Preparing E18 cortical rat neurons for compartmentalization in a microfluidic device

Department of Biomedical Engineering, University of California, Irvine, CA, USA.
Journal of Visualized Experiments (Impact Factor: 1.33). 02/2007; 8(8):305. DOI: 10.3791/305
Source: PubMed


In this video, we demonstrate the preparation of E18 cortical rat neurons. E18 cortical rat neurons are obtained from E18 fetal rat cortex previously dissected and prepared. The E18 cortex is, upon dissection, immediately dissociated into individual neurons. It is possible to store E18 cortex in Hibernate E buffer containing B27 at 4 degrees C for up to a week before the dissociation is performed. However, there will be a drop in cell viability. Typically we obtain our E18 Cortex fresh. It is transported to the lab in ice cold Calcium free Magnesium free dissection buffer (CMFM). Upon arrival, trypsin is added to the cortex to a final concentration of 0.125%. The cortex is then incubated at 37 degrees C for 8 minutes. DMEM containing 10% FBS is added to the cortex to stop the reaction. The cortex is then centrifuged at 2500 rpm for 2 minutes. The supernatant is removed and 2 ml of Neural Basal Media (NBM) containing 2% B27 (vol/vol) and 0.25% Glutamax (vol/vol) is added to the cortex which is then re-suspended by pipetting up and down. Next, the cortex is triturated with previously fire polished glass pipettes, each with a successive smaller opening. After triturating, the cortex is once again centrifuged at 2500 rpm for 2 minutes. The supernatant is then removed and the cortex pellet re-suspended with 2 ml of NBM containing B27 and Glutamax. The cell suspension is then passed through a 40 um nylon cell strainer. Next the cells are counted. The neurons are now ready for loading into the neuron microfluidic device.

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    • "The contents of the petri dish were transferred to a 15 mL sterile centrifuge tube. HG-DMEM was added to a volume of 10 mL, and a sterile fire-polished Pasteur pipette was used to triturate the cells gently for about 20 times while avoiding air bubbles that could result in oxidative damage to the cells [9]. A 100 μm sterile cell strainer was placed onto a 50 mL centrifuge tube to filter out any remaining tissue fragments after pipetting. "
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    ABSTRACT: The study explored a modified primary culture system for fetal rat cortical neurons. Day E18 embryos from pregnant Sprague Dawley rats were microdissected under a stereoscope. To minimize enzymatic damage to the cultured neurons, we applied a sequential digestion protocol using papain and Dnase I. The resulting sifted cell suspension was seeded at a density of 50,000 cells per cm(2) onto 0.1 mg/mL L-PLL-covered vessels. After a four-hour incubation in high-glucose Dulbecco's Modified Eagle's Medium (HG-DMEM) to allow the neurons to adhere, the media was changed to neurobasal medium that was refreshed by changing half of the volume after three days followed by a complete medium change every week. The cells displayed progressively robust neurite extension, and nonneuronal-like cells could barely be detected by five days in vitro (DIV); cell growth was still substantial at 14 DIV. Neurons were identified by β-tubulin III immunofluorescence, and neuronal purity within the cultures was assessed at over 95% by both flow cytometry and by dark-field counting of β-tubulin III-positive cells. These results suggest that the protocol was successful and that the high purity of neurons in this system could be used as the basis for generating various cell models of neurological disease.
    Full-text · Article · Oct 2012 · BioMed Research International
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    • "Primary fetal cortical neurons (FCN) were extracted from C57BL6 embryonic day 17-18 mice as previously described (Harris et al., 2007) and in accordance with Texas A&M College of Medicine IACUC. Briefly, cervical dislocation was used to euthanize pregnant females and embryos were dissected in order to get cerebral cortices. "
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    ABSTRACT: Brain extracellular matrix (ECM) is highly degraded after cerebral ischemia. The perlecan c-terminal fragment LG3 is generated at increased levels by proteolytic processing as long as 3 days after ischemia. It has previously been shown that oxygen-glucose deprivation (OGD), reperfusion and interleukin-1 α (IL-1α) stimulate brain cells to yield increased levels of LG3. This LG3, in turn, is neuroprotective against OGD, and may therefore represent one of the brain's defenses against ischemic injury. Here, we investigate whether, in neurons, this increased LG3 is the result of increased perlecan generation and cellular release, increased protease release (to generate LG3 from previous extracellularly deposited perlecan) or both. We found that pre-synthesized perlecan may be exocytosed by neurons during OGD and de novo synthesis of perlecan is increased during reperfusion, even 24 h after OGD. Furthermore, while cathepsin L activity was seen to be marginally important to generate LG3 during normoxic conditions, cathepsin B activity was found to be important to generate increased levels of LG3 following OGD and reperfusion. On the other hand, IL-1α treatment raised levels of cathepsin L in neuronal media, and both cathepsin L and cathepsin B were demonstrated to be important for increasing LG3 levels after IL-1α treatment.
    Preview · Article · Feb 2012 · Brain research
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    • "Primary neuronal cultures were prepared from CD1 mouse embryos at E17. Hippocampi were extracted, treated with trypsin (0.25%; Sigma) for 10 min at 378C and mechanically dissociated using glass pipettes. Cells were concentrated to 2 Â 10 6 cells/mL and 30 mL of cell suspension was loaded in the magnetic device as described by Harris et al. (2007), so as to achieve a final plating density of about 10 3 cells/mm 2 . To allow cell adhesion, the device was incubated for 4 h, and successively 800 mL of culture medium, composed of NBM, B-27 1Â (Invitrogen), 1% penicillin and streptomycin and glutamax 1 mM (Invitrogen), were added. "
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    ABSTRACT: In vitro recording of neuronal electrical activity is a widely used technique to understand brain functions and to study the effect of drugs on the central nervous system. The integration of microfluidic devices with microelectrode arrays (MEAs) enables the recording of networks activity in a controlled microenvironment. In this work, an integrated microfluidic system for neuronal cultures was developed, reversibly coupling a PDMS microfluidic device with a commercial flat MEA through magnetic forces. Neurons from mouse embryos were cultured in a 100 µm channel and their activity was followed up to 18 days in vitro. The maturation of the networks and their morphological and functional characteristics were comparable with those of networks cultured in macro-environments and described in literature. In this work, we successfully demonstrated the ability of long-term culturing of primary neuronal cells in a reversible bonded microfluidic device (based on magnetism) that will be fundamental for neuropharmacological studies.
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