Name: isolating and culturing cells from diffuse intrinsic pontine glioma
Uploaded: 2024-08-30T13:24:01.000+00:00
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Description: All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human ...

isolating and culturing cells from diffuse intrinsic pontine glioma

Isolating and Culturing Cells from Diffuse Intrinsic Pontine Glioma

In this procedure, centrifuge the conical tube containing the remaining tissue fragments for five minutes. After that, remove the supernatant and add the pre-warmed enzymatic digestion solution such that there is 5 milliliters of digestion solution for every 1 milliliter of tissue. Then, seal the conical tube lid with laboratory film and incubate the reaction on a rotator at 37 degrees Celsius for 30 minutes.
After incubation, triturate the samples gently. Using a 10-milliliter serological pipette, pipette the sample up and down for 6 to 8 times and avoid generating excessive air bubbles. Next, add a 1000-microliter pipette tip to the end of the pipette and triturate the sample for an additional 6 to 8 times.
Allow any remaining chunks to settle to the bottom of the tube. Then, remove and filter the supernatant with the cell still suspended through a 100-micron filter into a new 50-milliliter conical tube labeled enzymatic dissociation, and store it on ice. Following that, centrifuge the enzymatic dissociation tube for five minutes and continue to sucrose gradient centrifugation. If the sample is still suspended in solution, centrifuge it for five minutes.
Next, remove the supernatant, and resuspend the tissue in 20 milliliters of cold HBSS without calcium and magnesium. Then, bring the volume up to 25 milliliters with cold HBSS. Slowly, add 25 milliliters of 1.8 molar sucrose solution and invert the tube to mix. This results in a 0.9 molar sucrose gradient.
Subsequently, centrifuge the sample with no break for 10 minutes. Carefully aspirate the myelin debris and as much sucrose solution as possible. Then, wash the sample by adding 30 milliliters of cold HBSS without calcium and magnesium, and mixing gently. After that, centrifuge it for five minutes.
In this procedure, remove the wash supernatant. Add 5 milliliters of ACK lysis buffer and gently resuspend the cell pellet, swirling the tube for one minute at room temperature. Then, quench the lysis by adding 30 milliliters of cold HBSS without calcium and magnesium.
Subsequently, centrifuge it for five minutes. Resuspend the final cell pellet in 10 to 15 milliliters of warm complete TSM with growth factors, and quantify the viable cell density on a hemocytometer using trypan blue exclusion. Afterward, transfer the final cell suspension to a new T75 culture flask. Add additional growth factors every other day to maintain the overall growth factor levels and monitor for the development of tumor cell neurospheres.

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All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human welfare and have been reviewed by the local institutional review board.
1. Obtaining Tissue Samples
NOTE: A critical component of this protocol requires proper handling of the tissue donation starting immediately at the time of autopsy. The overall sample viability depends significantly on minimizing the Post-Mortem Interval (PMI), immediately transferring the tissue into the appropriate cold medium supplemented with antibiotics/antimycotics, keeping the sample on&#160;wet&#160;ice throughout transportation, and maintaining sterile technique throughout the protocol.
<ol>
	<li>Upon notification of an imminent donation, prepare a sample collection kit. Using a shipping box with an insulated container that can be directly used for return transportation of the tissue sample, include the following materials:
	<ol>
		<li>Include materials for sterile preparation, including sterile gloves, drapes, and scalpels.</li>
		<li>Include 8 - 10 sterile 50 mL conical tubes containing 30 mL shipping media in the insulated container with ice or cold packs.&#160;&#160;&#160;&#160;&#160;&#160;&#160; 
		NOTE: While any standard cell culture media can work, the best sample viability is obtained with Hibernate-A supplemented with antibiotic/antimycotic.</li>
		<li>Include any further sample tubes for other analyses (e.g.,&#160;fixatives).</li>
		<li>If the autopsy will not be performed at the investigator&#39;s site, include a document that clearly states how to collect the tumor sample. This includes details such as maintaining sterility, keeping sample tubes on wet ice, and including samples from the midbrain and medulla as tumor cells often invade these regions.</li>
	</ol>
	</li>
	<li>Maintain sterility during the autopsy. Consider the brain sterile inside the cranium, so observe sterile technique (including changing gloves) once the cranium is open. Avoiding contamination with skin and hair is critical.</li>
	<li>Dissect the tumor from the ventral side (the &#34;belly&#34; of the pons, see&#160;Figure 1). With a sterile scalpel, cut small 1 cm chunks from the tumor and immediately transfer into cold shipping media (see NOTE in step 1.1.2) and put on wet ice. Place 10 mL of tissue into each tube, resulting in a final volume of tissue and media in each tube of 40 mL.&#160;&#160;&#160;&#160;&#160;&#160;&#160; 
	NOTE: In the case of diffuse intrinsic pontine glioma (DIPG), the tumor diffusely infiltrates the pons and frequently the rest of the brainstem. As such, collect as much of the sample as possible, typically including 3 - 4 tubes (30 - 40 mL tissue) from pons and 1 - 2 tubes (10 - 20 mL tissue) each from midbrain and medulla.</li>
	<li>Collect samples from the midbrain and medulla juxtaposed to the pons, which often contain many tumor cells.</li>
	<li>After sample collection, ship the samples overnight (O/N) on&#160;wet&#160;ice in the insulated container. Do not ship the sample on dry ice, as freezing will kill the cells.</li>
</ol>
2. Preparation for Sample Processing
<ol>
	<li>Prior to beginning sample preparation, sterilize tissue culture hoods along with razor blades, curved hemostats, and any other non-sterile tools under ultraviolet (UV) light for 1 h. Perform all of the following steps under sterile conditions to prevent contamination of the cultures.</li>
	<li>Prepare and sterile filter solutions.
	<ol>
		<li>To prepare 1.8 M sucrose solution, dissolve 308.07 g sucrose in 300 mL distilled water. Add 50 mL 10x Hank&#39;s-buffered Saline Solution (HBSS) without calcium or magnesium and bring total volume to 500 mL using distilled water. Store at 4 &#176;C.</li>
		<li>To prepare enzymatic digestion solution, take 50 mL HBSS&#160;with&#160;calcium and magnesium for every 10 mL of minced tissue and add 500 &#956;L 5 mg/mL deoxyribonuclease I (final concentration 50 &#956;g/mL), 500 &#956;L 2.5 mg/mL collagenase I/II and dispase solution (final concentration 25 &#956;g/mL), and 500 &#956;L 1 M 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer. Prewarm digestion solution in a 37 &#176;C water bath.</li>
		<li>To prepare tumor stem media (TSM) base, mix 250 mL Neurobasal-A, 250 mL Dulbecco&#39;s Modified Eagle Medium: Nutrient Mixture F12 (DMEM/F12), 5 mL 100x antibiotic-antimycotic, 5 mL 200 mM L-alanyl-L-glutamine dipeptide (GlutaMAX-A), 5 mL HEPES buffer, 5 mL 100 mM sodium pyruvate, and 5 mL 100x MEM non-essential amino acids.</li>
		<li>To prepare complete TSM with growth factors, add the following supplements to TSM base: 50x B27 Supplement Minus Vitamin A (1:50), human Epidermal Growth Factor (H-EGF, 20 ng/mL), human Fibroblast Growth Factor (H-FGF, 20 ng/mL), human Platelet-derived Growth Factor AA (H-PDGF-AA, 10 ng/mL), human Platelet-derived Growth Factor BB (H-PDGF-BB, 10 ng/mL), and heparin solution (2 &#956;g/mL).</li>
	</ol>
	</li>
	<li>Precool a laboratory centrifuge equipped with a swinging-bucket rotor to 4 &#176;C.</li>
</ol>
3. Mechanical Dissociation
<ol>
	<li>Transfer the tissue into a high-walled 100 mm x 20 mm cell culture dish. Remove media leftover from shipping and replace with 10 - 15 mL cold culture media.</li>
	<li>Using the curved hemostats to grasp a razor blade, mince the tissue finely while removing obvious blood vessels or meninges.&#160;&#160;&#160;&#160; 
	NOTE: The final tissue fragments should be smaller than 1 mm (see&#160;Figure 2B).</li>
	<li>Transfer the tissue into a clean 50 mL conical tube. Wash the cell culture dish with an additional 5 mL cold culture media and transfer to the conical tube. Repeat this wash step as necessary to transfer remaining tissue.</li>
	<li>Using a 10 mL serological pipet, triturate gently (4 - 5 times). Allow larger tissue fragments to settle to the bottom of the tube. If necessary, briefly centrifuge the sample for 1 min&#160;(350 x g, 4 &#176;C).</li>
	<li>Collect the mechanically dissociated fraction.
	<ol>
		<li>Remove the supernatant and filter it through a 100 &#956;m filter into a new 50 mL conical tube labeled &#34;Mechanical Dissociation.&#34; Invert the filter over the original conical tube and wash with culture media to recover tissue fragments.</li>
		<li>Centrifuge the &#34;Mechanical Dissociation&#34; fraction for 5 min (350 x g, 4 &#176;C).</li>
		<li>Remove the supernatant from the pelleted &#34;Mechanical Dissociation&#34; fraction and resuspend the tissue in cold culture media. If there is more than 5 mL of tissue in the &#34;Mechanical Dissociation&#34; conical tube, split the tissue into other 50 mL conical tubes such that no tube has more than 5 mL tissue.</li>
	</ol>
	</li>
	<li>Store the mechanically dissociated fraction on ice until the sucrose gradient centrifugation step (Step 5). Alternatively, the mechanically dissociated fraction can proceed to Step 5 during the enzymatic dissociation incubation period (Step 4.4).</li>
</ol>
4. Enzymatic Dissociation
<ol>
	<li>If there is more than 5 mL of tissue in the tube containing the remaining larger tissue fragments, split the tissue into other new 50 mL conical tubes such that no tube has more than 5 mL tissue.</li>
	<li>Centrifuge the conical tube(s) containing the remaining tissue fragments for 5 min (350 x g, 4 &#176;C).</li>
	<li>Remove the supernatant and add the prewarmed enzymatic digestion solution, such that there is 5 mL digestion solution for every 1 mL tissue (e.g.,&#160;25 mL digestion solution for 5 mL tissue).</li>
	<li>Seal the conical tube lids with laboratory film and incubate the reaction on a rotator at 37 &#176;C for 30 min.</li>
	<li>After the incubation, triturate the samples gently (see&#160;Figure 2C).
	<ol>
		<li>Using a 10 mL serological pipet, pipet the sample up and down 6 - 8 times. Avoid generating excessive air bubbles.</li>
		<li>Add a 1,000 &#956;L pipet tip to the end of the pipet and triturate an additional 6 - 8 times.</li>
		<li>Allow any remaining chunks to settle to the bottom of the tube. Remove and filter the supernatant with the cells still suspended through a 100 &#956;m filter into a new 50 mL conical tube labeled &#34;Enzymatic Dissociation&#34; and store on ice.</li>
		<li>If significant tissue fragments remain, add an additional 10 mL HBSS with calcium and magnesium to the fragments and repeat steps 3.5.1 and 3.5.2. Filter this final solution through a 100 &#956;m filter into the &#34;Enzymatic Dissociation&#34; tube.</li>
	</ol>
	</li>
	<li>Centrifuge the &#34;Enzymatic Dissociation&#34; tube for 5 min (350 x g, 4 &#176;C) and continue to sucrose gradient centrifugation.</li>
</ol>
5. Sucrose Gradient Centrifugation
<ol>
	<li>If samples are still suspended in solution, centrifuge for 5 min (350 x g, 4 &#176;C).</li>
	<li>Remove the supernatant and resuspend tissue in 20 mL cold HBSS without calcium and magnesium. Bring volume up to 25 mL total with cold HBSS.</li>
	<li>Slowly add 25 mL 1.8 M sucrose solution and invert the tube to mix. This results in a 0.9 M sucrose gradient.</li>
	<li>Centrifuge&#160;with no brake&#160;for 10 min (800 x g, 4 &#176;C). Using a centrifugation brake will disrupt the gradient and reduce yield (see&#160;Figure 2D&#160;for an example of a sample before and after centrifugation).</li>
	<li>Carefully aspirate myelin debris and as much sucrose solution as possible. Wash the sample by adding 30 mL cold HBSS without calcium and magnesium and mixing gently. Centrifuge for 5 min (350 x g, 4 &#176;C).</li>
</ol>
6. ACK Red Blood Cell Lysis
<ol>
	<li>Remove the wash supernatant. Add 5 mL ACK lysis buffer and gently resuspend the cell pellet, swirling the tube for 1 min at RT.</li>
	<li>Quench the lysis by adding 30 mL cold HBSS without calcium and magnesium.</li>
	<li>Centrifuge for 5 min (350 x g, 4 &#176;C).</li>
</ol>
7. Initial Culture Maintenance
<ol>
	<li>Resuspend the final cell pellets in 10 - 15 mL warm complete TSM with growth factors and quantify viable cell density on a hemocytometser using trypan blue exclusion. 
	NOTE: The range of viable cells varies widely depending on the circumstances of the tissue donation, and quantification can be difficult at this early stage due to leftover cellular debris. In an ideal case, aim to have one million live cells per sample. In cases where excessive debris remains, plate at a lower density in a T175 flask.</li>
	<li>Transfer the final cell suspensions to a new T75 culture flask. Spike in additional growth factors every other day to maintain overall growth factor levels and monitor for development of tumor cell neurospheres (see&#160;Figure 2E&#160;and&#160;Figure 3).</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Hibernate-A</td>
			<td>Gibco</td>
			<td>A12475-01</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS with Calcium/Magnesium</td>
			<td>Gibco</td>
			<td>24020-117</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>Corning</td>
			<td>21-022-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Gibco</td>
			<td>15630-080</td>
			<td></td>
		</tr>
		<tr>
			<td>Liberase DH (Collagenase/Dispase)</td>
			<td>Roche</td>
			<td>5401054001</td>
			<td></td>
		</tr>
		<tr>
			<td>DNAse I</td>
			<td>Worthington Biochemical</td>
			<td>LS002007</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma</td>
			<td>S0389</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic/Antimycotic</td>
			<td>Gibco</td>
			<td>15240-096</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal-A</td>
			<td>Gibco</td>
			<td>10888-022</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>Gibco</td>
			<td>11330-032</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX-I (100X)</td>
			<td>Gibco</td>
			<td>35050-061</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Pyruvate (100mM)</td>
			<td>Gibco</td>
			<td>11360-070</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 Supplement Minus Vitamin A</td>
			<td>Gibco</td>
			<td>12587-010</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM Non-Essential Amino Acids (100X)</td>
			<td>Gibco</td>
			<td>11140-050</td>
			<td></td>
		</tr>
		<tr>
			<td>Human PDGF-AA</td>
			<td>Shenandoah Biotechnology</td>
			<td>100-16</td>
			<td></td>
		</tr>
		<tr>
			<td>Human PDGF-BB</td>
			<td>Shenandoah Biotechnology</td>
			<td>100-18</td>
			<td></td>
		</tr>
		<tr>
			<td>Human EGF</td>
			<td>Shenandoah Biotechnology</td>
			<td>100-26</td>
			<td></td>
		</tr>
		<tr>
			<td>Human FGF</td>
			<td>Shenandoah Biotechnology</td>
			<td>100-146</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile scalpel blades</td>
			<td>Bard Paker</td>
			<td>372610</td>
			<td></td>
		</tr>
		<tr>
			<td>Curved hemostats</td>
			<td>Fine Science Tools</td>
			<td>13013-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Razor blades</td>
			<td>VWR</td>
			<td>55411-050</td>
			<td></td>
		</tr>
		<tr>
			<td>100 x 20 mm cell culture dish</td>
			<td>Corning</td>
			<td>353003</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile forceps</td>
			<td>TWD Tradewinds</td>
			<td>DF8988-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile drapes</td>
			<td>3M</td>
			<td>2037</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile gloves</td>
			<td>Kimberly Clark</td>
			<td>1182307</td>
			<td></td>
		</tr>
		<tr>
			<td>ACK Lysis Buffer</td>
			<td>Gibco</td>
			<td>A1049201</td>
			<td></td>
		</tr>
		<tr>
			<td>100 micron mesh filter</td>
			<td>Fisher</td>
			<td>352360</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL conical tube</td>
			<td>Fisher</td>
			<td>1495949A</td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Lin, G. L. et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/55360">A Protocol for Rapid Post-mortem Cell Culture of Diffuse Intrinsic Pontine Glioma (DIPG).</a>&#160;J. Vis. Exp. (2017)
This video demonstrates the process of isolating and culturing glial progenitor cells from diffuse intrinsic pontine glioma, or DIPG-affected brainstem tissue. Glial cells isolated from tumor tissue via an enzymatic digestion are cultured in a neurobasal medium, which promotes their growth and the development of free-floating neurospheres.

<img alt="Figure 1" src="/files/ftp_upload/22428/22428_Figure1.JPG" /> 
Figure 1. Autopsy Sample of a Pontine Tumor. Immediate post-mortem appearance of a diffuse intrinsic pontine glioma. The tumor appears as a large, myelin-rich mass on the ventral surface of the pons.
<img alt="Figure 2" src="/files/ftp_upload/22428/22428_Figure2.jpg" /> 
Figure 2. Tissue Samples at Various Stages of Processing.&#160;(A) Obtain the sample through sterile autopsy procedures and overnight shipping on wet ice. (B) From left to right: (row 1) tubes containing tumor tissue in shipping media; fresh tissue in a 100 mm x 20 mm cell culture dish; initial mincing of tissue; (row 2) partially minced tissue; removal of meninges and blood vessels; final size of minced tissue pieces. (C) From left to right: (row 1) tissue incubated on a rotating table in a 37 &#176;C oven; (row 2) trituration of tissue through a 1000 &#956;L pipet tip attached to a 10 mL pipet; filtering of dissociated tissue through a 100 &#956;m nylon mesh filter. (D) From left to right: dissociated tissue suspended in 0.9 M sucrose solution prior to centrifugation; dissociated tissue separated across a sucrose gradient after centrifugation; a visible layer of myelin debris on top of the sucrose gradient; a final cell pellet after ACK lysis and wash. (E) The final sample can be placed into culture or used in other downstream analyses.&#160;
<img alt="Figure 3" src="/files/ftp_upload/22428/22428_Figure3.jpg" /> 
Figure 3. Images of Primary Cultures and Early Passage Cells. (A - B) Cultures plated immediately after dissociation (SU-DIPG-XXX, A), or that have not grown neurospheres yet (SU-DIPG-XXIX, B). (C - D) Early appearance of neurospheres in primary cultures of SU-DIPG-XXVIII (C) and SU-DIPG-XXIX (D). (E-F) First passage of the primary culture from D using a 100 &#956;m nylon filter, with the filtrate (E) and reverse filtrate (F) plated. (G - H) Mature neurospheres from the first passage of SU-DIPG-XXVII (G) and the third passage of SU-DIPG-XXV (H) growing in culture. Scale bar = 200 &#956;m.

All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human ...

Begin with brain stem tissue fragments containing diffuse intrinsic pontine glioma, a tumor derived from glial progenitor cells in a myelinated nerve-rich environment.
Treat with proteolytic enzymes to degrade the tissue&#8217;s extracellular matrix.
After incubation, repeatedly pipette the contents to facilitate cell dissociation and degradation of the myelin sheath.
Filter and centrifuge to collect the cells. Remove the supernatant and resuspend them in a cold buffer.
Add a sucrose solution for density gradient separation and mix thoroughly.
Next, centrifuge to separate the heavier cells from the lighter myelin debris.
Remove the myelin layer and add a RBC lysis buffer.
Add a cold buffer to stop lysis action. Centrifuge and remove the supernatant to eliminate the lysed RBCs.
Resuspend the cells in a warm neurobasal medium and culture them in a non-coated flask.
The absence of neurotropic factors causes neuronal death, while the glial progenitor cells utilize nutrients and growth factors, leading to proliferation. Add fresh medium to maintain growth factor levels.
Over time, these cells spontaneously aggregate into cell clusters, forming free-floating neurospheres.

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视频: 从弥漫性内源性脑桥神经胶质瘤中分离和培养细胞 - 实验

Evaluation of Biomarkers in Glioma by Immunohistochemistry on Paraffin-Embedded 3D Glioma Neurosphere Cultures

Analysis of protein expression in glioma is relevant for several aspects in the study of its pathology. Numerous proteins have been described as biomarkers with applications in diagnosis, prognosis, classification, state of tumor progression, and cell differentiation state. These analyses of biomarkers are also useful to characterize tumor neurospheres (NS) generated from glioma patients and glioma models. Tumor NS provide a valuable in vitro model to assess different features of the tumor from which they are derived and can more accurately mirror glioma biology. Here we describe a detailed method to analyze biomarkers in tumor NS using immunohistochemistry (IHC) on paraffin-embedded tumor NS.

Neurospheres grown as 3D cultures constitute a powerful tool to study glioma biology. Here we present a protocol to perform immunohistochemistry while maintaining the 3D structure of glioma neurospheres through paraffin embedding. This method enables the characterization of glioma neurosphere properties such as stemness and neural differentiation.

石蜡包埋三维胶质瘤神经球培养的免疫组织化学评价胶质瘤生物标志物

Analysis of protein expression in glioma is relevant for several aspects in the study of its pathology. Numerous proteins have been described as ...

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Co-culture of Glutamatergic Neurons and Pediatric High-Grade Glioma Cells Into Microfluidic Devices to Assess Electrical Interactions

Pediatric high-grade gliomas (pHGG) represent childhood and adolescent brain cancers that carry a rapid dismal prognosis. Since there is a need to overcome the resistance to current treatments and find a new way of cure, modeling the disease as close as possible in an in vitro setting to test new drugs and therapeutic procedures is highly demanding. Studying their fundamental pathobiological processes, including glutamatergic neuron hyperexcitability, will be a real advance in understanding interactions between the environmental brain and pHGG cells. Therefore, to recreate neurons/pHGG cell interactions, this work shows the development of a functional in vitro model co-culturing human-induced Pluripotent Stem (hiPS)-derived cortical glutamatergic neurons pHGG cells into compartmentalized microfluidic devices and a process to record their electrophysiological modifications. The first step was to differentiate and characterize human glutamatergic neurons. Secondly, the cells were cultured in microfluidic devices with pHGG derived cell lines. Brain microenvironment and neuronal activity were then included in this model to analyze the electrical impact of pHGG cells on these micro-environmental neurons. Electrophysiological recordings are coupled using multielectrode arrays (MEA) to these microfluidic devices to mimic physiological conditions and to record the electrical activity of the entire neural network. A significant increase in neuron excitability was underlined in the presence of tumor cells.

Recent works uncover the neuronal impact on high-grade pediatric glioma (pHGG) cells and their reciprocal interactions. The present work shows the development of an in vitro model co-culturing pHGG cells and glutamatergic neurons and recorded their electrophysiological interactions to mimic those interactivities.

将谷氨酸能神经元和儿科高级别胶质瘤细胞共培养到微流体装置中以评估电相互作用

Pediatric high-grade gliomas (pHGG) represent childhood and adolescent brain cancers that carry a rapid dismal prognosis. Since there is a need to ...

Live-3D-Cell Immunocytochemistry Assays of Pediatric Diffuse Midline Glioma

Cell migration and invasion are specific hallmarks of Diffuse Midline Glioma (DMG) H3K27M-mutant tumors. We have already modeled these features using three-dimensional (3D) cell-based invasion and migration assays. In this study, we have optimized these 3D assays for live-cell immunocytochemistry. An Antibody Labeling Reagent was used to detect in real-time the expression of the adhesion molecule CD44, on the plasma membrane of migrating and invading cells of a DMG H3K27M primary patient-derived cell line. CD44 is associated with cancer stem cell phenotype and tumor cell migration and invasion and is involved in the direct interactions with the central nervous system (CNS) extracellular matrix. Neurospheres (NS) from the DMG H3K27M cell line were embedded into the basal membrane matrix (BMM) or placed onto a thin coating layer of BMM, in the presence of an anti-CD44 antibody in conjunction with the antibody labeling reagent (ALR). The live-3D-cell immunocytochemistry image analysis was performed on a live-cell analysis instrument to quantitatively measure the overall CD44 expression, specifically on the migrating and invading cells. The method also allows visualizing in real-time the intermittent expression of CD44 on the plasma membrane of migrating and invading cells. Moreover, the assay also provided new insights into the potential role of CD44 in the mesenchymal to amoeboid transition in DMG H3K27M cells.

This study presents a protocol of live-3D-cell immunocytochemistry applied to a pediatric diffuse midline glioma cell line, useful to study in real-time the expression of proteins on the plasma membrane during dynamic processes like 3D cell invasion and migration.

小儿弥漫性中线胶质瘤的活-3D细胞免疫细胞化学测定

Cell migration and invasion are specific hallmarks of Diffuse Midline Glioma (DMG) H3K27M-mutant tumors. We have already modeled these features using ...

Isolating and Culturing Cells from Diffuse Intrinsic Pontine Glioma

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Isolating and Culturing Cells from Diffuse Intrinsic Pontine Glioma