Name: 비디오: Isolation and Culturing of Cells from Mouse Embryo Dorsolateral Telencephalons(쥐 배아에서 세포 분리 및 배양) - 개념
Uploaded: 2024-09-27T15:58:16.000+00:00
Duration: 1 min 18 s
Description: 38 조회수. 출처: Landeira, B. S., et al. 2D 배양 시스템을 사용한 일차 대뇌 피질 세포의 라이브 이미징. J. Vis. 특급 (2017) 이 비디오는 마우스 배아의 배측 뇌파에서 1차 대뇌 피질 세포를 분리하고 배양하는 프로토콜을 보여줍니다.

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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Dorsolateral Telencephalon Microdissection
<ol>
	<li>Prepare the dissection medium (100 mL): 98.5 mL Hank&#39;s Balanced Salt Solution (HBSS), 0.5 mL 5 M 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) pH 7.4, 1 mL penicillin/streptomycin (10,000 units/mL and 10,000 &#181;g/mL).</li>
	<li>Filter-sterilize the dissection medium.</li>
	<li>Remove embryonic day 14 (E14) embryos from a pregnant mouse C57/Bl6 (Mus musculus) under anesthesia with isoflurane. 
	NOTE: Consider E0 as the day of vaginal plug detection. Dissection should be completed no later than 1 h after removal from the pregnant mouse. This protocol also applies to embryos E11-E17.</li>
	<li>Remove the embryos by hysterectomy under sterile conditions.</li>
	<li>Transfer the embryos to a Petri dish with a cold dissection medium (4 &#176;C).</li>
	<li>Remove the brains from the skull by cutting along the longitudinal fissure. The number of embryos usually varies from five to ten. Each E14 brain yields approximately 2 x 106 cells, including progenitors and postmitotic neurons (1:1 ratio) 
	NOTE: Use the stereomicroscope for the following steps.</li>
	<li>Remove the meninges using dissection forceps. Split the telencephalon in the middle to separate the hemispheres. To isolate the dorsolateral telencephalon, cut along the dorsomedial curve and pallial-subpallial boundary using dissection forceps. Transfer the dorsolateral telencephalon to a 2 mL tube with a cold dissection medium (4 &#176;C) until all brains are dissected.</li>
</ol>
2. Cell Dissociation and Plating
<ol>
	<li>Prepare the proliferation medium (50 mL of DMEM+10% FCS): 44 mL Dulbecco Modified Eagle&#39;s Medium (DMEM), 5mL Fetal Calf Serum (FCS), 0.5 mL 40% glucose, 0.5 mL penicillin/streptomycin (10,000 units/mL and 10,000 &#181;g/mL). Filter-sterilize the proliferation medium.</li>
	<li>Prepare differentiation medium (50 mL of DMEM + 2% B27): 48 mL DMEM, 1 mL B27, 0.5 mL 40% glucose, 0.5 mL penicillin/streptomycin (10,000 units/mL and 10,000 &#181;g/mL). Filter-sterilize the differentiation medium.</li>
	<li>After micro-dissection of the dorsolateral telencephalon from E14 mice, centrifuge the tube with the collected tissue and cold dissection medium (for 5 min at 4 &#176;C, 340 x g) to precipitate the tissue.</li>
	<li>Remove the supernatant with a pipette and add 1 mL of pre-warmed (37 &#176;C) Trypsin-Ethylenediaminetetraacetic acid (EDTA) (0.05%) for chemical digestion. Incubate for 15 min at 37 &#176;C. Add 2 mL of proliferation medium to stop trypsin activity.</li>
	<li>Polish the tip of a glass Pasteur pipette over a gas burner or Bunsen burner for a few seconds to slightly narrow the aperture. Wash the pipette by aspirating FCS to coat the tip. To avoid bubbles, dissociate the cells mechanically with a fire-polished and FCS-coated Pasteur pipette.</li>
	<li>Centrifuge the cells (for 5 min at 4 &#176;C, 340 x g). Remove the supernatant with a pipette. Add 1 mL of proliferation medium and resuspend the cells using a pipette. Repeat this step once.</li>
	<li>Prepare a 1:1 dilution of the cell suspension using a 0.4% Trypan Blue solution. The non-viable cells will be blue. Using a Neubauer chamber, count the number of viable cells (unstained).</li>
	<li>Dilute the cells in a proliferation medium to get 106 cells/mL. Add 500 &#181;L of the cell suspension to each well of a 24-well tissue culture plate (approximately 2.5 &#215; 105 cells/cm2). Incubate the cells at 37 &#176;C and 5% CO2. 
	NOTE: Do not use glass coverslips, as they can move during the experiment and change the field of observation. Alternatively, use glass bottom tissue culture plates. 
	NOTE: If you are not using cell-culture-treated multidishes, it is important to pre-treat your plates with Poly-D-Lysin (50 &#181;g/ml) for 2 h at 37 &#176;C. PDL-treated plates can be stored at 4 &#176;C after washing with distilled water.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Hank&#39;s Balanced Salt Solution (HBSS)</td>
			<td>Invitrogen Life Technologies</td>
			<td>14175129</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H3375-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco Modified Eagle&#39;s Medium (DMEM)</td>
			<td>Gibco</td>
			<td>12400-024</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Calf Serum (FCS)</td>
			<td>Gibco</td>
			<td>10437028</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Gibco</td>
			<td>A2494001</td>
			<td></td>
		</tr>
		<tr>
			<td>B27</td>
			<td>Gibco</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.05%)</td>
			<td>Gibco</td>
			<td>25300054</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell observer microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation and culturing of cells from mouse embryo dorsolateral telencephalons

Source: Landeira, B. S., et al. <a href="https://app.jove.com/v/56063">Live Imaging of Primary Cerebral Cortex Cells Using a 2D Culture System.</a> J. Vis. Exp. (2017)
This video demonstrates a protocol for isolating and culturing primary cerebral cortex cells from the dorsolateral telencephalon of mouse embryos.

Isolation and Culturing of Cells from Mouse Embryo Dorsolateral Telencephalons

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

Take a mouse embryo in a buffer and separate the skull to isolate the brain.
Remove the meninges, the membranous layer covering the brain.
Dissect the brain to separate the hemispheres.
Isolate the dorsolateral telencephalons from the hemispheres and transfer them into a tube.
The dorsolateral telencephalons are rich in neurons and neural progenitor cells.
Centrifuge and remove the supernatant.
Add an enzyme solution. The enzymes break down the tissue matrix, loosening the cells.
Introduce a proliferation medium containing serum. The serum proteins inhibit enzyme activity.
Use a pipette to mechanically dissociate the tissue, forming a cell suspension.&#160;&#160;
Centrifuge and remove the supernatant containing inactivated enzymes and tissue debris.
Resuspend the cells in the proliferation medium.
Transfer the cells onto a cell culture substrate-coated microplate.
Incubate for the cells to adhere to the substrate. The medium facilitates progenitor cell proliferation.

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38 조회수. 출처: Landeira, B. S., et al. 2D 배양 시스템을 사용한 일차 대뇌 피질 세포의 라이브 이미징. J. Vis. 특급 (2017) 이 비디오는 마우스 배아의 배측 뇌파에서 1차 대뇌 피질 세포를 분리하고 배양하는 프로토콜을 보여줍니다. 

비디오: Isolation and Culturing of Cells from Mouse Embryo Dorsolateral Telencephalons(쥐 배아에서 세포 분리 및 배양) - 개념

A Triple Primary Cell Culture Model of the Human Blood-Brain Barrier for Studying Ischemic Stroke In Vitro

Ischemic stroke is a major cause of death and disability worldwide with limited therapeutic options. The neuropathology of ischemic stroke is characterized by an interruption in blood supply to the brain leading to cell death and cognitive dysfunction. During and after ischemic stroke, blood-brain barrier (BBB) dysfunction facilitates injury progression and contributes to poor patient recovery. Current BBB models primarily include endothelial monocultures and double co-cultures with either astrocytes or pericytes.
Such models lack the ability to fully imitate a dynamic brain microenvironment, which is essential for cell-to-cell communication. Additionally, commonly used BBB models often contain immortalized human endothelial cells or animal-derived (rodent, porcine, or bovine) cell cultures that pose translational limitations. This paper describes a novel well-insert-based BBB model containing only primary human cells (brain microvascular endothelial cells, astrocytes, and brain vascular pericytes) enabling the investigation of ischemic brain injury in vitro. 
The effects of oxygen-glucose deprivation (OGD) on barrier integrity were assessed by passive permeability, transendothelial electrical resistance (TEER) measurements,and direct visualization of hypoxic cells. The presented protocol offers a distinct advantage inmimicking the intercellular environment of the BBB in vivo, serving as a more realistic in vitro BBB model for developing new therapeutic strategies in the setting of ischemic brain injury.

A Triple Primary Cell Culture Model of the Human Blood-Brain Barrier for Studying Ischemic Stroke In Vitro

Here, we describe the method for establishing a triple cell culture model of the blood-brain barrier based on primary human brain microvascular endothelial cells, astrocytes, and pericytes. This multicellular model is suitable for studies of neurovascular unit dysfunction during ischemic stroke in vitro or for the screening of drug candidates.

시험관 내 허혈성 뇌졸중 연구를 위한 인간 혈액-뇌 장벽의 삼중 일차 세포 배양 모델

Ischemic stroke is a major cause of death and disability worldwide with limited therapeutic options. The neuropathology of ischemic stroke is ...

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A Simplified Method for Ultra-Low Density, Long-Term Primary Hippocampal Neuron Culture

Culturing primary hippocampal neurons in vitro facilitates mechanistic interrogation of many aspects of neuronal development. Dissociated embryonic hippocampal neurons can often grow successfully on glass coverslips at high density under serum-free conditions, but low density cultures typically require a supply of trophic factors by co-culturing them with a glia feeder layer, preparation of which can be time-consuming and laborious. In addition, the presence of glia may confound interpretation of results and preclude studies on neuron-specific mechanisms. Here, a simplified method is presented for ultra-low density (~2,000 neurons/cm2), long-term (&#62;3 months) primary hippocampal neuron culture that is under serum free conditions and without glia cell support. Low density neurons are grown on poly-D-lysine coated coverslips, and flipped on high density neurons grown in a 24-well plate. Instead of using paraffin dots to create a space between the two neuronal layers, the experimenters can simply etch the plastic bottom of the well, on which the high density neurons reside, to create a microspace conducive to low density neuron growth. The co-culture can be easily maintained for &#62;3 months without significant loss of low density neurons, thus facilitating the morphological and physiological study of these neurons. To illustrate this successful culture condition, data are provided to show profuse synapse formation in low density cells after prolonged culture. This co-culture system also facilitates the survival of sparse individual neurons grown in islands of poly-D-lysine substrates and thus the formation of autaptic connections.

Low density cultures of primary hippocampal neurons usually require glia feeder layer to supply neurotrophic factors and sustain longevity. We describe here a simplified method to culture ultra-low density neurons on glass coverslips in the presence of a high density neuronal feeder layer, which facilitates investigation of specific neuronal-autonomous mechanisms.

매우 낮은 밀도에 대한 단순화 된 방법, 장기 차 해마 신경 세포 문화

Culturing primary hippocampal neurons in vitro facilitates mechanistic interrogation of many aspects of neuronal development. Dissociated embryonic ...

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Microdissection of Mouse Brain into Functionally and Anatomically Different Regions

The brain is the command center for the mammalian nervous system and an organ with enormous structural complexity. Protected within the skull, the brain consists of an outer covering of grey matter over the hemispheres known as the cerebral cortex. Underneath this layer reside many other specialized structures that are essential for multiple phenomenon important for existence. Acquiring samples of specific gross brain regions requires quick and precise dissection steps.&#160;It is understood that at the microscopic level, many sub-regions exist and likely cross the arbitrary regional boundaries that we impose for the purpose of this dissection.
Mouse models are routinely used to study human brain functions and diseases. Changes in gene expression patterns may be confined to specific brain areas targeting a particular phenotype depending on the diseased state. Thus, it is of great importance to study regulation of transcription with respect to its well-defined structural organization. A complete understanding of the brain requires studying distinct brain regions, defining connections, and identifying key differences in the activities of each of these brain regions. A more comprehensive understanding of each of these distinct regions may pave the way for new and improved treatments in the field of neuroscience. Herein, we discuss a step-by-step methodology for dissecting the mouse brain into sixteen distinct regions. In this procedure, we have focused on male mouse C57Bl/6J (6-8 week old) brain removal and dissection into multiple regions using neuroanatomical landmarks to identify and sample discrete functionally-relevant and behaviorally-relevant&#160;brain regions. This work will help lay a strong foundation in the field of neuroscience, leading to more focused approaches in the deeper understanding of brain function.

We present a hands-on, step-by-step, rapid protocol for mouse brain removal and dissection of discrete regions from fresh brain tissue. Obtaining brain regions for molecular analysis has become routine in many neuroscience labs. These brain regions are immediately frozen to obtain high quality transcriptomic data for system level analysis.

The brain is the command center for the mammalian nervous system and an organ with enormous structural complexity. Protected within the skull, the brain ...

Isolation and Culturing of Cells from Mouse Embryo Dorsolateral Telencephalons

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Isolation and Culturing of Cells from Mouse Embryo Dorsolateral Telencephalons