Name: isolation and culture expansion of tumor-specific endothelial cells
Uploaded: 2015-10-14T15:00:00.000+00:00
Duration: 10 min 15 s
Description: Watch this Scientific Journal Video about Isolation and Culture Expansion of Tumor-specific Endothelial Cells at JoVE.com

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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T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+cell+diversity+revealed+by+global+expression+profiling.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+signatures+of+tissue-specific+microvascular+endothelial+cell+heterogeneity+in+organ+maintenance+and+regeneration.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vessel+wall-derived+endothelial+cells+rapidly+proliferate+because+they+contain+a+complete+hierarchy+of+endothelial+progenitor+cells.">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

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...

Watch this Scientific Journal Video about Isolation and Culture Expansion of Tumor-specific Endothelial Cells at JoVE.com

Isolation and Culture Expansion of Tumor-specific Endothelial Cells

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

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Medicine

11.1K Views.  University of North Carolina at Chapel Hill. We report a reliable method to isolate and culture primary tumor-specific endothelial cells from genetically engineered mouse models. 

Video: Isolation and Culture Expansion of Tumor-specific Endothelial Cells

Ex vivo Expansion of Tumor-reactive T Cells by Means of Bryostatin 1/Ionomycin and the Common Gamma Chain Cytokines Formulation

It was reported that breast cancer patients have pre-existing immune responses against their tumors1,2. However, such immune responses fail to provide complete protection against the development or recurrence of breast cancer. To overcome this problem by increasing the frequency of tumor-reactive T cells, adoptive immunotherapy has been employed. A variety of protocols have been used for the expansion of tumor-specific T cells. These protocols, however, are restricted to the use of tumor antigens ex vivo for the activation of antigen-specific T cells. Very recently, common gamma chain cytokines such as IL-2, IL-7, IL-15, and IL-21 have been used alone or in combination for the enhancement of anti-tumor immune responses3. However, it is not clear what formulation would work best for the expansion of tumor-reactive T cells. Here we present a protocol for the selective activation and expansion of tumor-reactive T cells from the FVBN202 transgenic mouse model of HER-2/neu positive breast carcinoma for use in adoptive T cell therapy of breast cancer. The protocol includes activation of T cells with bryostatin-1/ionomycin (B/I) and IL-2 in the absence of tumor antigens for 16 hours. B/I activation mimics intracellular signals that result in T cell activation by increasing protein kinase C activity and intracellular calcium, respectively4. This protocol specifically activates tumor-specific T cells while killing irrelevant T cells. The B/I-activated T cells are cultured with IL-7 and IL-15 for 24 hours and then pulsed with IL-2. After 24 hours, T cells are washed, split, and cultured with IL-7 + IL-15 for additional 4 days. Tumor-specificity and anti-tumor efficacy of the ex vivo expanded T cells is determined.

Ex vivo Expansion of Tumor-reactive T Cells by Means of Bryostatin 1/Ionomycin and the Common Gamma Chain Cytokines Formulation

An efficient protocol for the ex vivo expansion of tumor-reactive T cells from tumor-draining lymph nodes or other secondary lymphoid tissues of tumor-bearing hosts is described. This protocol selectively expands tumor-specific T cells for use in adoptive immunotherapy of breast cancer.

It was reported that breast cancer patients have pre-existing immune responses against their tumors1,2. However, such immune responses fail to provide ...

Immunology and Infection

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

Primary cells isolated from human carcinomas are valuable tools to identify pathogenic mechanisms contributing to disease development and progression. In particular, endothelial cells (EC) constituting the inner surface of vessels, directly participate in oxygen delivery, nutrient supply, and removal of waste products to and from tumors, and are thereby prominently involved in the constitution of the tumor microenvironment (TME). Tumor endothelial cells (TECs) can be used as cellular biosensors of the intratumoral microenvironment established by communication between tumor and stromal cells. TECs also serve as targets of therapy. Accordingly, in culture these cells allow studies on mechanisms of response or resistance to anti-angiogenic treatment. Recently, it was found that TECs isolated from human colorectal carcinoma (CRC) exhibit memory-like effects based on the specific TME they were derived from. Moreover, these TECs actively contribute to the establishment of a specific TME by the secretion of different factors. For example, TECs in a prognostically favorable Th1-TME secrete the anti-angiogenic tumor-suppressive factor secreted protein, acidic and rich in cysteine-like 1 (SPARCL1). SPARCL1 regulates vessel homeostasis and inhibits tumor cell proliferation and migration. Hence, cultures of pure, viable TECs isolated from human solid tumors are a valuable tool for functional studies on the role of the vascular system in tumorigenesis. Here, a new up-to-date protocol for the isolation of primary EC from the normal colon as well as CRC is described. The technique is based on mechanical and enzymatic tissue digestion, immunolabeling, and fluorescence activated cell sorting (FACS)-sorting of triple-positive cells (CD31, VE-cadherin, CD105). With this protocol, viable TEC or normal endothelial cell (NEC) cultures could be isolated from colon tissues with a success rate of 62.12% when subjected to FACS-sorting (41 pure EC cultures from 66 tissue samples). Accordingly, this protocol provides a robust approach to isolate human EC cultures from normal colon and CRC.

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.

Primary cells isolated from human carcinomas are valuable tools to identify pathogenic mechanisms contributing to disease development and progression. In ...

Cancer Research

Isolation of Endothelial Progenitor Cells from Human Umbilical Cord Blood

The existence of endothelial progenitor cells (EPCs) in peripheral blood and its involvement in vasculogenesis was first reported by Ashara and colleagues1. Later, others documented the existence of similar types of EPCs originating from bone marrow2,3. More recently, Yoder and Ingram showed that EPCs derived from umbilical cord blood had a higher proliferative potential compared to ones isolated from adult peripheral blood4,5,6. Apart from being involved in postnatal vasculogenesis, EPCs have also shown promise as a cell source for creating tissue-engineered vascular and heart valve constructs7,8. Various isolation protocols exist, some of which involve the cell sorting of mononuclear cells (MNCs) derived from the sources mentioned earlier with the help of endothelial and hematopoietic markers, or culturing these MNCs with specialized endothelial growth medium, or a combination of these techniques9. Here, we present a protocol for the isolation and culture of EPCs using specialized endothelial medium supplemented with growth factors, without the use of immunosorting, followed by the characterization of the isolated cells using Western blotting and immunostaining.

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.

The existence of endothelial progenitor cells (EPCs) in peripheral blood and its involvement in vasculogenesis was first reported by Ashara and ...

Developmental Biology

Isolation of Primary Human Colon Tumor Cells from Surgical Tissues and Culturing Them Directly on Soft Elastic Substrates for Traction Cytometry

Cancer cells respond to matrix mechanical stiffness in a complex manner using a coordinated, hierarchical mechano-chemical system composed of adhesion receptors and associated signal transduction membrane proteins, the cytoskeletal architecture, and molecular motors1, 2. Mechanosensitivity of different cancer cells in vitro are investigated primarily with immortalized cell lines or murine derived primary cells, not with primary human cancer cells. Hence, little is known about the mechanosensitivity of primary human colon cancer cells in vitro. Here, an optimized protocol is developed that describes the isolation of primary human colon cells from healthy and cancerous surgical human tissue samples. Isolated colon cells are then successfully cultured on soft (2 kPa stiffness) and stiff (10 kPa stiffness) polyacrylamide hydrogels and rigid polystyrene (~3.6 GPa stiffness) substrates functionalized by an extracellular matrix (fibronectin in this case). Fluorescent microbeads are embedded in soft gels near the cell culture surface, and traction assay is performed to assess cellular contractile stresses using free open access software. In addition, immunofluorescence microscopy on different stiffness substrates provides useful information about primary cell morphology, cytoskeleton organization and vinculin containing focal adhesions as a function of substrate rigidity.

A protocol is described to extract primary human cells from surgical colon tumor and normal tissues. The isolated cells are then cultured on soft elastic substrates (polyacrylamide hydrogels) functionalized by an extracellular matrix protein, and embedded with fluorescent microbeads. Traction cytometry is performed to assess cellular contractile stresses.

Cancer cells respond to matrix mechanical stiffness in a complex manner using a coordinated, hierarchical mechano-chemical system composed of adhesion ...

Bioengineering

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

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult human peripheral blood within a mean of 12 days. After large scale expansion &gt;1x108 ECFCs can be obtained for further tests. Advantageously by using pHPL the contact of human cells with bovine serum antigens can be excluded. By flow cytometry and immunohistochemistry the isolated cells can be characterized as ECFC and their in vitro functionality to form vascular like structures can be tested in a matrigel assay. Further these cells can be subcutaneously injected in a mouse model to form functional, perfused vessels in vivo. After long term expansion and cryopreservation proliferation, function and genomic stability appear to be preserved. 3,4
This animal-protein free isolation and expansion method is easily applicable to generate a large quantity of ECFCs.

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.

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult ...

Biology

Isolation and Culture of Endothelial Cells from the Embryonic Forebrain

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two vascular networks of the embryonic forebrain, pial and periventricular, are spatially distinctive and have different origins and growth patterns. Endothelial cells from the pial and periventricular vascular networks have unique gene expression profiles and functions. Here we present a step-by-step protocol for isolation, culture, and verification of pure populations of endothelial cells from the periventricular vascular network (PVECs) of the embryonic forebrain (telencephalon). In this approach, telencephalon devoid of pial membrane obtained from embryonic day 15 mice is minced, digested with collagenase/dispase, and dispersed mechanically into a single cell suspension. PVECs are purified from cell suspension using positive selection with anti-CD-31/PECAM-1 antibody conjugated to MicroBeads using a strong magnetic separation method. Purified cells are cultured on collagen 1&#160;coated culture dishes in endothelial cell culture medium until they become confluent and further subcultured. PVECs obtained with this protocol exhibit cobblestone and spindle shaped phenotypes, as visualized by phase-contrast light microscopy and fluorescence microscopy. Purity of PVEC cultures was established with endothelial cell markers. In our hands, this method reliably and consistently yields pure populations of PVECs. This protocol will benefit studies aimed at gaining mechanistic insights into forebrain angiogenesis, understanding PVEC interactions, and cross-talks with neuronal cell types and holds tremendous potential for therapeutic angiogenesis.

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.

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two ...

Neuroscience

A Patient-Derived Xenograft Model for Venous Malformation

Venous malformation (VM) is a vascular anomaly that arises from impaired development of the venous network resulting in dilated and often dysfunctional veins. The purpose of this article is to carefully describe the establishment of a murine xenograft model that mimics human VM and is able to reflect patient heterogeneity. Hyper-activating non-inherited (somatic) TEK (TIE2) and PIK3CA mutations in endothelial cells (EC) have been identified as the main drivers of pathological vessel enlargement in VM. The following protocol describes the isolation, purification and expansion of patient-derived EC expressing mutant TIE2 and/or PIK3CA. These EC are injected subcutaneously into the back of immunodeficient athymic mice to generate ectatic vascular channels. Lesions generated with TIE2 or PIK3CA-mutant EC are visibly vascularized within 7&#8210;9 days of injection and recapitulate histopathological features of VM patient tissue. This VM xenograft model provides a reliable platform to investigate the cellular and molecular mechanisms driving VM formation and expansion. In addition, this model will be instrumental for translational studies testing the efficacy of novel drug candidates in preventing the abnormal vessel enlargement seen in human VM.

We present a detailed protocol to generate a murine xenograft model of venous malformation. This model is based on the subcutaneous injection of patient-derived endothelial cells containing hyper-activating TIE2 and/or PIK3CA gene mutations. Xenograft lesions closely recapitulate the histopathological features of VM patient tissue.

Venous malformation (VM) is a vascular anomaly that arises from impaired development of the venous network resulting in dilated and often dysfunctional ...

Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells (HUVECs) have shown to be convenient, easy to obtain and culture, and thus are the most widely studied endothelial cells. However, for research focused on processes like angiogenesis, permeability or many others, microvascular endothelial cells (ECs) are a much more physiologically relevant model to study 2. Furthermore, ECs isolated from knockout mice provide a useful tool for analysis of protein function ex vivo. Several approaches to isolate and culture microvascular ECs of different origin have been reported to date 3-7, but consistent isolation and culture of pure ECs is still a major technical problem in many laboratories. Here, we provide a step-by-step protocol on a reliable and relatively simple method of isolating and culturing mouse lung endothelial cells (MLECs). In this approach, lung tissue obtained from 6- to 8-day old pups is first cut into pieces, digested with collagenase/dispase (C/D) solution and dispersed mechanically into single-cell suspension. MLECS are purified from cell suspension using positive selection with anti-PECAM-1 antibody conjugated to Dynabeads using a Magnetic Particle Concentrator (MPC). Such purified cells are cultured on gelatin-coated tissue culture (TC) dishes until they become confluent. At that point, cells are further purified using Dynabeads coupled to anti-ICAM-2 antibody. MLECs obtained with this protocol exhibit a cobblestone phenotype, as visualized by phase-contrast light microscopy, and their endothelial phenotype has been confirmed using FACS analysis with anti-VE-cadherin 8 and anti-VEGFR2 9 antibodies and immunofluorescent staining of VE-cadherin. In our hands, this two-step isolation procedure consistently and reliably yields a pure population of MLECs, which can be further cultured. This method will enable researchers to take advantage of the growing number of knockout and transgenic mice to directly correlate in vivo studies with results of in vitro experiments performed on isolated MLECs and thus help to reveal molecular mechanisms of vascular phenotypes observed in vivo.

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.

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells ...

Isolation of CD133+ Liver Stem Cells for Clonal Expansion

Liver stem cell, or oval cells, proliferate during chronic liver injury, and are proposed to differentiate into both hepatocytes and cholangiocytes. In addition, liver stem cells are hypothesized to be the precursors for a subset of liver cancer, Hepatocellular carcinoma. One of the primary challenges to stem cell work in any solid organ like the liver is the isolation of a rare population of cells for detailed analysis. For example, the vast majority of cells in the liver are hepatocytes (parenchymal fraction), which are significantly larger than non-parenchymal cells. By enriching the specific cellular compartments of the liver (i.e. parenchymal and non-parenchymal fractions), and selecting for CD45 negative cells, we are able to enrich the starting population of stem cells by over 600-fold.The proceduresdetailed in this report allow for a relatively rare population of cells from a solid organ to be sorted efficiently. This process can be utilized to isolateliver stem cells from normal murine liver as well as chronic liver injury models, which demonstrate increased liver stem cell proliferation. This method has clear advantages over standard immunohistochemistry of frozen or formalin fixed liver as functional studies using live cells can be performed after initial co-localization experiments. To accomplish the procedure outlined in this report, a working relationship with a research based flow-cytometry core is strongly encouraged as the details of FACS isolation are highly dependent on specialized instrumentation and a strong working knowledge of basic flow-cytometry procedures. The specific goal of this process is to isolate a population of liver stem cells that can be clonally expanded in vitro.

Here we describe the isolation of CD133 expressing liver stem cells and cancer stem cells from whole murine liver, a process that requires tissue digestion, cell enrichment, and flow cytometry isolation. We include methods for advanced single cell isolation and clonal expansion.

Liver stem cell, or oval cells, proliferate during chronic liver injury, and are proposed to differentiate into both hepatocytes and cholangiocytes. In ...

Isolation of Immune Cells from Primary Tumors

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various chemokines and growth factors 1,2. However, once in the TME, these cells lose the ability to promote anti-tumor immunity and begin to support tumor growth and down-regulate anti-tumor immune responses 3-4. Studies on tumor-associated leukocytes have mainly focused on cells isolated from tumor-draining lymph nodes or spleen due to the inherent difficulties in obtaining sufficient cell numbers and purity from the primary tumor. While identifying the mechanisms of cell activation and trafficking through the lymphatic system of tumor bearing mice is important and may give insight to the kinetics of immune responses to cancer, in our experience, many leukocytes, including dendritic cells (DCs), in tumor-draining lymph nodes have a different phenotype than those that infiltrate tumors 5,6 . Furthermore, we have previously demonstrated that adoptively-transferred T cells isolated from the tumor-draining lymph nodes are not tolerized and are capable of responding to secondary stimulation in vitro unlike T cells isolated from the TME, which are tolerized and incapable of proliferation or cytokine production 7,8. Interestingly, we have shown that changing the tumor microenvironment, such as providing CD4+ T helper cells via adoptive transfer, promotes CD8+ T cells to maintain pro-inflammatory effector functions 5. The results from each of the previously mentioned studies demonstrate the importance of measuring cellular responses from TME-infiltrating immune cells as opposed to cells that remain in the periphery. To study the function of immune cells which infiltrate tumors using the Miltenyi Biotech isolation system9, we have modified and optimized this antibody-based isolation procedure to obtain highly enriched populations of antigen presenting cells and tumor antigen-specific cytotoxic T lymphocytes. The protocol includes a detailed dissection of murine prostate tissue from a spontaneous prostate tumor model (TRansgenic Adenocarcinoma of the Mouse Prostate -TRAMP) 10 and a subcutaneous melanoma (B16) tumor model followed by subsequent purification of various leukocyte populations.

In this report, we describe a protocol for isolating highly purified populations of leukocytes that infiltrate tumors. This protocol is adapted from the Miltenyi Biotech protocol to enhance yield and purity for isolating cells from complex tumor tissue.

Isolation and Culture Expansion of Tumor-specific Endothelial Cells

Summary

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Isolation of Immune Cells from Primary Tumors