The authors would like to acknowledge Hans de Graaf and Tomas Veenendaal for their technical assistance in culturing the ECs.

Ethics statement: Blood vessels used in this study were harvested as surplus material obtained from fresh canine cadavers (n= 4) from healthy dogs euthanized for other unrelated research (University 3R policy). Aberrant blood vessels (intra- and extrahepatic portosystemic shunts, n= 1 each) were harvested post-mortem after informed consent of the owners from dogs presented to the University Clinic for Companion Animals of Utrecht University.
1. Isolation and Culture of Primary Canine Endothelial Cells
<ol>
	<li>Pre-coat 6-well plates with 2 ml/well 0.5% w/v gelatin and leave for 2 hr in an incubator with a humidified atmosphere of 5% CO2 at 37 &#176;C. Remove excess gelatin solution prior to seeding the primary cells.</li>
	<li>Aseptically remove the blood vessel(s) of interest (e.g., aorta, vena cava, vena porta) from a fresh canine cadaver (Figure 1A). Maintain the anatomy of the vessel system by placing straight forceps on both ends of the vessel of interest. Avoid unnecessary manipulation of the tissue with forceps to prevent damage to the endothelial cells.
	<ol>
		<li>Cut the clamped vessel on both ends with surgical scissors and remove from the cadaver. Transport in Hank&#39;s Balanced Salt Solution (HBSS) on ice. 
		NOTE: A vessel length of approximately 5 cm is preferred for this procedure, but those that measure 1 cm will also provide enough cells to culture.</li>
	</ol>
	</li>
	<li>Transfer the blood vessel to a Petri dish filled with ice-cold HBSS. Using surgical scissors, remove any adherent tissue and fat from the outside of the vessel, keeping the vessel itself intact (Figure 1B). Make sure the vessel is clearly visible at all times when cutting: pull aside the surrounding tissue with a clamp or forceps for optimal view.
	<ol>
		<li>Close any branches of the vessel with ligatures (e.g., polyglactin 3-0) and subsequently remove them with surgical scissors or a scalpel.</li>
	</ol>
	</li>
	<li>Carefully enter a vessel end with a curved Halsted mosquito forceps, clamp the tip of the forceps at the other end of the vessel and then retract, slowly inverting the vessel until it is completely inside out (Figure 1C-G). The outside now consists of the endothelial cell layer. If any difficulty is encountered upon inverting the vessel, submerge in HBSS again to reduce friction.</li>
	<li>Place purse-string sutures at both ends of the vessel to close it completely and to prevent exposure of any non-endothelial vascular tissue to the digestion procedure (Figure 1H). Use the ligature ends to manipulate the inverted vessel.</li>
	<li>If digestion and culture is performed later, cryopreserve the inverted vessel prior to the digestion protocol (step 1.7).
	<ol>
		<li>Place the inverted vessel in a cryovial and fill with cell culture freezing medium. Freeze down to -80 &#176;C using a freezing container. Store at -80 &#176;C if vessels are to be used within one week. For long term storage, place cryovials at -180 &#176;C. When performing the EC isolation, thaw the cryovials rapidly in a water bath (37 &#176;C) and immediately place in ice-cold HBSS. Proceed as indicated in 1.7. 
		NOTE: Take into account that the total yield of ECs after isolation will be lower due to loss of viability during the freezing process.</li>
	</ol>
	</li>
	<li>Transfer the vessel to a 50 ml tube and rinse it twice in HBSS to remove erythrocytes (or residual freezing medium in case of thawing) (Figure 1I).</li>
	<li>Digest the vessel in a 50 ml tube with a solution of 30 ml collagenase type II (0.15 U/ml) and dispase (0.15 U/ml) in Canine Endothelial Cells Growth Medium (CECGM) for 1 hr at 37 &#176;C with intermittent gentle agitation.</li>
	<li>Remove the vessel from the tube and centrifuge the cell suspension for 5 min at 250 x g.</li>
	<li>Resuspend the cell pellet in CECGM and seed 2 ml cell suspension per well (1 - 3 wells depending on vessel size). Culture the cells at 37 &#176;C in a humidified atmosphere with 5% CO2 in air and change the culture medium twice a week.</li>
	<li>Passage the cells when a confluency of 70 - 80% is reached.
	<ol>
		<li>Wash the cells with pre-warmed HBSS to remove dead or non-attached cells. Add 200 &#181;l recombinant cell-dissociation enzyme (1x) to each well and place back in the incubator for 5 min or until all cells are detached.</li>
		<li>Transfer the cells to a 15 ml tube and stop trypsinization by adding 10 ml of culture media (CEGM) including 10% Fetal Calf Serum (FCS). Centrifuge for 5 min at 250 x g. Continue the culture in a gelatin pre-coated plate or flask.</li>
	</ol>
	</li>
</ol>
2. Characterization
<ol>
	<li>Culture Canine Aortic Endothelial Cells (CnAOECs) and compare the morphology of the primary and commercial cell lines twice weekly with a microscope. 
	NOTE: The typical growing pattern of ECs in patches can best be observed when a confluency of &#62; 70% is reached.
	<ol>
		<li>Culture CnAOECs in CECGM on a 0.5% w/v gelatin pre-coated T75 flask. Passage cells once a week when the confluency is 70 - 80%.
		<ol>
			<li>For passaging, wash the cells once with pre-warmed HBSS and add 1 ml recombinant cell-dissociation enzyme (1x). Place the flask back in the incubator for 5 min or until all cells are detached. Inactivate trypsin by adding 10 ml culture medium (CEGM) including 10% FCS. Centrifuge the cell suspension for 5 min at 250 x g, discard the supernatant and resuspend the cells in 1 ml culture medium.</li>
			<li>Take a 10 &#181;l aliquot of the cell suspension and dilute 1:1 with 0.4% trypan blue. Count the cells using an automated cell counter, and plate out 4.0 x 105 viable cells in a new T75 flask. Add 10 ml of pre-warmed CECGM to the flask and culture at 37 &#176;C in a humidified atmosphere with 5% CO2.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Isolate RNA from passage 1 of the cultured CaPECs and CnAOECs.
	<ol>
		<li>Collect at least 1 x 103 cells as a pellet, add 20 &#181;l of sample preparation reagent (SPR) and incubate for 1 min to lyse the cells. After incubation, carefully collect the cell lysate containing total RNA and store at -70 &#176;C.</li>
	</ol>
	</li>
	<li>Convert mRNA to cDNA using a cDNA synthesis kit (e.g., iScript) following the instructions of the manufacturer. Store cDNA at 4 &#176;C up to one week, or at -20 &#176;C for long term until ready to perform the qPCR.</li>
	<li>Measure gene expression of the endothelial marker CD31 to confirm endothelial cell origin. Perform experimental setup and quantification using the MIQE pr&#233;cis guidelines10. For normalization, measure expression of reference genes GAPDH, RPS19, and B2MG11.
	<ol>
		<li>Prepare a 10-fold dilution of the obtained cDNA in nuclease free water.</li>
		<li>Prepare a 4-fold dilution from pooled cDNA samples for the standard line and use nuclease free water as a non-template control. Dilute the samples five times to reach a 50-fold total dilution for qPCR reactions. Pipette 10 &#181;l reactions in duplicate in a 384-well format using 4 &#181;l cDNA and 6 &#181;l of the fluorophore mixed with 20 pmol of reverse and forward primer (Table 1).</li>
		<li>Set the program to 95 &#176;C for 5 min for Taq polymerase activation, followed by 39 cycles of 10 sec at 95 &#176;C for denaturation, and 30 sec at Tm for annealing and elongation. The Tm for each primer set is shown in Table 1.</li>
		<li>Perform melting curve analyses following every run to ensure only one product is amplified. Normalize the expression levels of the samples using the average relative amount of the reference genes, and calculate the &#916;Ct if reaction efficiency is between 95% and 105%.</li>
	</ol>
	</li>
	<li>Assess EC functionality with an angiogenesis assay.
	<ol>
		<li>Add 10 &#181;l of extracellular matrix to each well of a pre-cooled angiogenesis slide and spread with a pipette tip to cover the surface of the well. Keep the extracellular matrix on ice and avoid the introduction of air bubbles into the gel. Solidify the gel by placing the slide in a Petri dish with water soaked paper towels in an incubator at 37 &#176;C with 5% CO2 for 30 min.</li>
		<li>Add 1.0 x 104 primary ECs in 50 &#181;l Endothelial Growth Medium per well. Incubate the slide for 6 hr at 37 &#176;C with 5% CO2.</li>
		<li>After 6 hr take photographs with a 20X magnification, making sure to include the whole well in the image.</li>
	</ol>
	</li>
</ol>

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The authors have no competing financial interests.

In studies focusing on canine ECs the CnAOEC primary line is used to model the endothelial lineages of the dog3, 12, 13. In human studies, the HUVEC culture is still considered the gold standard. Clearly, focusing merely on ECs derived from umbilical cord is a firm restriction in cardiovascular research. Endothelial cells have a specific gene expression pattern determining arteriovenous specification. In order to account for these differences in postnatal vessels we present this novel isolation method based on...

Dogs are used as large animal model for cardiovascular disease research and can also suffer from inborn (genetic) vascular abnormalities1, 2. To study these diseases commercial endothelial cell lines are often used to assess endothelial cell (EC) functionality. For dogs there is one commercial endothelial cell line available (CnAOEC), derived from canine aorta. This cell line is mostly used in studies as control normal ECs3-5. In human cardiovascular research the most commonly used endothelial cell lines are Human Umbilical Vein Endothelial Cells (HUVECs) and Human Umbilical Artery Endothelial Cells (HUAECs) derived from human umbilical cord vein and artery, respectively. HUVECs have been used as the golden standard in vascular research since the 1980s6. They are considered to be the classic model system to study endothelial function and disease adaptation. Endothelial cells isolated from different blood vessels vary in appearance and functionality due to genetic background and exposure to the microenvironment7. In addition, HUVECs and HUAECs are derived from umbilical cord, a developmental vascular structure that might not fully mimic adult blood vessels with respect to the conditions they are exposed to and response to disease. Hence, translating results found in HUVECs and HUAECs to cardiovascular disease in general is inadequate.
When studying adaptation and behavior of adult ECs, primary ECs from the vessel of interest should be used as a more direct approach. To isolate these cells, several methods have been reported. A widely described method, which is also used for HUVECs, is flushing the vessel with an enzymatic digestion solution8. This often results in contamination with non-ECs such as smooth muscle cells and fibroblasts9. Another frequently used method for isolation is enzymatic digestion of minced vessel tissue followed by fluorescence-activated cell sorting (FACS) based on endothelial cell marker Cluster of Differentiation (CD)317, 8. FACS sorting and subsequent cell culture requires relatively large amounts of cells and is therefore not suitable for the isolation of endothelium from small blood vessels. We therefore aimed at developing a new robust method for isolating a pure endothelial cell population from various canine blood vessels with high purity. To test the efficiency of the new isolation method, we isolated and obtained pure Canine Primary Endothelial Cell (CaPEC) cultures from different canine arteries and veins, both large and small. This method also enables the culture of endothelial cells originating from diseased and/or aberrant vessels such as inborn intra- or extra-hepatic portosystemic shunts, a common disease in dogs2. The method allows the isolation of additional relevant cell types of the same vessel such as vascular smooth muscle cells since most of the vessel remains intact during the procedure.

<table border="0" cellpadding="0" cellspacing="0" width="904">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Collagenase type II</td>
			<td style="width:164px;">Life Technologies</td>
			<td style="width:211px;">17101-015</td>
			<td style="width:201px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dispase</td>
			<td style="width:164px;">Life Technologies</td>
			<td style="width:211px;">17105-041</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">DMEM (1X) + GlutaMAX</td>
			<td style="width:164px;">Life Technologies</td>
			<td style="width:211px;">31966-021</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Hank&#39;s Balanced Salt Solution</td>
			<td style="width:164px;">Life Technologies</td>
			<td style="width:211px;">14025-050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Canine Endothelial Cells Growth Medium&#160;</td>
			<td style="width:164px;">Cell Applications</td>
			<td style="width:211px;">Cn211-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">CnAOECs</td>
			<td style="width:164px;">Cell Applications</td>
			<td>Cn304-05</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Fetal Calf Serum (FCS)&#160;</td>
			<td style="width:164px;">GE Healthcare</td>
			<td style="width:211px;">16000-044</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">TrypLE Express</td>
			<td style="width:164px;">Life Technologies</td>
			<td style="width:211px;">12604-013</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">SPR</td>
			<td style="width:164px;">Bio-Rad</td>
			<td>170-8898</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">iScript synthesis kit</td>
			<td style="width:164px;">Bio-Rad</td>
			<td style="width:211px;">170-8891</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">SYBR green super mix</td>
			<td style="width:164px;">Bio-Rad</td>
			<td style="width:211px;">170-8886</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Recovery Cell Freezing Medium</td>
			<td style="width:164px;">Gibco/Life Technologies</td>
			<td style="width:211px;">12648-010</td>
			<td>Keep on ice prior to use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Freezing container, Nalgene Mr. Frosty</td>
			<td style="width:164px;">Sigma-Aldrich</td>
			<td style="width:211px;">C1562</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Gelatin</td>
			<td style="width:164px;">Sigma-Aldrich</td>
			<td style="width:211px;">G1890</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Surgical scissors (Mayo or Metzenbaum)</td>
			<td style="width:164px;">B. Braun Medical</td>
			<td style="width:211px;">BC555R</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mosquito forceps&#160;</td>
			<td style="width:164px;">B. Braun Medical</td>
			<td style="width:211px;">FB440R</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Mosquito forceps curved</td>
			<td style="width:164px;">B. Braun Medical</td>
			<td style="width:211px;">FB441R</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">polyglactin 3-0</td>
			<td style="width:164px;">Ethicon</td>
			<td style="width:211px;">VCP311H</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Trypan blue</td>
			<td style="width:164px;">Bio-Rad</td>
			<td style="width:211px;">145-0013</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Automated counting chamber</td>
			<td style="width:164px;">Bio-Rad</td>
			<td style="width:211px;">145-0102</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Counting Slides, Dual Chamber</td>
			<td style="width:164px;">Bio-Rad</td>
			<td style="width:211px;">145-0011</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Matrigel</td>
			<td style="width:164px;">BD Biosciences</td>
			<td style="width:211px;">BD356231</td>
			<td>Slowly thaw on ice</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">&#181;-Slide Angiogenesis</td>
			<td style="width:164px;">Ibidi</td>
			<td style="width:211px;">81501</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Endothelial Growth Medium</td>
			<td style="width:164px;">Lonza</td>
			<td style="width:211px;">CC-3156</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">EGM-2 SingleQuot Kit&#160;</td>
			<td style="width:164px;">Lonza</td>
			<td style="width:211px;">CC-4176</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation and culture of primary endothelial cells from canine arteries and veins

Cardiovascular disease is studied in both human and veterinary medicine. Endothelial cells have been used extensively as an in vitro model to study vasculogenesis, (tumor) angiogenesis, and atherosclerosis. The current standard for in vitro research on human endothelial cells (ECs) is the use of Human Umbilical Vein Endothelial Cells (HUVECs) and Human Umbilical Artery Endothelial Cells (HUAECs). For canine endothelial research, only one cell line (CnAOEC) is available, which is derived from canine aortic endothelium. Although currently not completely understood, there is a difference between ECs originating from either arteries or veins. For a more direct approach to in vitro functionality studies on ECs, we describe a new method for isolating Canine Primary Endothelial Cells (CaPECs) from a variety of vessels. This technique reduces the chance of contamination with fast-growing cells such as fibroblasts and smooth muscle cells, a problem that is common in standard isolation methods such as flushing the vessel with enzymatic solutions or mincing the vessel prior to digestion of the tissue containing all cells. The technique we describe was optimized for the canine model, but can easily be utilized in other species such as human.

Different blood vessels were successfully subjected to the described isolation protocol (Figure 2). It was possible to dissect and invert aorta, vena cava, vena porta, and coronary artery from healthy dogs (all vessels from each dog, n= 4). With the same approach ECs were isolated from two congenital portosystemic shunts (extrahepatic and intrahepatic, n= 1 each). Although aorta was easily inverted, thoracic aorta segments were more challenging than abdominal aorta. In th...

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Isolation and Culture of Primary Endothelial Cells from Canine Arteries and Veins

Novel isolation methods of primary endothelial cells from blood vessels are needed. This protocol describes a new technique that completely inverts blood vessels of interest, exposing only the endothelial side to enzymatic digestion. The resulting pure endothelial cell culture can be used to study cardiovascular diseases, disease modelling, and angiogenesis.

Cardiovascular disease is studied in both human and veterinary medicine. Endothelial cells have been used extensively as an in vitro model to study ...

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11.2K Views.  Utrecht University. Novel isolation methods of primary endothelial cells from blood vessels are needed. This protocol describes a new technique that completely inverts blood vessels of interest, exposing only the endothelial side to enzymatic digestion. The resulting pure endothelial cell culture can be used to study cardiovascular diseases, disease modelling, and angiogenesis.

Video: Isolation and Culture of Primary Endothelial Cells from Canine Arteries and Veins

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for progression-free and overall survival. Therefore, translational research needs to provide further molecular evidence to improve targeted therapies for malignant melanomas. In the past, oncogenic mechanisms related to melanoma were extensively studied in established cell lines. On the way to more personalized treatment regimens based on individual genetic profiles, we propose to use patient-derived cell lines instead of generic cell lines. Together with high quality clinical data, especially on patient follow-up, these cells will be instrumental to better understand the molecular mechanisms behind melanoma progression.
Here, we report the establishment of primary melanoma cultures from dissected fresh tumor tissue. This procedure includes mincing and dissociation of the tissue into single cells, removal of contaminations with erythrocytes and fibroblasts as well as primary culture and reliable verification of the cells' melanoma origin.
Recent reports revealed that melanomas, like the majority of tumors, harbor a small subpopulation of cancer stem cells (CSCs), which seem to exclusively fuel tumor initiation and progression towards the metastatic state. One of the key markers for CSC identification and isolation in melanoma is CD133. To isolate CD133+ CSCs from primary melanoma cultures, we have modified and optimized the Magnetic-Activated Cell Sorting (MACS) procedure from Miltenyi resulting in high sorting purity and viability of CD133+ CSCs and CD133- bulk, which can be cultivated and functionally analyzed thereafter.

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

This article describes the preparation of freshly obtained melanoma tissue into primary cell cultures, and how to remove contaminations of erythrocytes and fibroblasts from the tumor cells. Finally, we describe how CD133+ putative melanoma stem cells are sorted from the CD133- bulk using Magnetic Activated Cell Sorting (MACS).

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for ...

Primary Culture of Human Vestibular Schwannomas

Vestibular schwannomas (VSs) represent Schwann cell (SC) tumors of the vestibular nerve, compromising 10% of all intracranial neoplasms. VSs occur in either sporadic or familial (neurofibromatosis type 2, NF2) forms, both associated with inactivating defects in the NF2 tumor suppressor gene. Treatment for VSs is generally surgical resection or radiosurgery, however the morbidity of such procedures has driven investigations into less invasive treatments. Historically, lack of access to fresh tissue specimens and the fact that schwannoma cells are not immortalized have significantly hampered the use of primary cultures for investigation of schwannoma tumorigenesis. To overcome the limited supply of primary cultures, the immortalized HEI193 VS cell line was generated by transduction with HPV E6 and E7 oncogenes. This oncogenic transduction introduced significant molecular and phenotypic alterations to the cells, which limit their use as a model for human schwannoma tumors. We therefore illustrate a simplified, reproducible protocol for culture of primary human VS cells. This easily mastered technique allows for molecular and cellular investigations that more accurately recapitulate the complexity of VS disease.

Vestibular Schwannomas (VSs) are non-malignant tumors of Schwann cell (SC) origin, associated with mutations in the NF2 tumor suppressor gene. We report a reproducible, efficient protocol for primary human VS cell culture that allows for molecular and cellular experimental manipulation and analysis and recapitulates the heterogeneous nature of human disease.

Vestibular schwannomas (VSs) represent Schwann cell (SC) tumors of the vestibular nerve, compromising 10% of all intracranial neoplasms. VSs occur in ...

Method for Obtaining Primary Ovarian Cancer Cells From Solid Specimens

Reliable tools for investigating ovarian cancer initiation and progression are urgently needed. While the use of ovarian cancer cell lines remains a valuable tool for understanding ovarian cancer, their use has many limitations. These include the lack of heterogeneity and the plethora of genetic alterations associated with extended in vitro passaging.&#160;Here we describe a method that allows for rapid establishment of primary ovarian cancer cells form solid clinical specimens collected at the time of surgery. The method consists of subjecting clinical specimens to enzymatic digestion for 30 min. The isolated cell suspension is allowed to grow and can be used for downstream application including drug screening. The advantage of primary ovarian cancer cell lines over established ovarian cancer cell lines is that they are representative of the original specific clinical specimens they are derived from and can be derived from different sites whether primary or metastatic ovarian cancer.

This study describes a detailed method for isolation and characterization of primary ovarian cancer cells from solid clinical specimens. Ovarian cancer clinical specimens are subjected to enzymatic digestion to obtain viable, fibroblast-free epithelial ovarian cancer (EOC) cells highly suitable for downstream applications.

Reliable tools for investigating ovarian cancer initiation and progression are urgently needed. While the use of ovarian cancer cell lines remains a ...

Isolation, Cryopreservation and Culture of Human Amnion Epithelial Cells for Clinical Applications

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a potential source of cells for regenerative medicine. The reason for this interest is evidence that these cells have highly multipotent differentiation ability, low immunogenicity, and anti-inflammatory functions. These properties have prompted researchers to investigate the potential of hAECs to be used to treat a variety of diseases and disorders in pre-clinical animal studies with much success.
hAECs have found widespread application for the treatment of a range of diseases and disorders. Potential clinical applications of hAECs include the treatment of stroke, multiple sclerosis, liver disease, diabetes and chronic and acute lung diseases. Progressing from pre-clinical animal studies into clinical trials requires a higher standard of quality control and safety for cell therapy products. For safety and quality control considerations, it is preferred that cell isolation protocols use animal product-free reagents. 
We have developed protocols to allow researchers to isolate, cryopreserve and culture hAECs using animal product-free reagents. The advantage of this method is that these cells can be isolated, characterized, cryopreserved and cultured without the risk of delivering potentially harmful animal pathogens to humans, while maintaining suitable cell yields, viabilities and growth potential. For researchers moving from pre-clinical animal studies to clinical trials, these methodologies will greatly accelerate regulatory approval, decrease risks and improve the quality of their therapeutic cell population.

We describe a protocol to isolate and culture human amnion epithelial cells (hAECs) using animal product-free reagents in accordance with current good manufacturing practices (cGMP) guidelines.

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a ...

Isolation of Human Lymphatic Endothelial Cells by Multi-parameter Fluorescence-activated Cell Sorting

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity but little is known about their biology. Isolating highly pure human lymphatic endothelial cells (LECs) from diseased and healthy tissue would facilitate studies of the lymphatic endothelium at genetic, molecular and cellular levels. It is anticipated that these investigations may reveal targets for new therapies that may change the clinical management of these conditions. A protocol describing the isolation of human foreskin LECs and lymphatic malformation lymphatic endothelial cells (LM LECs) is presented. To obtain a single cell suspension tissue was minced and enzymatically treated using dispase II and collagenase II. The resulting single cell suspension was then labelled with antibodies to cluster of differentiation (CD) markers CD34, CD31, Vascular Endothelial Growth Factor-3 (VEGFR-3) and PODOPLANIN. Stained viable cells were sorted on a fluorescently activated cell sorter (FACS) to separate the CD34LowCD31PosVEGFR-3PosPODOPLANINPos LM LEC population from other endothelial and non-endothelial cells. The sorted LM LECs were cultured and expanded on fibronectin-coated flasks for further experimental use.

The goal of this protocol is to isolate lymphatic endothelial cells lining human lymphatic malformation cyst-like vessels and foreskins using fluorescence-activated cell sorting (FACS). Subsequent cell culturing and expansion of these cells permits a new level of experimental sophistication for genetic, proteomic, functional and cell differentiation studies.

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity ...

Isolation and Propagation of Circulating Tumor Cells from a Mouse Cancer Model

Cancer metastasis is the foremost cause of cancer-associated deaths. Recent studies have shown that circulating tumor cells (CTCs) are important in cancer metastasis. Indeed, the number of CTCs correlates with tumor size. Here, a detailed description is provided of a methodology for isolation and propagation of CTCs from a syngeneic mouse model of hepatocellular carcinoma (HCC) which allows for downstream analysis of potentially important molecular mechanisms of solid organ tumor metastasis. This method is efficient and reproducible. It is a non-invasive technique and, therefore, has potential to replace the invasive biopsy of tissues from humans which may be associated with complications. Therefore, the method discussed here allows for the isolation and propagation of CTCs from whole blood samples such that they can be examined and characterized. This has potential for future adaptation for clinical applications such as diagnosis, and personalized targeted therapy.

Circulating tumor cells (CTCs) have been shown to play an important role in tumor metastasis. Here, a method for the isolation and propagation of CTCs from the whole blood of a syngeneic mouse tumor model of hepatocellular carcinoma (HCC) metastasis is described.

Cancer metastasis is the foremost cause of cancer-associated deaths. Recent studies have shown that circulating tumor cells (CTCs) are important in cancer ...

Isolation and Culture Expansion of Tumor-specific Endothelial Cells

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model for developing new angiogenesis inhibitors for cancer. However, long-term in vitro expansion of murine endothelial cells (EC) is challenging due to phenotypic drift in culture (endothelial-to-mesenchymal transition) and contamination with non-EC. This is especially true for TEC which are readily outcompeted by co-purified fibroblasts or tumor cells in culture. Here, a high fidelity isolation method that takes advantage of immunomagnetic enrichment coupled with colony selection and in vitro expansion is described. This approach generates pure EC fractions that are entirely free of contaminating stromal or tumor cells. It is also shown that lineage-traced Cdh5cre:ZsGreenl/s/l reporter mice, used with the protocol described herein, are a valuable tool to verify cell purity as the isolated EC colonies from these mice show durable and brilliant ZsGreen fluorescence in culture.

We report a reliable method to isolate and culture primary tumor-specific endothelial cells from genetically engineered mouse models.

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model ...

Isolation of Endothelial Progenitor Cells from Healthy Volunteers and Their Migratory Potential Influenced by Serum Samples After Cardiac Surgery

Endothelial progenitor cells (EPCs) are recruited from the bone marrow under pathological conditions like hypoxia and are crucially involved in the neovascularization of ischemic tissues. The origin, classification and characterization of EPCs are complex; notwithstanding, two prominent sub-types of EPCs have been established: so-called &#34;early&#34; EPCs (subsequently referred to as early-EPCs) and late-outgrowth EPCs (late-EPCs). They can be classified by biological properties as well as by their appearance during in vitro culture. While &#34;early&#34; EPCs appear in less than a week after culture of peripheral blood-derived mononuclear cells in EC-specific media, late-outgrowth EPCs can be found after 2-3 weeks. Late-outgrowth EPCs have been recognized to be directly involved in neovascularization, mainly through their ability to differentiate into mature endothelial cells, whereas &#34;early&#34; EPCs express various angiogenic factors as endogenous cargo to promote angiogenesis in a paracrine manner. During myocardial ischemia/reperfusion (I/R), various factors control the homing of EPCs to regions of blood vessel formation.
Macrophage migration inhibitory factor (MIF) is a chemokine-like pro-inflammatory and ubiquitously expressed cytokine and was recently described to function as key regulator of EPCs migration at physiological concentrations1. Interestingly, MIF is stored in intracellular pools and can rapidly be released into the blood stream after several stimuli (e.g. myocardial infarction). 
This protocol describes a method for the reliable isolation and culture of early-EPCs from adult human peripheral blood based on CD34-positive selection with subsequent culture in medium containing endothelial growth factors on fibronectin-coated plates for use in in vitro migration assays against serum samples of cardiac surgical patients. Furthermore, the migratory influence of MIF on chemotaxis of EPCs compared to other well-known angiogenesis-stimulating cytokines is demonstrated.

Endothelial progenitor cells (EPCs) are crucially involved in the neovascularization of ischemic tissues. This method describes the isolation of human EPCs from peripheral blood, as well as the identification of their migratory potential against serum samples of cardiac surgical patients.

Endothelial progenitor cells (EPCs) are recruited from the bone marrow under pathological conditions like hypoxia and are crucially involved in the ...

A Cell Culture Model of Resistance Arteries

The myoendothelial junction (MEJ), a unique signaling microdomain in small diameter resistance arteries, exhibits localization of specific proteins and signaling processes that can control vascular tone and blood pressure. As it is a projection from either the endothelial or smooth muscle cell, and due to its small size (on average, an area of ~1 &#181;m2), the MEJ is difficult to study in isolation. However, we have developed a cell culture model called the vascular cell co-culture (VCCC) that allows for in vitro MEJ formation, endothelial cell polarization, and dissection of signaling proteins and processes in the vascular wall of resistance arteries. The VCCC has a multitude of applications and can be adapted to suit different cell types. The model consists of two cell types grown on opposite sides of a filter with 0.4 &#181;m pores in which the in vitro MEJs can form. Here we describe how to create the VCCC via plating of cells and isolation of endothelial, MEJ, and smooth muscle fractions, which can then be used for protein isolation or activity assays. The filter with intact cell layers can be fixed, embedded, and sectioned for immunofluorescent analysis. Importantly, many of the discoveries from this model have been confirmed using intact resistance arteries, underscoring its physiological relevance.

A cell culture model of resistance arteries is described, allowing for the dissection of signaling pathways in endothelium, smooth muscle, or between endothelium and smooth muscle (the myoendothelial junction). The selective application of agonists or protein isolation, electron microscopy, or immunofluorescence can be utilized using this cell culture model.

The myoendothelial junction (MEJ), a unique signaling microdomain in small diameter resistance arteries, exhibits localization of specific proteins and ...