a belligerent carcinoma that is unresponsive to targeted receptor therapies. Development of new treatment strategies would benefit from an expanded repertoire of in vitro cell culture systems, such as those that support tridimensional growth in the presence of hydrogel scaffolds. To this end, we established protocols for maintenance of the TNBC cell line HCC70 in monolayer culture and in a commercially available basement membrane matrix hydrogel. We evaluated the general morphology of cells grown in both conditions with light microscopy, and examined their subcellular organization using transmission electron microscopy (TEM). Phase contrast and confocal microscopy showed the prevalence of irregularly shaped flattened cells in monolayer cultures, while cells maintained in hydrogel organized into multi-layered spheroids. A quantitative ultrastructural analysis comparing cells from the two culture conditions revealed that cells that formed spheroids comprised a greater number of mitochondria, autophagic vacuoles and intercellular junctions than their monolayer counterparts, within the equivalent area of sampled tissue. These observations suggest that triple negative breast cancer cells in culture can alter their organelle content, as well as their morphology, in response to their microenvironment. Methods presented here may be useful for those who intend to image cell cultures with TEM, and for investigators who seek to implement diverse in vitro models in the search for therapeutic molecular targets for TNBC.

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