scholarly journals Simple Method to Isolation and Culture of Neuron Progenitor Cells (NPCs) from Whole Brain Post-Natal Rat

2018 ◽  
Vol 9 (2) ◽  
pp. 63-69
Author(s):  
ARIYANI NOVIANTARI ◽  
Masagus Zainuri ◽  
Ratih Rinendyaputri ◽  
Ni Ketut Susilarini
Keyword(s):  
Progenitor Cells ◽  
Neuronal Cell ◽  
Neonatal Rat ◽  
Sprague Dawley ◽  
Simple Method ◽  
Whole Brain ◽  
Culture Cells ◽  
Result Show ◽  
Neuronal Cell Lines

Background: Using of neuron cells for in vitro neurobiology study is needed. Neuron cell can be obtained from a primary neuron or neuronal cell lines, depend on the aim of the study because both are not equivalent. Various methods are performed to obtain primary neurons from the cortical, hippocampal and whole brain of pre or neonatal rat. The limitations of neuron cells to proliferate so that is necessary to develop a method to isolate neuron progenitor cells (NPCs). The aim of the present study was to isolate NPCs from whole brain post-natal rat.   Methods: Whole brain were obtained from neonates Sprague Dawley rat. There are 2 step to get NSC; first isolation by taking the brain into the 15 ml of tube with 1 ml of  0,05% trypsin EDTA for 400g brain (incubated in the 370C, 5% CO2 for 10 minutes),  tirturation with adding 1 ml culture medium  and 5 ml HBSS-glucose then filtered by 70μm pore size membrane and centrifuged  2000 rpm for 10 minutes. Second: remove of supernatant with add 1 ml of HBSS-Glucose and taking it into a tube with  35% and 65% concentration of Ficoll then centrifuged at 1800 g for 10 minutes then supernatant were replated twice with poly D lysine (100µg/ml). Characterization of progenitor neuron immunotype was checked by immunohistochemistry with positive marker (NeuN and MAP2) and flow cytometry (PSANCAM+ and A2B5 -). Results: In this study, our result show that this method does not take longer than one hours and > 95% cells that obtained are expressing PSANCAM+.  After 4 days culture, cells exhibit positive for neuron marker as MAP2 and NeuN.   Conclusion: The method that our develope to isolate neuron progenitor cell from whole-brain are more effective and more simple with high viability and purity.

scholarly journals Human Neuronal Cell Lines as An In Vitro Toxicological Tool for the Evaluation of Novel Psychoactive Substances

2021 ◽  
Vol 22 (13) ◽  
pp. 6785
Author(s):  
Valeria Sogos ◽  
Paola Caria ◽  
Clara Porcedda ◽  
Rafaela Mostallino ◽  
Franca Piras ◽  
...  
Keyword(s):  
Cell Death ◽  
Neuronal Cell ◽  
In Vitro Study ◽  
Dependent Manner ◽  
Novel Psychoactive Substances ◽  
Psychoactive Substances ◽  
Concentration Dependent Manner ◽  
Mechanisms Of Toxicity ◽  
Neuronal Cell Lines

Novel psychoactive substances (NPS) are synthetic substances belonging to diverse groups, designed to mimic the effects of scheduled drugs, resulting in altered toxicity and potency. Up to now, information available on the pharmacology and toxicology of these new substances is very limited, posing a considerable challenge for prevention and treatment. The present in vitro study investigated the possible mechanisms of toxicity of two emerging NPS (i) 4′-methyl-alpha-pyrrolidinoexanophenone (3,4-MDPHP), a synthetic cathinone, and (ii) 2-chloro-4,5-methylenedioxymethamphetamine (2-Cl-4,5-MDMA), a phenethylamine. In addition, to apply our model to the class of synthetic opioids, we evaluated the toxicity of fentanyl, as a reference compound for this group of frequently abused substances. To this aim, the in vitro toxic effects of these three compounds were evaluated in dopaminergic-differentiated SH-SY5Y cells. Following 24 h of exposure, all compounds induced a loss of viability, and oxidative stress in a concentration-dependent manner. 2-Cl-4,5-MDMA activates apoptotic processes, while 3,4-MDPHP elicits cell death by necrosis. Fentanyl triggers cell death through both mechanisms. Increased expression levels of pro-apoptotic Bax and caspase 3 activity were observed following 2-Cl-4,5-MDMA and fentanyl, but not 3,4-MDPHP exposure, confirming the different modes of cell death.

scholarly journals Matching target dose to target organ

F1000Research ◽  
2017 ◽  
Vol 5 ◽  
pp. 2785
Author(s):  
Desmond I. Bannon ◽  
Marc A. Williams
Keyword(s):  
Neuronal Cell ◽  
Lead Toxicity ◽  
Target Organ ◽  
Target Dose ◽  
Neuronal Cell Lines ◽  
Stems Cells ◽  
Benchmark Doses ◽  
Molecular Toxicity

In vitro assays have become a mainstay of modern approaches to toxicology with the promise of replacing or reducing the number of in vivo tests required to establish benchmark doses, as well as increasing mechanistic understanding. However, matching target dose to target organ is an often overlooked aspect of in vitro assays, and the calibration of in vitro exposure against in vivo benchmark doses is often ignored, inadvertently or otherwise.  An example of this was recently published in Environmental Health Perspectives by Wagner et al (2016), where neural stems cells were used to model the molecular toxicity of lead.  On closer examination of the in vitro work, the doses used in media reflected in vivo lead doses that would be at the highest end of lead toxicity, perhaps even lethal.  Here we discuss the doses used and suggest more realistic doses for future work with stem cells or other neuronal cell lines.

Coverslip hypoxia: a novel method for studying cardiac myocyte hypoxia and ischemia in vitro

2004 ◽  
Vol 287 (4) ◽  
pp. H1801-H1812 ◽  
Author(s):  
Kelly R. Pitts ◽  
Christopher F. Toombs
Keyword(s):  
Metabolic Activity ◽  
Cardiac Myocyte ◽  
Experimental Models ◽  
Membrane Dynamics ◽  
Neonatal Rat ◽  
Simple Method ◽  
Glass Coverslip ◽  
Media Volume

In vitro experimental models designed to study the effects of hypoxia and ischemia typically employ oxygen-depleted media and/or hypoxic chambers. These approaches, however, allow for metabolites to diffuse away into a large volume and may not replicate the high local concentrations that occur in ischemic myocardium in vivo. We describe herein a novel and simple method for creating regional hypoxic and ischemic conditions in neonatal rat cardiac myocyte monolayers. This method consists of creating a localized diffusion barrier by placing a glass coverslip over a portion of the monolayer. The coverslip restricts covered myocytes to a thin film of media while leaving uncovered myocytes free to access the surrounding bulk media volume. Myocytes under the coverslip undergo marked morphology changes over time as assessed by video microscopy. Fluorescence microscopy shows that these changes are accompanied by alterations in mitochondrial membrane potential and plasma membrane dynamics and eventually result in myocyte death. We also show that the metabolic activity of myocytes drives cell necrosis under the coverslip. In addition, the intracellular pH of synchronously contracting myocytes under the coverslip drops rapidly, which further implicates metabolic activity in regulating cell death under the coverslip. In contrast with existing models of hypoxia/ischemia, this technique provides a simple and effective way to create hypoxic/ischemic conditions in vitro. Moreover, we conclude that myocyte death is hastened by the combination of hypoxia, metabolites, and acidosis and is facilitated by a reduction in media volume, which may better represent ischemic conditions in vivo.

scholarly journals Fetal and Neonatal Iron Deficiency Reduces Thyroid Hormone-Responsive Gene mRNA Levels in the Neonatal Rat Hippocampus and Cerebral Cortex

Endocrinology ◽  
2012 ◽  
Vol 153 (11) ◽  
pp. 5668-5680 ◽  
Author(s):  
Thomas W. Bastian ◽  
Jeremy A. Anderson ◽  
Stephanie J. Fretham ◽  
Joseph R. Prohaska ◽  
Michael K. Georgieff ◽  
...  
Keyword(s):  
Cerebral Cortex ◽  
Thyroid Hormone ◽  
Neonatal Rat ◽  
Fe Deficiency ◽  
Common Mechanism ◽  
Mrna Levels ◽  
Micronutrient Deficiencies ◽  
Sprague Dawley ◽  
Whole Brain ◽  
Responsive Gene

Abstract Copper (Cu), iron (Fe), and thyroid hormone (TH) deficiencies produce similar defects in late brain development including hypomyelination of axons and impaired synapse formation and function, suggesting that these micronutrient deficiencies share a common mechanism contributing to these derangements. We previously demonstrated that fetal/neonatal Cu and Fe deficiencies lower circulating TH concentrations in neonatal rats. Fe deficiency also reduces whole-brain T3 content, suggesting impaired TH action in the developing Fe-deficient brain. We hypothesized that fetal/neonatal Cu and Fe deficiencies will produce mild or moderate TH deficiencies and will impair TH-responsive gene expression in the neonatal cerebral cortex and hippocampus. To test this hypothesis, we rendered pregnant Sprague Dawley rats Cu-, Fe-, or TH-deficient from early gestation through postnatal d 10 (P10). Mild and moderate TH deficiencies were induced by 1 and 3 ppm propylthiouracil treatment, respectively. Cu deficiency did not significantly alter serum or tissue TH concentrations or TH-responsive brain mRNA expression. Fe deficiency significantly lowered P10 serum total T3 (45%), serum total T4 (52%), whole brain T3 (14%), and hippocampal T3 (18%) concentrations, producing a mild TH deficiency similar to 1 ppm propylthiouracil treatment. Fe deficiency lowered Pvalb, Enpp6, and Mbp mRNA levels in the P10 hippocampus. Fe deficiency also altered Hairless, Dbm, and Dio2 mRNA levels in the P10 cerebral cortex. These results suggest that some of the brain defects associated with Fe deficiency may be mediated through altered thyroidal status and the concomitant alterations in TH-responsive gene transcription.

scholarly journals Matching target dose to target organ

F1000Research ◽  
2016 ◽  
Vol 5 ◽  
pp. 2785 ◽  
Author(s):  
Desmond I. Bannon ◽  
Marc A. Williams
Keyword(s):  
Neuronal Cell ◽  
Lead Toxicity ◽  
Target Organ ◽  
Target Dose ◽  
Neuronal Cell Lines ◽  
Stems Cells ◽  
Benchmark Doses ◽  
Molecular Toxicity

In vitro assays have become a mainstay of modern approaches to toxicology with the promise of replacing or reducing the number of in vivo tests required to establish benchmark doses, as well as increasing mechanistic understanding. However, matching target dose to target organ is an often overlooked aspect of in vitro assays, and the calibration of in vitro exposure against in vivo benchmark doses is often ignored, inadvertently or otherwise.  An example of this was recently published in Environmental Health Perspectives by Wagner et al., where neural stems cells were used to model the molecular toxicity of lead.  On closer examination of the in vitro work, the doses used in media reflected in vivo lead doses that would be at the highest end of lead toxicity, perhaps even lethal.  Here we discuss the doses used and suggest more realistic doses for future work with stem cells or other neuronal cell lines.

Abstract 13832: Cardiac Mesenchymal Cells (CMCs) Cultured at Physiologic Oxygen Tension Have Superior Therapeutic Efficacy in Mice With Heart Failure Caused by Myocardial Infarction

Circulation ◽  
2020 ◽  
Vol 142 (Suppl_3) ◽  
Author(s):  
Robi Bolli ◽  
Chandrashekhar Dasari ◽  
Amr Alkasir ◽  
Asma Arshia ◽  
Yibing Nong ◽  
...  
Keyword(s):  
Myocardial Infarction ◽  
Progenitor Cells ◽  
Therapeutic Efficacy ◽  
Telomerase Activity ◽  
Severe Hypoxia ◽  
Functional Improvement ◽  
Lv Function ◽  
Culture Cells ◽  
Almost All

Stem/progenitor cells are usually cultured at atmospheric O 2 tension (21%); however, since physiologic O 2 tension in the heart is ~5%, using 21% O 2 may cause oxidative stress and toxicity. CMCs, a newly-discovered and promising type of progenitor cells, are effective in improving LV function after myocardial infarction (MI). To determine if 5% O 2 enhances therapeutic efficacy of CMCs, murine CMCs were cultured at 21% or 5% O 2 . Compared with 21% O 2 , culture at 5% O 2 significantly ( P <0.001) increased cell proliferation, telomerase activity, telomere length, and resistance to severe hypoxia (1% O 2 for 24 h) in vitro . Then, LV dysfunction was produced in 48 mice by a 60 min MI; 30 days later, mice received vehicle or CMCs cultured at 21% O 2 or 5% O 2 . After 35 days, the improvement in LV ejection fraction effected by 5% O 2 CMCs was >3 times greater than by 21% O 2 CMCs (5.2 vs. 1.5 units, P <0.01) (Figs. A-B). Hemodynamic studies (Millar catheter) yielded similar results both for load-dependent (LV dP/dt) and load-independent (end-systolic elastance) indices (Figs. C-D). Thus, 2 independent methods (echo and hemodynamics) demonstrated that compared with 21% O 2 , using 5% O 2 to culture CMCs results in greater functional improvement in the failing heart. Further, 5% O 2 CMCs produced greater reduction in myocardial fibrosis and exhibited much longer survival after transplantation ( P <0.01 for both). In conclusion, culturing CMCs at physiologic (5%) O 2 tension results in more rapid proliferation (reducing time and cost to achieve target cell numbers), less senescence, greater resistance to severe hypoxia (making cells better able to survive in scarred regions where O 2 is very low [1-2%]), and superior therapeutic efficacy in promoting cardiac repair after MI. Thus far, almost all preclinical and clinical studies of cell therapy have used 21% O 2 to culture cells. Our data challenge this paradigm and support the need to change the methods used to culture CMCs and possibly other progenitor cells.

Sign in / Sign up

Export Citation Format

Share Document