Name: Isolation and Culture Expansion of Tumor-specific Endothelial Cells (Video) | JoVE Journal
Uploaded: 2015-10-14T15:00:00.000+00:00
Duration: 10 min 15 s
Description: Learn to isolate tumor-specific endothelial cells using magnetic bead enrichment and cloning ring selection from mouse tumor tissue. This gentle method enables high-purity clonal expansion for cancer vascular research.

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5 minutes

ACD is supported by a grant from the National Institute of Health (R01-CA177875). LX is a fellow in the HHMI-funded translational medicine program at UNC Chapel Hill. JVM is supported by a T32 pre-doctoral fellowship from the Integrative Vascular Biology Program at UNC Chapel Hill. We thank Clayton Davis for assistance with confocal microscopy.

The following protocol is carried out according to guidelines established by the Department of Laboratory Animal Medicine at the University of North Carolina at Chapel Hill.
1. Prepare the Following Material and Reagents Before Starting
<ol>
	<li>Prepare EC media by supplementing 400 ml&#160;low glucose (1 g/L D-glucose or LG) Dulbecco&#39;s Modified Eagle&#39;s medium (DMEM) with 50 ml heat-inactivated fetal bovine serum, 50 ml Nu-Serum IV, 5 ml antibiotic-antimycotic, and the hFGF, VEGF, hEGF, R3-IGF-1, and heparin components from the commercial kit.</li>
	<li>Prepare 500 ml FACS buffer (0.5% BSA and 2 mM EDTA in PBS); filter through a sterile 0.22 &#181;m filter cup.</li>
	<li>Sterilize or disinfect dissecting board.</li>
	<li>Sterilize dissection pins, surgical scissors, and dissectors.</li>
</ol>
2. EC Isolation (Day 1, ~5 hr)
<ol>
	<li>Euthanize mouse with carbon dioxide or other methods compliant with the Institutional Animal Care and Use Committee (IACUC) policies. 
	Note: Multiple mammary tumors varying in size (5 - 15 mm in diameter) may develop in a single genetically modified mouse such as C3-TAg, be sure to harvest all of them for TEC isolation. If using tumors that are orthotopically engrafted in mice, pool two to three 1 cm3 tumors for a single EC isolation. Here we use mammary tumors as a demonstration, but the protocol can be modified for other types of tumors.</li>
	<li>Disinfect mouse by spraying or wiping the mouse ventral side with ample amount of 75% v/v ethanol.</li>
	<li>Resect tumors with one pair of scissors and dissectors using aseptic techniques in a sterile hood or at a clean bench.
	<ol>
		<li>Stretch out and pin the limbs of the mice on a dissecting board. Make a midline ventral incision with the scissors without opening the peritoneum. Dissect laterally between the skin and the peritoneum towards the mammary glands where tumors are located. Do not open the peritoneum.</li>
		<li>Excise only tumor tissue from the mammary glands, leaving out normal mammary margins. Carefully trim off non-tumor tissues such as skin and muscles, and place dissected tumors in a conical tube containing 30 ml of LG-DMEM on ice.</li>
	</ol>
	</li>
	<li>Bring tumor samples to tissue culture hood; wash tissues with sterile LG-DMEM 1 - 2 times.</li>
	<li>Transfer tumors from the conical tube to a sterile tissue-culture petri dish, add 2 ml of LG-DMEM in the dish, and mince with a pair of sterile scissors into pieces &#60;5 mm.</li>
	<li>Add 5 ml of collagenase (stock = 2 mg/ml in Hank&#8217;s Balanced Salt Solution, hereafter HBSS), 1 ml of dispase (stock = 2.5 U/ml in HBSS) and 75 &#956;l of deoxyribonuclease (stock = 1 mg/ml in PBS) into the petri dish. Total volume is now ~10 ml.</li>
	<li>Transfer the collagenase/tissue mix from the petri dish to a tissue-dissociator tube and run on a tissue dissociator for 60 sec twice (pre-set dissociation program on the dissociator: 1,270 total rounds per run). Incubate with light shaking on a shaker for 75 min at 37 &#176;C.</li>
	<li>Filter digested tissue through a 100 &#956;m cell strainer over a 50 ml conical tube. Rinse filter with 5 ml of FACS buffer to wash any remaining cells. Spin at 280 x g for 5 min and carefully aspirate the supernatant without disturbing the cell pellet.</li>
	<li>Dilute 1 ml of stock RBC lysis buffer (10x) in 9 ml of sterile water. Lyse red blood cells with 10 ml of lysis buffer (1x), and immediately spin 5 min at 280 x g. Note: This step can be skipped if little blood is visible.</li>
	<li>Resuspend in 10 ml of FACS buffer. Mix 10 &#956;l of cell suspension with 10 &#956;l of trypan blue, and count live cells using a hemocytometer .</li>
	<li>Resuspend cells at ~107 cells/100 &#956;l. Add 10 &#956;l of FcR block per 100 &#956;l of cell suspension, and incubate on ice for 10 min.</li>
	<li>Add rat-anti mouse PE-conjugated CD31 antibody according to Table 1. Incubate on ice for 10 - 15 min and flick tube occasionally.</li>
	<li>Add 10 ml of FACS buffer to the tube and spin at 280 x g for 5 min. Carefully remove the supernatant, and wash the cell pellet again with 5 ml of FACS buffer. Centrifuge to pellet cells. Aspirate supernatant without disturbing cell pellet.</li>
	<li>Add FACS buffer and anti-PE microbeads according to Table 2. Incubate on ice for 10 - 15 min. Flick tube occasionally.</li>
	<li>Add 10 ml of FACS buffer and spin samples at 280 x g for 5 min; wash once with 5 ml of FACS buffer and centrifuge again. Aspirate supernatant without disturbing pellet.</li>
	<li>Bring volume to 300 &#956;l in FACS buffer. Spin through 35 &#956;m cell-strainer capped tube 5 min at 280 x g. Use 2 tubes and a larger volume if needed.</li>
	<li>Set up magnetic multistand and magnetic columns in hood, attach a column to the separator and equilibrate the column with 2 ml of FACS buffer.</li>
	<li>Aspirate the supernatant and resuspend the cell pellet in 0.5 - 1 ml of FACS buffer.</li>
	<li>Pass the cell suspension through the equilibrated magnetic column.</li>
	<li>Wash the column three times with 2 ml of FACS buffer, and collect flow-through (FT) in a 15 ml tube (FT fraction).</li>
	<li>Take the column off the separator and elute with 2 ml of FACS buffer into another 15 ml tube (eluate fraction). Use plunger to ensure all cells are off the column. Repeat the elution two more times with 2 ml of FACS buffer each time.</li>
	<li>Spin the eluate at 280 x g for 5 min.</li>
	<li>Remove the supernatant and resuspend the pellet in 10 ml of EC media.</li>
	<li>Equally divide the eluate fraction (~ 6 ml) into 10 cm gelatin-coated dishes. For three 1 cm3 tumors, plate eluted cells in at least four plates. Alternatively, plate eluted cells at different concentrations in multiple plates (e.g., seed 0.5 ml, 1 ml, 1.5 ml, and 3 ml of the eluate into four plates) to ensure that at least one plate is sparsely seeded with eluted cells. Check that the confluency of the attached cells is at ~1% the next day (i.e., approximately 1.0 - 2.0 X 105 attached cells). 
	Note: The cells need to be plated sparse so that EC colonies can form without being contaminated by other cell types.</li>
	<li>Plate FT fraction in one 10 cm dish to let cells recover O/N. Check that the plate is 80&#160;- 100% confluent the next day. Freeze down the cells (-80 &#176;C) in 250 &#181;l cell-freezing media in a cryotube and store them in liquid nitrogen the next day.&#160;Note: FT fraction can be used for tumor cell isolation at a later stage and/or as a negative control for EC gene expression analysis of the isolated EC clones.</li>
	<li>Change media every 2 - 3 days. Colonies start to form after 7 - 10 days. Small EC colonies can be identified as early as day 3. Mark the colonies with a fine-tip marker on the bottom of the dish.</li>
	<li>Scrape off non-specific cells surrounding the identified colonies with a sterile 200 &#181;l pippet tip.</li>
</ol>
3. Colony Selection Using Cloning Rings
<ol>
	<li>Change media every 2 - 3 days. EC purity can be checked by LDL (DiI-Ac-LDL) addition at about 5 - 7 days. Add 50 &#181;l of LDL per 10 ml EC medium and incubate for 3 - 4 hr before checking the cells under fluorescence microscope. 
	Note: Multiple LDL+ EC clones could be observed at ~day 7.</li>
	<li>Start harvesting EC colonies when they reach diameters of 3 - 5 mm in size. (Choose big colonies that are packed with small LDL+ cells for best results.)</li>
	<li>Before harvesting EC with cloning rings, pre-coat a few 6-well plates with 0.5% gelatin. Aspirate gelatin, add 2 ml of EC media to each well, and keep the plates in an incubator until needed.</li>
	<li>Scrape off non-EC on the edges of the colonies to make sure no other cell types will be trapped within the cloning ring.</li>
	<li>Using a phase-contrast microscope (4X or 10X objective), outline with a fine-tip marker on the bottom of the culture dish the areas containing EC colonies.</li>
	<li>Wash the plate with 10 ml of PBS and leave a very thin layer of PBS in the plate when aspirating. (Important: a small amount (~0.5 ml) of PBS will keep cells alive during the cloning-ring procedure; also, tissue adhesive needs water to bond.)</li>
	<li>Choose a cloning ring of appropriate size. Use a pair of dissecting forceps to pick up a ring, and with a 10 &#181;l&#160;pipet tip evenly apply a small amount of tissue adhesive onto the cloning ring. 
	Note: use only minimal amount (~0.2 &#956;l for a small ring) of tissue adhesive on cloning rings, and make sure tissue adhesive is spread out evenly around the bottom surface to ensure good sealing. Excessive tissue adhesive produces heat and forms films that may kill cells.</li>
	<li>Place the cloning ring over the EC colony. Gently press down the cloning ring to glue the ring onto the plate. Ensure that the colonies are not dried out before gluing the ring.</li>
	<li>Immediately pipet 25 &#181;l&#160;of enzymatic cell detachment solution into the cloning ring and incubate ~1 min or until the cells are loosely attached.</li>
	<li>Pipet cells in the cloning ring drop-wise into one well of a 6-well plate containing pre-warmed EC media. (Important: Do not shake the plate to disperse cells; EC prefer to grow in tight clusters.) Wash the cloning ring with 50 - 100 &#181;l&#160;EC media to collect as many cells as possible, and transfer all washes into the same 6-well.</li>
	<li>If some colonies in the 10 cm dish are too small to harvest, add 10 ml fresh media, let the colonies grow for a few more days, and repeat the cloning ring procedure again.</li>
	<li>Grow harvested colonies in 6-well plates until 80 - 100% confluent, and transfer cells to 3 wells of a 6-well plate, before expanding them in a 10 cm tissue culture dish. Scrape off contaminant cells. Repeat another round of cloning ring procedure (Steps 3.5 - 3.10) if necessary. Keep cells relatively confluent (~60 - 70%) when expanding. EC may stop growing if plated too sparse.</li>
	<li>Characterize EC by FACS, staining, PCR, etc. Note that Dil-Ac-LDL is fluorescent and may interfere with PE or other fluorescent antibodies for FACS.</li>
</ol>

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<li>Chi, J. -T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Endothelial+cell+diversity+revealed+by+global+expression+profiling%5BTitle%5D)+AND+%22Proc.+Natl.+Acad.+Sci%22%5BJournal%5D)">Endothelial cell diversity revealed by global expression profiling.</a> Proc. Natl. Acad. Sci. 100 (19), 10623-10628 (2003).</li>
<li>Nolan, D. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Molecular+signatures+of+tissue-specific+microvascular+endothelial+cell+heterogeneity+in+organ+maintenance+and+regeneration%5BTitle%5D)+AND+%22Dev.+Cell%22%5BJournal%5D)">Molecular signatures of tissue-specific microvascular endothelial cell heterogeneity in organ maintenance and regeneration.</a> Dev. Cell. 26 (2), 204-219 (2013).</li>
<li>Ingram, D. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Vessel+wall-derived+endothelial+cells+rapidly+proliferate+because+they+contain+a+complete+hierarchy+of+endothelial+progenitor+cells%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">Vessel wall-derived endothelial cells rapidly proliferate because they contain a complete hierarchy of endothelial progenitor cells.</a> Blood. 105 (7), 2783-2786 (2005).</li>
</ol>

The authors have nothing to disclose.

Due to the difficulties in obtaining pure primary TEC cultures, many in vitro studies substitute TEC with commercially available EC lines or primary EC such as human umbilical vein EC (HUVEC) 13. However, these EC populations from normal tissues may only serve as a proxy for TEC which differ markedly from their normal counterparts. For example, TEC are phenotypically and functionally abnormal in vivo and some of these abnormalities may be transmittable in vitro 14-18. TEC ...

Endothelial cells (EC) are essential during the development of solid tumors. From initiation of the angiogenic switch in dormant tumors to dissemination and seeding of metastases at distant sites, EC form the conduits that provide blood, oxygen, and nutrients to sustain tumor growth 1. As recently suggested, EC also have perfusion-independent functions and form a niche that supports the growth of cancer stem cells and other tumor stromal cells 2-5. Thus, highly purified tumor-specific EC (TEC) for in vitro culture allows for routine functional studies that will shed light on novel molecular mechanisms mediating tumor angiogenesis and cross talk with tumor cells.
EC are highly specialized depending on the tissue of origin 6. Due to the heterogeneous nature of different tumor types and the tumor microenvironment, TEC may also display unique features that reflect a tumor-specific specialization of the vasculature. For example, there is striking variability in the gene expression signatures in TEC isolated from different types or grades of tumors 7,8. However, frequent co-purification of non-EC, especially tumor-associated fibroblasts and tumor cells, with TEC can confound genome-wide expression analyses. These unwanted cell types are especially problematic in studies that rely on long-term in vitro expansion of TEC cultures.
Described here is a high-fidelity method that consistently produces pure EC cultures from tumors and other tissues. Following immunomagnetic column enrichment of EC fractions and removal of co-purified non-EC, an additional cloning-ring step to capture pure EC colonies is used 9. Each colony can be expanded in culture for multiple passages without the emergence of contaminating non-EC. This method also yields multiple EC clones from a single isolation procedure, which is ideal for the study of endothelial heterogeneity. In addition, it is shown that Cdh5cre:ZsGreenl/s/l reporter mice are a valuable tool for generating &#8220;fate-mapped&#8221; and indelibly-marked EC which maintain ZsGreen fluorescence in culture 10. With minor adjustments to the protocol, this method should be adaptable to different tumor types or normal tissues.

<table><tbody><tr><td>Antibiotic-Antimycotic&amp;nbsp;</td><td>Sigma-Aldrich</td><td>A5955</td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle&#039;s medium (1 g/L D-glucose) (LG-DMEM)</td><td>Gibco</td><td>11885-084</td><td></td></tr><tr><td>EGM-2 Bullet Kit&amp;nbsp;</td><td>Lonza</td><td>CC4176</td><td>Not all components used</td></tr><tr><td>Fetal bovine serum (Hyclone)</td><td>Thermo Scientific</td><td>SH30071.03</td><td>Heat inactivated at 56 &amp;deg;C for 30 min</td></tr><tr><td>Nu-Serum IV</td><td>Corning</td><td>CB-51004</td><td></td></tr><tr><td>Hank&#039;s Balanced Salt Solution (HBBS)</td><td>Gibco</td><td>14175-095</td><td></td></tr><tr><td>Phosphate-buffered saline (PBS)</td><td>Gibco</td><td>14190-144</td><td></td></tr><tr><td>FACS buffer&amp;nbsp;</td><td></td><td></td><td>0.5 % BSA and 2 mM EDTA in PBS, filtered through a 0.22 &amp;mu;m filter</td></tr><tr><td>75% v/v ethanol for disinfection</td><td></td><td></td><td></td></tr><tr><td>Anti-PE microbeads&amp;nbsp;</td><td>Miltenyi Biotech</td><td>130-048-801</td><td></td></tr><tr><td>Bovine serum albumin (BSA) fraction V, 7.5%</td><td>Gibco</td><td>15260-37</td><td></td></tr><tr><td>Cell freezing media (Bambanker)</td><td>Wako Chemicals</td><td>302-14681</td><td></td></tr><tr><td>Collagenase type II&amp;nbsp;&amp;nbsp;</td><td>Worthington Biochemical</td><td>LS004176</td><td>Make stock concentration 2 mg/ml in HBSS</td></tr><tr><td>Deoxyribonuclease I (DNase)</td><td>Worthington Biochemical</td><td>LS002004</td><td>Make stock concentration 1 mg/ml in PBS</td></tr><tr><td>Dil-Ac-LDL</td><td>Biomedical Technologies</td><td>BT-902</td><td></td></tr><tr><td>EDTA, 0.5M, pH 8.0</td><td>Cellgro</td><td>46-034-CL</td><td></td></tr><tr><td>Enzymatic cell detachment solution (Accutase)</td><td>Sigma-Aldrich</td><td>A6964-100ML</td><td></td></tr><tr><td>Gelatin, 2% in water, tissue culture grade</td><td>Sigma-Aldrich</td><td>G1393-100ML</td><td>Dilute in PBS to make 0.5% gelatin solution</td></tr><tr><td>Mouse FcR Blocking Reagent&amp;nbsp;</td><td>Miltenyi Biotech</td><td>130-092-575</td><td></td></tr><tr><td>Neutral protease (Dispase)</td><td>Worthington Biochemical</td><td>LS02104</td><td>Make stock concentration 2.5 U/ml in HBSS</td></tr><tr><td>PE-rat anti-mouse CD31 antibody</td><td>BD Pharmingen</td><td>553373</td><td></td></tr><tr><td>RBC lysis buffer (BD Pharm Lyse)</td><td>BD Pharmingen</td><td>555899</td><td></td></tr><tr><td>Sterile water</td><td></td><td></td><td></td></tr><tr><td>Trypan blue, 0.4 %&amp;nbsp;</td><td>Life Technologies</td><td>15250-061</td><td></td></tr><tr><td>10 mm tissue culture dishes</td><td>Corning</td><td></td><td></td></tr><tr><td>15 ml conical tubes (sterile)</td><td>Corning</td><td></td><td></td></tr><tr><td>50 ml conical tubes (sterile)&amp;nbsp;</td><td>Corning</td><td></td><td></td></tr><tr><td>6-well tissue culture plates</td><td>Corning</td><td></td><td></td></tr><tr><td>Tissue-dissociator tubes (gentleMACS) C tubes)&amp;nbsp;</td><td>Miltenyi Biotech</td><td>130-093-237</td><td></td></tr><tr><td>Cell Separator&amp;nbsp; (MidiMACS)</td><td>Miltenyi Biotech</td><td>130-042-302</td><td></td></tr><tr><td>Cell strainer 100 &amp;mu;m&amp;nbsp;</td><td>Corning</td><td>352360</td><td></td></tr><tr><td>Cloning rings (assorted sizes)</td><td>Bel-Art Products</td><td>378470000</td><td></td></tr><tr><td>Cryotubes</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>Dissecting board</td><td></td><td></td><td>Sterilize or disinfect with 75% v/v ethanol before use&amp;nbsp;</td></tr><tr><td>Dissecting forceps and scissors</td><td></td><td></td><td>Sterilize before use&amp;nbsp;</td></tr><tr><td>Dissecting pins 2&quot;</td><td></td><td></td><td>Sterilize before use&amp;nbsp;</td></tr><tr><td>FACS tubes with 35 &amp;mu;m filter cap</td><td>Corning</td><td>352235</td><td></td></tr><tr><td>Filter cup (Stericup, 0.22 &amp;mu;m)</td><td>Millipore</td><td>SCGPU05RE</td><td></td></tr><tr><td>Fine-tip marker</td><td></td><td></td><td></td></tr><tr><td>Hemocytometer</td><td></td><td></td><td></td></tr><tr><td>LS Columns</td><td>Miltenyi Biotech</td><td>130-042-401</td><td></td></tr><tr><td>Magnetic Multistand</td><td>Miltenyi Biotech</td><td>130-042-303</td><td></td></tr><tr><td>Tissue adhesive (Vetbond)</td><td>3M</td><td>1469SB</td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>5810R</td><td>Or a centrifuge with similar capacity for 15 ml and 50 ml conical tube centrifugation</td></tr><tr><td>Tissue culture hood</td><td></td><td></td><td></td></tr><tr><td>Tissue dissociator (gentleMACS)</td><td>Miltenyi Biotech</td><td>130-093-235</td><td>Preset program &quot;m_impTumor_01&quot; used for tissue dissociation&amp;nbsp;</td></tr><tr><td>Liquid nitrogen freezer</td><td></td><td></td><td></td></tr><tr><td>Microplate or rotary shaker</td><td></td><td></td><td></td></tr><tr><td>Phase contrast light microscope</td><td></td><td></td><td></td></tr></tbody></table>

isolation and culture expansion of tumor-specific endothelial cells

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.

EC represent only minor a fraction of the total cell population in most adult tissues 11. It is therefore important to fully digest the harvested tissue into a single-cell suspension that ensures the maximal release of EC from extracellular matrix (ECM) and connective tissues. In our experience, CD31-mediated immunomagnetic selection only provides enriched but not pure EC fractions; therefore, another crucial step is the physical removal of co-purified non-EC and selection/expansion of EC colonies using clonin...

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 ...

The overall goal of this procedure is to isolate tumor endothelial cells using magnetic columns and cloning rings. This is accomplished by first digesting tumor tissue dissected from mice to generate a single cell suspension. The second step is to enrich the endothelial cell population using a magnetic bead column.Next, the endothelial cell colonies are grown and selected using a cloning ring method. The final step is to expand and characterize the selected endothelial cell colonies. Ultimately, immunofluorescence microscopy using specific endothelial cell markers is used to show the purity of the clonal populations.The main advantage of this technique over existing methods like fact sorting, is that it is gentler on the cells, which allows for greater recovery of viable endothelial cells and subsequent colony expansion. This method can help answer key questions in the tumor angiogenesis field, such as how vascular endothelial cell dysfunction in tumors contributes to tumor progression. Generally, individuals new to this method will struggle because endothelial cells are frequently contaminated with other cell types from the tumor microenvironment, and thus one must be diligent in quickly removing these contaminating cells from the cultures Following euthanasia.Bring the mouse to a clean bench and disinfect the ventral side by spraying it with an ample amount of 75%volume to volume. Ethanol next, pin the limbs of the mouse on the dissecting board using proper aseptic technique. Make a midline ventral incision without opening the peritoneum while keeping the peritoneum intact.Dissect laterally between the skin and peritoneum towards the MA glands where the tumors are located. Excise only tumor tissue from the memory glands by carefully trimming non-tumor tissues, including skin and muscles. Place the dissected tumors in a conical tube containing 30 milliliters of low glucose D echos, modified eagles medium, or LG DMEM To begin tumor dissociation.Transfer the tissue in sterile L-G-D-M-E-M to a tissue culture dish and wash one to two times with L-G-D-M-E-M. After the washes, add two milliliters of L-G-D-M-E-M and mince the tumors with a pair of sterile scissors. Add five milliliters of collagenase, one milliliter of disc pace, 75 microliters of deoxy ribonuclease, and two milliliters of L-G-D-M-E-M to the minced tumor.Transfer the collagenase tissue mix from the Petri dish to a tissue dissociation tube. Using a preset dissociation program, perform two 62nd rounds of dissociation. Incubate the tube with light shaking for 75 minutes at 37 degree Celsius.Following incubation, filter the digested tissue through a 100 micron cell strainer positioned over a 50 milliliter conical tube. Rinse the filter with five milliliters of fax buffer to collect any remaining cells and centrifuge the tube at 280 times G for five minutes. Carefully remove the supernatant without disturbing the cell pellet.Next, dilute one milliliter of 10 x stock. Red blood cell lysis buffer in nine milliliters of sterile water and resuspend the pellet to lyse any contaminating red blood cells immediately centrifuge the cells at 280 times G for five minutes. Remove the supernatant and resuspend the cells in 10 milliliters of fax buffer.To begin the immuno magnetic separation count live cells using a hemo cytometer and dilute to 10 to the seven cells per 100 microliters. In fax buffer, add 10 microliters of FCR blocking solution per 100 microliters and incubate on ice for 10 minutes After blocking, add rat anti mouse PE conjugated CD 31 antibody to the cells and incubate them on ice for 10 to 15 minutes. With occasional agitation, add 10 milliliters of fax buffer to the tube and centrifuge at 280 times G for five minutes.Carefully remove the snat and wash the cell pellet with five milliliters of fax buffer centrifuge again at 280 times G for five minutes to pellet the cells and then aspirate the supernatant. Now add fax buffer and anti PE microbeads to the cells and incubate them on ice for 10 to 15 minutes. With occasional agitation, add 10 milliliters of fax, buffer and centrifuge the samples at 280 times G for five minutes.Wash the cells with five milliliters of fax buffer centrifuge and remove the supernatant After bringing the volume to 300 microliters. With fax buffer centrifuge the cells through a 35 micron cell strainer capped tube at 280 times G for five minutes. Aspirate the supernatant for magnetic separation.Set up a magnetic multis stand and magnetic columns. In the cell culture hood, attach a column to the magnetic separator and then equate the column with two milliliters of fax buffer. Next, re suspend the cell palate in half a milliliter of fax buffer and pass the cell suspension through the equilibrated magnetic column.Wash the column three times with two milliliters of fax buffer while collecting the flow through in a 15 milliliter conical tube. Remove the column from the magnetic separator and elute with two milliliters of fax buffer using a plunger to ensure all of the cells are removed from the column. Collect the eluate in a new 15 milliliter conical tube.Centrifuge the eluate fraction at 280 times G for five minutes and remove the supernatant resus. Suspend the cells in 10 milliliters of endothelial cell media equally. Divide the cell suspension into at least four 10 centimeter gelatin coated cell culture dishes and incubate at 37 degrees Celsius and 5%carbon dioxide for an expected co fluency of approximately 1%The next day.Endothelial cell colonies should start to form after seven to 10 days and grow to three to five millimeters in diameter as colonies appear after seven to 10 days, remove any nonspecific surrounding cells with a sterile 200 microliter pipette tip. Once the colonies have grown to the correct size, view the dishes using a phase contrast microscope at four x or 10 x magnification and outline individual colonies on the bottom of the culture dish. Using a fine tip marker, wash the plate with 10 milliliters of PBS and aspirate leaving a small amount of PBS to keep the cells moist.Using, using a pair of dissecting forceps, pick up a cloning ring and pipette approximately half a microliter of tissue adhesive to the bottom. Spread the tissue adhesive around the bottom of the cloning ring evenly with a 10 microliter pipette tip. Place the cloning ring over the endothelial cell colony using the marks as a guide.Gently press down on the cloning ring to glue the ring onto the plate. Next, pipette 25 microliters of enzymatic cell detachment solution into the cloning ring and incubate for approximately one minute or until the cells are loosely attached. Pipette the cells from each cloning ring into one well of a six.Well plate. Wash the ring with 50 microliters of endothelial cell media to collect the remaining cells and transfer them to the same well in the six well plate grow isolated colonies in the six well plate until they reach 80 to 100%co fluency before transferring the cells to a new dish. Immuno magnetic endothelial cell isolation alone often results in culture contamination with non endothelial cells.Here endothelial cells specifically stained with dye, I-A-C-L-D-L can be seen commingling with unstained non endothelial cells. After using clonal rings for colony, even cell populations that have been expanded show uniform DI IAC LDL staining indicating that they are pure endothelial cell populations comparing three separately isolated colonies. Distinct uniform CD 31 positive populations are apparent.CD 31 is an endothelial cell marker while IgG serves as an isotype negative control for non-specific antibody binding. These cell populations also exhibited increased expression of endothelial cell marker genes between 200 to 7, 000 times higher than mouse embryonic fibroblast controls. Finally isolated normal or tumor endothelial cells retain endothelial cell function as evidenced by spontaneous vessel leg structure formation in culture.After watching this video, you should have a good understanding of how to perform a high fidelity isolation of tumor specific endothelial cells using immuno magnetic enrichment, coupled with colony selection and in vitro expansion.

isolation-and-culture-expansion-of-tumor-specific-endothelial-cells

Research

JoVE Journal

Medicine

Endothelial colony forming progenitor cells (ECFCs) are a promising tool to study vascular homeostasis and repair.1,2 This paper introduces a novel animal-serum free method for isolation and expansion of ECFC from heparinised adult human peripheral blood with pooled human platelet lysate (pHPL) diminishing the risk of anti-bovine immunisation.

Isolation and Large Scale Expansion of Adult Human Endothelial Colony Forming Progenitor Cells

Here, we describe a protocol for isolation and culture of murine pulmonary endothelial cells. This method comprises mechanic and enzymatic lung tissue dissociation as well as a 2-step purification process using anti-PECAM-1 and anti-ICAM-2 antibodies conjugated to magnetic beads, which produces a pure endothelial cell population of mostly microvascular origin.

Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice

A simple method is described for analyzing effects of tissue fibroblasts on associated epithelial cells.  The combination of this method and three-dimensional tissue culture can facilitate analysis of cells after isolation from 3D.  The technique is applicable to cells of varying malignant potential, allowing systematic study of effects of tumor-associated stroma on tumor cells.

Isolation of Mammary Epithelial Cells from Three-dimensional Mixed-cell Spheroid Co-culture

Methods to create human 3D tumor tissues as test systems are described. These technologies are based on a decellularized Biological Vascularized Scaffold (BioVaSc), primary human cells and a tumor cell line, which can be cultured under static as well as under dynamic conditions in a flow bioreactor.

Tissue Engineering of a Human 3D in vitro Tumor Test System

Primary cell culture is a useful technique for analyzing specific populations of cells, particularly from transgenic mouse embryos at specific developmental stages. Herein, embryonic ventricles are dissected and dissociated, and antibody-conjugated beads recognize and separate out the endothelial cells for further analysis.

Isolation of Embryonic Ventricular Endothelial Cells

This video demonstrates an easy and reliable strategy for preparation of pure cultures of endothelial cells from the embryonic forebrain within 10-12 days and will be useful for research focused on many aspects of cerebral angiogenesis.

Isolation and Culture of Endothelial Cells from the Embryonic Forebrain

Tumor-initiating cells (TICs) may represent a viable therapeutic target for the treatment of ovarian cancer, a highly recurrent and fatal disease. We present a protocol for culture conditions that enrich for this highly tumorigenic population of cells.

In vitro Enrichment of Ovarian Cancer Tumor-initiating Cells

The goal of this protocol is to isolate endothelial progenitor cells from umbilical cord blood. Some of the applications include using these cells as a biomarker for identifying patients with cardiovascular risk, treating ischemic diseases, and creating tissue-engineered vascular and heart valve constructs.

Isolation of Endothelial Progenitor Cells from Human Umbilical Cord Blood

Tumor endothelial cells are important determinants of the tumor microenvironment and the course of the disease. Here, a protocol for the isolation of pure and viable endothelial cells from human colorectal carcinoma and normal colon to be used in drug testing and pathogenesis research is described.

Isolation of Human Endothelial Cells from Normal Colon and Colorectal Carcinoma - An Improved Protocol

We describe an approach to detect and capture invasive cell subpopulations in real-time. The experimental design uses Real-Time Cellular Analysis by monitoring changes in the electric impedance of cells. Invasive cancer, immune, endothelial or stromal cells in complex tissues can be captured, and the impact of co-cultures can be assessed.

Real-Time Detection and Capture of Invasive Cell Subpopulations from Co-Cultures

Learn to isolate tumor-specific endothelial cells using magnetic bead enrichment and cloning ring selection from mouse tumor tissue. This gentle method enables high-purity clonal expansion for cancer vascular research.

Isolation and Culture Expansion of Tumor-specific Endothelial Cells

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