centrifugation-assisted mitochondrial transfer to glioblastoma stem cells

Centrifugation-Assisted Mitochondrial Transfer to Glioblastoma Stem Cells

To transfer isolated MSC mitochondria to GSCs, add 200 microliters of precooled GSC proliferation medium to the mitochondrial pellet. Dilute the mitochondrial preparation in GSC proliferation medium to consistently allow the addition of 20 microliters of mitochondrial suspension to the GSCs.
Next, slowly add the mitochondria close to the bottom of the well. Centrifuge the plates at 1,500 times G at 4 degrees Celsius for 15 minutes. Then, immediately place the cultures at 37 degrees Celsius. Finally, carry out analysis of mitochondria transfer, FACS analysis, and confocal imaging according to the text protocol.

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Day 1
1. Labeling of the Mesenchymal Stem Cell (MSC) Mitochondria 
<ol>
	<li>Two days before the mitochondria preparation, seed human MSCs in a 100 mm cultAure dish, in 10 ml (Minimum Essential Medium Eagle)/FBS (Fetal bovine serum) 10%, so as to have 4 x 105 MSCs in culture on Day 1.</li>
	<li>Rinse MSCs with PBS (4 ml) and add 4 ml &#945;MEM/FBS 1% (prewarmed to 37 &#176;C).</li>
	<li>Add the required amount of mitochondria vital dye and incubate cells for 30 min in the 37 &#176;C incubator.</li>
	<li>Remove the mitochondria dye solution, rinse cells twice with 4 ml prewarmed (37 &#176;C) &#945;MEM/FBS 1% and add back 4 ml &#945;MEM/FBS 10%. Incubate cells at 37 &#176;C.</li>
	<li>Change the culture medium (10 ml &#945;MEM/FBS 10%) after 30 min and, another time, 2 hr later.</li>
</ol>
Day 1
2. Labeling of the Glioblastoma Stem Cells (GSC) 
<ol>
	<li>Dissociate GSCs Gb4 cell line (10 x 106 cells) grown as neurospheres on poly-HEMA coated cell culture flasks</li>
	<li>Seed GSCs in a 48-well plate at 105 cells/well in GSC proliferation medium (500 &#181;l) (see Table 1).</li>
	<li>Centrifuge the plate at 270 x g for 5 min at 20 &#176;C.</li>
	<li>Add the required amount of cell vital dye and incubate for 30 min at 37 &#176;C.</li>
	<li>Add 500 &#181;l of GSC basal medium (Table 1) per well of the 48-well plate.</li>
	<li>Centrifuge the plate at 270 x g at 20 &#176;C for 5 min. Aspirate the supernatant.</li>
	<li>Repeat steps 2.5 to 2.6.</li>
	<li>Add 500 &#181;l of GSC basal medium and incubate at 37 &#176;C for 30 min.</li>
	<li>Centrifuge the plate at 270 x g at 20 &#176;C for 5 min. Aspirate the supernatant.</li>
	<li>Add 500 &#181;l of GSC proliferation medium per well of the 48-well plate and incubate in the 37 &#176;C incubator. 
	Note: The amount of GSCs indicated (10 x 106 cells) allows performing the different dose-response experiments and FACS controls. Once the experimental conditions are more precisely defined this amount can be scaled down.</li>
</ol>
3. Seeding of Glioblastoma Stem Cells
<ol>
	<li>Collect the GSC (Glioblastoma stem cells ) neurospheres (10 x 106 cells) by centrifugation at 270 x g for 5 min, at 20 &#176;C in a 50 ml tube.</li>
	<li>Wash cells with 5 ml of HBSS (Hanks&#39; Balanced Salt Solution), and centrifuge at 270 x g for 5 min at 20 &#176;C.</li>
	<li>Aspirate the supernatant.</li>
	<li>Gently resuspend the GSC pellet in 100 &#181;l trypsin-EDTA(Ethylenediamine tetraacetic acid)&#160; (0.25%) (per 10 x 106 cells)</li>
	<li>Incubate at 37 &#176;C for 3 min.</li>
	<li>Add 10 &#181;l CaCl2 (20 mM) and 2 &#181;l DNase I (10 mg/ml).</li>
	<li>Dissociate the neurospheres by gently pipetting up and down (30-50x) with a P200 pipette. Avoid bubbles. Check under the microscope that all GSCs are dissociated.</li>
	<li>Add 10 &#181;l trypsin inhibitor (5%) and 10 ml HBSS.</li>
	<li>Centrifuge GSCs at 270 x g for 7 min at 20 &#176;C.</li>
	<li>Discard the supernatant and add 10 ml GSC basal medium .</li>
	<li>Count GSCs with a Thoma counting chamber and then centrifuge the cells at 270 x g for 7 min at 20 &#176;C.</li>
	<li>Add the appropriate volume of GSC proliferation medium to reach the cellular concentration of 106 GSCs/ml.</li>
	<li>Seed 105 GSCs (100 &#181;l of the cell suspension) per well of a 96-well plate.</li>
	<li>Centrifuge the plates at 270 x g for 7 min at 20 &#176;C to get GSCs at the bottom of wells.</li>
	<li>Incubate the 96-well plate at 37 &#176;C.</li>
</ol>
Day 2
4. MSC (Mesenchymal stem cells) Mitochondria Isolation
<ol>
	<li>Adjust the microtube centrifuge temperature to 4 &#176;C.</li>
	<li>Prepare two 1.5 ml tubes &#34;A&#34; and &#34;C&#34; containing the reagents for the mitochondria extraction (200 &#181;l reagent A and 400 &#181;l reagent C, both containing the EDTA-free protease inhibitors). Prepare 2 other tubes, labeled &#34;MSC&#34; and &#34;Mito&#34;. Keep all tubes on ice. Also prepare tubes for the mitochondria serial dilutions.</li>
	<li>Wash MSCs with 10 ml prewarmed (37 &#176;C) PBS (Phosphate buffered saline).</li>
	<li>Wash MSCs with 2 ml trypsin (no EDTA) for 10 sec and add 1 ml trypsin (no EDTA). Incubate cells for 5-10 min at 37 &#176;C.</li>
	<li>Recover the MSCs by adding 10 ml &#945;MEM /FBS 10%, transfer to a 50 ml tube.</li>
	<li>Centrifuge cells at 270 x g for 5 min at 20 &#176;C.</li>
	<li>Discard the supernatant and add 10 ml &#945;MEM/FBS 10% to the cell pellet.</li>
	<li>Count the MSCs with a Malassez counting chamber.</li>
	<li>Centrifuge MSCs (4-5 x 105) at 270 x g for 5 min at 20 &#176;C.</li>
	<li>Discard the supernatant, add 1 m ice-cold &#945;MEM/FBS 10% to the cell pellet and transfer the cells to the &#34;MSC&#34;-labeled tube. Keep the tube on ice.</li>
	<li>Centrifuge the tube containing the MSCs at 900 x g for 5 min, at 4 &#176;C.</li>
	<li>Remove all residual medium from the tube.</li>
	<li>Add 200 &#181;l of Mitochondria Isolation Reagent A (containing the EDTA-free protease inhibitors). Vortex at medium speed for 5 sec and leave tubes on ice for exactly 2 min.</li>
	<li>Add 2.5 &#181;l of Mitochondria Isolation Reagent B. Vortex at maximum speed for 10 sec, and then leave tubes on ice. Repeat every 30 sec for 5 min.</li>
	<li>Add 200 &#181;l of Mitochondria Isolation Reagent C (containing the EDTA-free protease inhibitors). Mix by tilting the tube (roughly 30 times, do not vortex). Centrifuge the tube at 700 x g for 10 min at 4 &#176;C.</li>
	<li>Transfer the supernatant (containing the MSC mitochondria) to the &#34;Mito&#34; tube.</li>
	<li>Centrifuge at 3,000 x g, 15 min at 4 &#176;C, to get the mitochondria pellet. Discard the supernatant. The pellet contains the isolated mitochondria.</li>
	<li>Rinse the mitochondria pellet with 200 &#181;l of the Reagent C. Then, centrifuge at 12,000 x g, 5 min at 4 &#176;C, to get the mitochondria pellet.</li>
</ol>
5. Transfer of Isolated MSC Mitochondria to GSCs (MitoCeption)
<ol>
	<li>Add 200 &#181;l of the pre-cooled (0 &#176;C) GSC proliferation medium to the mitochondria pellet isolated from MSCs (4 x 105).</li>
	<li>Dilute the mitochondria preparation (in GSC proliferation medium) to consistently add 20 &#181;l of mitochondrial suspension to the GSCs.</li>
	<li>Add the volume of isolated mitochondria into the wells of the 96-well plate containing the GSCs , at the desired concentration (0.1 to 10 &#181;g). Add the mitochondria slowly, close to the bottom of the well, covering the entire surface at least once.</li>
	<li>Centrifuge the 96-well plate containing the mitochondria recipient GSCs with the MSC mitochondria and the control plate at 1,500 x g for 15 min at 4 &#176;C.</li>
	<li>Place the culture plates in the 37 &#176;C cell incubator immediately after centrifugation. 
	Note: The mitochondria transfer protocol relies on the centrifugation of the mitochondria suspension on the cultured cells at the adequate centrifugation force, with a number of centrifugations that can be adjusted depending on the system of mitochondria donor/recipient cells.</li>
</ol>
Table 1: Culture Media.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Cell culture medium</td>
			<td>Composition</td>
		</tr>
		<tr>
			<td rowspan="5">GSC basal medium</td>
			<td>DMEM/F-12 supplemented with</td>
		</tr>
		<tr>
			<td>Insulin 20 mg/ml</td>
		</tr>
		<tr>
			<td>N2 supplement 1x</td>
		</tr>
		<tr>
			<td>Glucose 3 g/L</td>
		</tr>
		<tr>
			<td>L-glutamine 2 mM</td>
		</tr>
		<tr>
			<td rowspan="9">GSC proliferation medium</td>
			<td>Add to the basal medium:</td>
		</tr>
		<tr>
			<td>B27 supplement 1x</td>
		</tr>
		<tr>
			<td>EGF 10 ng/ml</td>
		</tr>
		<tr>
			<td>bFGF 10 ng/ml</td>
		</tr>
		<tr>
			<td>Fungine 10 mg/ml</td>
		</tr>
		<tr>
			<td>Fungizone 0.25 mg/ml</td>
		</tr>
		<tr>
			<td>Heparin 2 mg /ml</td>
		</tr>
		<tr>
			<td>Ciprofloxacin 2 &#956;g / ml</td>
		</tr>
		<tr>
			<td>Gentamicin 2 &#956;g/ml</td>
		</tr>
		<tr>
			<td rowspan="4">MSC proliferation medium</td>
			<td>&#945;MEM supplemented with</td>
		</tr>
		<tr>
			<td>L-glutamine 2 mM</td>
		</tr>
		<tr>
			<td>10% FBS</td>
		</tr>
		<tr>
			<td>bFGF 2 ng/ml</td>
		</tr>
	</tbody>
</table>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Mitochondria Isolation Kit for Tissue</td>
			<td>Fisher Scientific</td>
			<td>10579663</td>
			<td></td>
		</tr>
		<tr>
			<td>N-2 Supplement (100X)</td>
			<td>Fisher Scientific</td>
			<td>11520536</td>
			<td></td>
		</tr>
		<tr>
			<td>B-27 Supplement W/O VIT A (50X)</td>
			<td>Fisher Scientific</td>
			<td>11500446</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS w/o Ca2+ w/o Mg2+</td>
			<td>Sigma</td>
			<td>H4385</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly Heme</td>
			<td>Sigma</td>
			<td>P3932</td>
			<td></td>
		</tr>
		<tr>
			<td>aMEM w/o glutamine</td>
			<td>Ozyme</td>
			<td>BE12-169F</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F-12 without glutamine,</td>
			<td>Fisher Scientific</td>
			<td>11540566</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Invitrogen</td>
			<td>25030-024</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma</td>
			<td>G7021</td>
			<td></td>
		</tr>
		<tr>
			<td>Insuline</td>
			<td>Sigma</td>
			<td>I 1882</td>
			<td></td>
		</tr>
		<tr>
			<td>Human bFGF</td>
			<td>R&#38;D Systems</td>
			<td>233-FB-025</td>
			<td></td>
		</tr>
		<tr>
			<td>Human EGF</td>
			<td>Peprotech</td>
			<td>AF-100-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Sigma</td>
			<td>H3149</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>MERCK</td>
			<td>2382</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsine Inhibitor</td>
			<td>Sigma</td>
			<td>T9003</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase I</td>
			<td>SIGMA</td>
			<td>10104159001</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsine 0.25% /EDTA 1 mM</td>
			<td>Invitrogen</td>
			<td>25200056</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Gibco</td>
			<td>15090-046</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease inhibitors EDTA free</td>
			<td>Sigma</td>
			<td>4693159001</td>
			<td></td>
		</tr>
		<tr>
			<td>Ciprofloxacine</td>
			<td>Sigma</td>
			<td>17850-5G-F</td>
			<td></td>
		</tr>
		<tr>
			<td>Fungine</td>
			<td>Invivogen</td>
			<td>ant-fn-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Fungizone</td>
			<td>Thermofisher</td>
			<td>15290018</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamycin</td>
			<td>Euromedex</td>
			<td>EU0410</td>
			<td></td>
		</tr>
		<tr>
			<td>MitoTracker Green FM</td>
			<td>Molecular Probes</td>
			<td>M7514</td>
			<td></td>
		</tr>
		<tr>
			<td>MitoTracker Red CMXRos</td>
			<td>Molecular Probes</td>
			<td>M7512</td>
			<td></td>
		</tr>
		<tr>
			<td>MitoTracker Deep Red FM</td>
			<td>Molecular Probes</td>
			<td>M22426</td>
			<td></td>
		</tr>
		<tr>
			<td>CellTracker Green CMFDA</td>
			<td>Molecular Probes</td>
			<td>C7025</td>
			<td></td>
		</tr>
		<tr>
			<td>CellTracker Blue CMF2HC</td>
			<td>Molecular Probes</td>
			<td>C12881</td>
			<td></td>
		</tr>
		<tr>
			<td>RIPA</td>
			<td>Santa Cruz</td>
			<td>sc-24948</td>
			<td></td>
		</tr>
		<tr>
			<td>FluoroDish Sterile Culture Dish</td>
			<td>World Precision Instruments</td>
			<td>FD35-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemacytometer</td>
			<td>Fisher Scientific</td>
			<td>267110</td>
			<td></td>
		</tr>
		<tr>
			<td>FACS tubes</td>
			<td>Beckman Coulter</td>
			<td>25,23,749</td>
			<td></td>
		</tr>
		<tr>
			<td>FACS apparatus</td>
			<td>Gallios</td>
			<td>3L 10C</td>
			<td></td>
		</tr>
		<tr>
			<td>LC FAST START DNA MASTER PLUS</td>
			<td>Roche</td>
			<td>3515885001</td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Mombo, B. N., et al.&#160;<a href="https://app.jove.com/v/55245">MitoCeption: transferring isolated human MSC mitochondria to glioblastoma stem cells.</a> J. Vis. Exp. (2017)
In this video, a method is demonstrated to enable the transfer of fluorescently labeled mitochondria into glioblastoma stem cells (GSCs) through centrifugation. The successful uptake and functional integration of the donor mitochondria are confirmed by detecting the fluoresce using confocal microscopy.

<img alt="Figure 1" src="/files/ftp_upload/23209/23209_Figure1.jpg" /> 
Figure 1: Workflow for the transfer of isolated MSC mitochondria to GSCs by MitoCeption. The 7 steps for the MitoCeption of MSC mitochondria to GSCs and the subsequent GSC analyses are shown. The step numbers are indicated as in the protocol section. If MSC and GSC labeling is not performed, as for functional analyses, steps 1 and 2 can be omitted, and the protocol can be followed from day 2 on. Phase contrast images represent Gb4 GSCs as neurospheres (day 1), as unicellular culture ready for the MSC mitochondria transfer (day 2), and 24 hr after the mitochondria transfer (day 3). Scale bar = 100 &#181;m. In step 4, a representative photomicrograph of isolated MSC mitochondria, prelabeled with MitoTracker Red CMXRos before isolation from MSCs. Scale bar = 5 &#181;m.

Source: Mombo, B. N., et al.&#160;MitoCeption: transferring isolated human MSC mitochondria to glioblastoma stem cells. J. Vis. Exp. (2017)
In this video, ...

Start with a tube containing deep-red fluorescently labeled donor mitochondria.
Add a cold proliferation medium to disperse the mitochondria.
Dispense the mitochondrial solution near the bottom of the well containing modified glioblastoma stem cells or GSCs.
These modified brain cancer cells are labeled with a blue fluorescent dye and contain red-fluorescently labeled endogenous mitochondria.
The suspension covers the entire surface of the well, uniformly distributing the mitochondria.
Centrifuge the culture plate to bring the mitochondria closer to the cells for their interaction.
Now, incubate the culture plate.
The cell membrane forms large vesicles that engulf extracellular material, including the donor mitochondria.
The cells then internalize these vesicles, releasing the donor mitochondria into their cytoplasm, potentially altering GSC metabolism and functions.
Observe the cells under a confocal microscope.
After image analysis, the presence of blue cells containing green donor mitochondria along with red endogenous mitochondria confirms successful mitochondrial transfer.

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视频: Centrifugation-Assisted Mitochondrial Transfer to Glioblastoma Stem Cells - 实验

Preparation of Mitochondria from Ovarian Cancer Tissues and Control Ovarian Tissues for Quantitative Proteomics Analysis

Ovarian cancer is a common gynecologic cancer with high mortality but unclear molecular mechanism. Most ovarian cancers are diagnosed in the advanced stage, which seriously hampers therapy. Mitochondrial changes are a hallmark of human ovarian cancers, and mitochondria are the centers of energy metabolism, cell signaling, and oxidative stress. In-depth insights into the changes of the mitochondrial proteome in ovarian cancers compared to control ovarian tissue will benefit in-depth understanding of the molecular mechanisms of ovarian cancer, and the discovery of effective and reliable biomarkers and therapeutic targets. An effective mitochondrial preparation method coupled with an isobaric tag for relative and absolute quantification (iTRAQ) quantitative proteomics are presented here to analyze human ovarian cancer and control mitochondrial proteomes, including differential-speed centrifugation, density gradient centrifugation, quality assessment of mitochondrial samples, protein digestion with trypsin, iTRAQ labeling, strong cation exchange fractionation (SCX), liquid chromatography (LC), tandem mass spectrometry (MS/MS), database analysis, and quantitative analysis of mitochondrial proteins. Many proteins have been successfully identified to maximize the coverage of the human ovarian cancer mitochondrial proteome and to achieve the differentially expressed mitochondrial protein profile in human ovarian cancers.

This article presents a protocol of differential-speed centrifugation in combination with density gradient centrifugation to separate mitochondria from human ovarian cancer tissues and control ovarian tissues for quantitative proteomics analysis, resulting in a high-quality mitochondrial sample and high-throughput and high-reproducibility quantitative proteomics analysis of a human ovarian cancer mitochondrial proteome.

卵巢癌组织线粒体的制备与控制卵巢组织进行定量蛋白质组学分析

Ovarian cancer is a common gynecologic cancer with high mortality but unclear molecular mechanism. Most ovarian cancers are diagnosed in the advanced ...

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癌症研究

Isolation of mRNAs Associated with Yeast Mitochondria to Study Mechanisms of Localized Translation

Most of mitochondrial proteins are encoded in the nucleus and need to be imported into the organelle. Import may occur while the protein is synthesized near the mitochondria. Support for this possibility is derived from recent studies, in which many mRNAs encoding mitochondrial proteins were shown to be localized to the mitochondria vicinity. Together with earlier demonstrations of ribosomes&#8217; association with the outer membrane, these results suggest a localized translation process. Such localized translation may improve import efficiency, provide unique regulation sites and minimize cases of ectopic expression. Diverse methods have been used to characterize the factors and elements that mediate localized translation. Standard among these is subcellular fractionation by differential centrifugation. This protocol has the advantage of isolation of mRNAs, ribosomes and proteins in a single procedure. These can then be characterized by various molecular and biochemical methods. Furthermore, transcriptomics and proteomics methods can be applied to the resulting material, thereby allow genome-wide insights. The utilization of yeast as a model organism for such studies has the advantages of speed, costs and simplicity. Furthermore, the advanced genetic tools and available deletion strains facilitate verification of candidate factors.

Many mRNAs encoding mitochondrial proteins are associated with the mitochondria outer membrane. We describe a subcellular fractionation procedure aimed at isolation of yeast mitochondria with its associated mRNAs and ribosomes. This protocol can be applied to cells grown under diverse conditions&#160;in order to reveal mechanisms of mRNA localization and localized translation near the mitochondria.

Most of mitochondrial proteins are encoded in the nucleus and need to be imported into the organelle. Import may occur while the protein is synthesized ...

生物学

Spatial Measurement of Tumor Interstitial Fluid Pressure: A Method to Measure the Interstitial Fluid Pressure

Source: Stapleton et. al., <a href="https://www.jove.com/v/54226/spatial-measurements-perfusion-interstitial-fluid-pressure-liposomes">Measurements of Perfusion, Interstitial Fluid Pressure and Liposomes Accumulation in Solid Tumors.</a> J. Vis. Exp. (2016).
This video describes a technique of measuring the interstitial fluid pressure within a tumor by perfusion imaging using an image-guided robotic system. Measurements are compared to the intra-tumoral accumulation of liposomes, within a solid tumor.

肿瘤间质液压力的空间测量:一种测量间质液压力的方法

Source: Stapleton et. al., Measurements of Perfusion, Interstitial Fluid Pressure and Liposomes Accumulation in Solid Tumors. J. Vis. Exp. (2016).
This ...

Centrifugation-Assisted Mitochondrial Transfer to Glioblastoma Stem Cells

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Centrifugation-Assisted Mitochondrial Transfer to Glioblastoma Stem Cells