The work was supported by grants from Natural Science Foundation of Guangdong Province, Grant/Award Number: 2016A030313028; Medical Scientific Research Foundation of Guangdong Province, Grant/Award Number: B2018003; Shenzhen Foundation of Science and Technology, Grant/Award Number: JCYJ20180306172449376, JCYJ20180306172459580, JCJY20160229204849975, GJHZ20170314171357556, JCYJ20160425103000011 and JCYJ20160428142040945; Shenzhen Longhua District Foundation of Science and Technology, Grant/Award Number: 2017013; National Key R&#38;D Program of China, Grant/Award Number: 2017YFC1103704; Sanming Project of Medicine in Shenzhen, Grant/Award Number: SZSM201412020; Special Funds for the Construction of High Level Hospitals in Guangdong Province (2019); Fund for High Level Medical Discipline Construction of Shenzhen, Grant/Award Number: 2016031638; Shenzhen Foundation of Health and Family Planning Commission, Grant/Award Number: SZXJ2017021 and SZXJ2018059. We thank Hancheng Zhang and Zhicheng Zou from Shenzhen University for assisting in the preparation of the manuscript.

The use of animals was approved by the Ethics Review Committee of Shenzhen Second People&#39;s Hospital, in accordance with the principles of animal welfare.
1. Preparation of animals, medium, and buffers
<ol>
	<li>Prepare the miniature pig. 
	NOTE: All miniature pigs were Chinese Wuzhishan or Bama pigs (male). The age and weight of pigs were 100 days &#177; 8 days and 5.7 kg &#177; 1.0 kg (mean &#177; SD, n = 3), respectively.</li>
	<li>Prepare the culture medium: endothelial cell medium (ECM) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS), 1% (v/v) penicillin/streptomycin (P/S) and 1% (v/v) endothelial cell growth supplement (ECGS).</li>
	<li>Prepare the washing buffer: 1x PBS solution (pH 7.4) with 1% (v/v) P/S.</li>
	<li>Prepare a 0.005% collagenase IV digestive solution: 1 mg of collagenase IV powder in 20 mL of PBS solution. Pre-warm the collagenase digestive solution at 37 &#176;C prior to the digestion.</li>
	<li>Prepare the stopping buffer: Dulbecco&#8217;s modified Eagle&#8217;s medium (DMEM) supplemented with 10% heat-inactivated FBS and 1% P/S.</li>
</ol>
2. Surgery and preparation for porcine aortic endothelial cell isolation
<ol>
	<li>Disinfecting the operating room with UV light for 1 h and medical disinfectant 84 liquid (available chlorine content is 4.0% - 5.0%, Table of Materials) before performing surgery.</li>
	<li>After the miniature pig received an intramuscular injection formed by 15 mg/kg Ketamine, 15 mg/kg Xylazine hydrochloride and 0.05 mg/kg Phenacetin hydrochloride( Volumes: 100&#956;L/kg, Table of Materials) (Figure 1A), confirm the animal&#8217;s anesthesia status with checking porcine painful stimulus (eg. pinching one ear or shaking one forelimb) and physiological index (eg. blood pressure, heart and respiratory rate). 
	NOTE: Another half dose injection could be given to the pig if it shows the signs of waking up.&#160;</li>
	<li>Cut the abdomen with a scalpel, expose the inferior vena cava and consequently inject heparin sodium (5,000 U, Table of Materials) into the inferior vena cava with 5 mL syringe.</li>
	<li>Five minutes later, insert a catheter to the abdominal aorta to blood, cut the skin of chest and incise the diaphragm with a scalpel and subsequently cut the left ventricle off. 
	NOTE: Make sure that pig is euthanized by checking aortic pulse after blooding and cutting the heart.&#160;</li>
	<li>Excise the ribs with bone forceps (Table of Materials), and expose the heart and lungs. Find the aorta posterior to the heart and lungs and clamp the two ends. Wash the aorta once with pre-cooled washing buffer (Figure 1B,C).</li>
	<li>Cut out the aorta with scissors and keep the aorta clamped at each end. Excise excess tissue around the aorta with sterile forceps and scissors. Put the aorta into a 50 mL centrifuge tube, wash the aorta with pre-cooled washing buffer 3x (30 mL per wash), and transfer the aorta to the laboratory (Figure 1D).</li>
</ol>
3. Isolation of porcine aortic endothelial cells
<ol>
	<li>Under aseptic conditions, remove the pig aorta from the washing buffer, and place it on a 150 mm Petri dish (Figure 2A).</li>
	<li>Gently cut off the two ends of the aorta and excise excess tissue around aorta with sterile forceps and scissors again (Figure 2B). Wash the outside and inside of the aorta (over the culture dish at room temperature) with 20&#8722;50 mL of washing buffer. 
	NOTE: Try to only cut the area where the clamps were placed during surgery since some ECs were damaged in this area, and remove excess tissues and arterial side branches.</li>
	<li>Pass a surgical suture through the aorta and then tie one end of the aorta using the surgical suture (5-0, 90cm, Table of Materials). Keep the surgical suture in the inside of the aorta (Figure 2C,D).</li>
	<li>Gently fix the aorta near the tied end with forceps, and then pull the surgical suture slowly to reverse the aorta until the endothelial surface of the aorta is exposed (Figure 2E-G). 
	NOTE: Ensure to fix the aorta near the tied end and do not fix the aorta tightly, or else the aorta cannot be reversed by pulling the surgical suture.</li>
	<li>Wash the endothelial surface of the aorta with washing buffer 3x (10 mL per wash), and then discard this solution. Place the aorta into a 15 mL centrifuge tube, and cover the aorta with 10 mL of 0.005% collagenase IV digestive solution in the tube (Figure 2H). 
	NOTE: Pre-warm the 0.005% collagenase IV digestive solution at 37 &#176;C before digestion.</li>
	<li>Incubate at 37 &#176;C for 15 min. Place the digested aorta and digestive solution into a 100 mm culture dish and stop the effects of 0.005% collagenase IV by adding 10&#8722;15 mL of pre-cooled stopping buffer. 
	NOTE: The recommended digestion time is between 10 and 20 minutes. Make sure the endothelial surface of the aorta is covered by the stopping buffer.</li>
	<li>Gently scrape pAECs off the inside surface of the aorta using a cell scraper (Figure 2I). Wash the scraped aorta 3x with washing buffer (5 mL per time). Put the solution from the culture dish into a 50 mL centrifuge tube. Wash the bottom of the culture dish 2x with washing buffer (5 mL per time), and put the solution into a 50 mL centrifuge tube again. 
	NOTE: Scrape in one direction gently and do not compress. Do not touch the tissue near the edges and holes. Scraping 5&#8722;8x is recommended.</li>
	<li>Centrifuge the tube at 600 x g for 6 min at 4 &#176;C. Discard the supernatant and leave 10 mL of solution at the bottom of a 50 mL centrifuge tube. Add 20 mL of washing buffer to the 50 mL centrifuge tube, and resuspend the pellets. Avoid bubbles during this step.</li>
	<li>Centrifuge at 600 x g for 6 min at 4 &#176;C. Slowly discard the supernatant. Resuspend the cell pellets with 1 mL of culture medium and mix well. 
	NOTE: The obtained average number of ECs per cm aorta is 1.9 x 105 &#177; 1.4 x 104 (mean &#177; SD, n = 3).</li>
	<li>Count the cells with a cell counter. Plate and culture the cells in a vessel without coating any materials according to the cell number. If the cell number is less than or equal to 1.0 x 106, culture cells in a 25 cm2 flask. If the cell number is larger than 1.0 x 106, culture cells in several 25 cm2 flasks (1.0 x 106 cells per flask). Place the flask in an incubator (without shaking) at 37 &#176;C with 5% CO2 and replace the medium every 2&#8722;3 days. 
	NOTE: Some cells are damaged by the cell scraper. A cell viability assay found the percentage of live cells to be 95.7% &#177; 1.7% (mean &#177; SD, n = 3). The doubling time of the isolated cells is about 1&#8722;2 days.</li>
</ol>
4. Harvest and characterization of pure endothelial cells
<ol>
	<li>Leave the cells grow for about 4&#8722;5 days. When the cells reach 80% confluence (Figure 3A), digest the cells with 0.25% trypsin and passage the cells. Then, collect some of the isolated cells (Day 3, Passage 1 and Passage 2) and identify by flow cytometry (with an anti-CD31 antibody).</li>
	<li>Label isolated pAECs (cell number: 1 x 105) (Day 3, Passage 1 and Passage 2) with anti-porcine CD31-fluorescein isothiocyanate (FITC) conjugated antibody (10 &#181;L antibody for per 100 &#181;L cells, stained for 30 min at 4 &#176;C) for flow cytometry analysis.</li>
	<li>Gate total live cell population with a forward scattered plot, unstained sample as a negative control. Then present the CD31 positive cells of the gated cells with histogram. 
	NOTE: The efficacy of CD31-positive cells is 97.4% &#177; 1.2% (Day 3), 94.4% &#177; 1.1% (P1) and 92.4% &#177; 1.7% (P2) (mean &#177; SD), respectively (Figure 3B).</li>
</ol>

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The authors have nothing to disclose.

Endothelial cells are commonly used in research of vascular dysfunction, diabetes, tissue regeneration, transplantation, and cancers14,15,16,17,18. To understand and characterize the biology and function of ECs in these diseases, numerous methods to isolate the ECs of different diseased organs or tissues have been reported8,<...

The shortage of available organs for transplantation is an outstanding problem world-wide1. According to the Red Cross Society of China, only a small number of patients with end-stage organ failure received a suitable organ in China in 2018.
Xenotransplantation is a promising way to resolve the problem of organ shortage. Pig organs are considered to be the most suitable organs for humans because of anatomic and physiologic similarities2,3. The failure of a pig xenograft is largely related to the primate immune rejection and coagulation responses. Porcine endothelial cells (ECs) are critical since these cells are the first to interact with the primate immune system, which includes antibody, complement, cytokines, and immune cells (e.g., T cells, B cells, and macrophages)4,5. Porcine ECs play a vital role in pig organ and islet xenotransplantation6,7. Thus, ECs are important cells for investigating the rejection and coagulation responses to a pig graft. Isolation of high-quality porcine ECs is required for xenotransplantation research.
In attempts to isolate ECs from different organs (e.g., heart, kidney, liver and aorta), several protocols have been reported in a xenotransplant setting8,9,10,11,12. However, keeping an ultrapure culture of isolated ECs is an outstanding problem in standard protocols. The increased concentration of the digested solution, inappropriate digestion time and scrape intensity may contribute to the increased fibroblast contamination in current studies8,10,13. Besides, the method of isolated pAECs from miniature pigs is less studied. Here, we describe an optimized enzymatic method to isolate highly-purified pAECs from miniature pigs (Wuzhishan or Bama). Several steps in the protocol have been designed to reduce fibroblast contamination and obtain high-purity ECs.

<table><tbody><tr><td>BD FACSAria II</td><td>BD Bioscience</td><td></td><td></td></tr><tr><td>Boneforceps</td><td>Beijing HeLi KeChuang Technology Development CO.,Ltd. China</td><td>HL-YGQ&amp;nbsp;&amp;nbsp;</td><td></td></tr><tr><td>BOON Disposable Syringe (10ml)</td><td>Jiangsu Yile medical Article Co., Ltd. China</td><td></td><td></td></tr><tr><td>CD31-FITC antibody</td><td>Bio-Rad</td><td>MCA1746F</td><td></td></tr><tr><td>Cell scraper</td><td>Corning&amp;nbsp;</td><td>3010#</td><td></td></tr><tr><td>Collagenase IV</td><td>Sigma-Aldrich</td><td>C5138#-1G</td><td></td></tr><tr><td>Compound ketamine injection&amp;nbsp;</td><td>Veterinary Pharmaceutical Factory of Shenyang, China</td><td></td><td>Ketamine Hydrochloride?0.3g/2ml,Xylazine hydrochloride:0.3g/2ml, Phenacetin hydrochloride?1mg/2ml</td></tr><tr><td>DAPI (4&#039;,6-Diamidino-2-Phenylindole, Dihydrochloride)</td><td>Life Technologies</td><td>D1306#</td><td></td></tr><tr><td>DMEM</td><td>Life Technologies</td><td>11965118#</td><td></td></tr><tr><td>ECM</td><td>Sciencell</td><td>1001#</td><td></td></tr><tr><td>ECGS</td><td>Sciencell</td><td>1052#</td><td></td></tr><tr><td>Eppendorf Snap-Cap Microtube(1.5mL)&amp;nbsp;</td><td>AXYGEN</td><td>MCT-150-C#</td><td></td></tr><tr><td>Falcon 100mm Cell Culture Dish</td><td>Corning</td><td>353003#</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>GIBCO</td><td>10270-106#</td><td></td></tr><tr><td>Flowjo v10.0</td><td></td><td></td><td></td></tr><tr><td>Forceps&amp;nbsp;</td><td>ShangHai medical instruments Co.,Ltd.China</td><td></td><td></td></tr><tr><td>Heparin sodium</td><td>Jiangsu WanBang biopharmaceutical Co.,Ltd.China</td><td></td><td></td></tr><tr><td>Iodine tincture</td><td>Guilin LiFeng Medical Supplies Co.,Ltd.China</td><td></td><td></td></tr><tr><td>Miniature Pig (Bama or Wuzhishan)</td><td>Kang Yi Ecological Agriculture Co., Ltd, China</td><td></td><td></td></tr><tr><td>Mshot microscope&amp;nbsp;</td><td>Guangzhou Micro-shot Technology Co., Ltd.</td><td>M152</td><td></td></tr><tr><td>Petri Dishes (150 x 15 mm)</td><td>Biologixgroup</td><td>66-1515#</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>Life Technologies</td><td>15070063#</td><td></td></tr><tr><td>Rectangular Canted Neck Cell Culture Flask with Vent Cap ?T25?</td><td>Corning&amp;nbsp;</td><td>3289#</td><td></td></tr><tr><td>Scissors</td><td>ShangHai medical instruments Co.,Ltd.China</td><td></td><td></td></tr><tr><td>Serological Transfer Pipettes (10ml)</td><td>JET Biofil</td><td>GSP010010#&amp;nbsp;</td><td></td></tr><tr><td>Sterile Pasteur Pipette</td><td>GeneBrick</td><td>GY0025#</td><td></td></tr><tr><td>Sterile Syringe Filter (0.22&amp;micro;m)</td><td>Millipore</td><td>SLGV033RS#</td><td></td></tr><tr><td>Surgical scalpel</td><td>ShangHai medical instruments Co.,Ltd.China</td><td>22#</td><td></td></tr><tr><td>Surgical suture</td><td>Shanghai Pudong Jinhuan Medical Supplies Co., Ltd</td><td>5-0#</td><td></td></tr><tr><td>Syringe?5mL?</td><td>Shengguang Medical Instrument Co., Ltd.China</td><td></td><td></td></tr><tr><td>Trypsin-EDTA (0.25%), phenol red</td><td>GIBCO</td><td>25200056#</td><td></td></tr><tr><td>75% Medical alcohol</td><td>Guilin LiFeng Medical Supplies Co.,Ltd.China</td><td></td><td></td></tr><tr><td>20 x PBS solution (pH 7.4,Nuclease free)</td><td>Sangon Biotech</td><td>B540627#</td><td></td></tr><tr><td>Medical disinfectant 84 liquid</td><td>Sichuan Province Yijieshi Medical Technology Co., Ltd</td><td>450ml/bottle</td><td></td></tr></tbody></table>

isolation and culture of primary aortic endothelial cells from miniature pigs

Xenotransplantation is a promising way to resolve the shortage of human organs for patients with end-stage organ failure, and the pig is considered as a suitable organ source. Immune rejection and coagulation are two major hurdles for the success of xenotransplantation. Vascular endothelial cell (EC) injury and dysfunction are important for the development of the inflammation and coagulation responses in xenotransplantation. Thus, isolation of porcine aortic endothelial cells (pAECs) is necessary for investigating the immune rejection and coagulation responses. Here, we have developed a simple enzymatic approach for the isolation, characterization, and expansion of highly purified pAECs from miniature pigs. First, the miniature pig was anaesthetized with ketamine, and a length of aorta was excised. Second, the endothelial surface of aorta was exposed to 0.005% collagenase IV digestive solution for 15 min. Third, the endothelial surface of the aorta was scraped in only one direction with a cell scraper (&#60;10 times), and was not compressed during the process of scraping. Finally, the isolated pAECs of Day 3, and after passage 1 and passage 2, were identified by flow cytometry with an anti-CD31 antibody. The percentages of CD31-positive cells were 97.4% &#177; 1.2%, 94.4% &#177; 1.1%, and 92.4% &#177; 1.7% (mean &#177; SD), respectively. The concentration of Collagenase IV, the digestive time, the direction, and frequency and intensity of scraping are critical for decreasing fibroblast contamination and obtaining high-purity and a large number of ECs. In conclusion, our enzymatic method is a highly-effctive method for isolating ECs from the miniature pig aorta, and the cells can be expanded in vitro to investigate the immune and coagulation responses in xenotransplantation.

Our method aims to provide an effective way to isolate highly-purified ECs from the aortas from miniature pigs. The process of aorta surgery is shown in Figure 1. The first step is that the whole aorta is excised from the pig. Preventing other cell or bacterial contamination is very important. Thus, do not injure other organs or tissues in case untargeted cells or bacteria contaminate the aorta, and wash the aorta with pre-cooled washing buffer 3x (Figure 1<stro...

Watch this Scientific Journal Video about Isolation and Culture of Primary Aortic Endothelial Cells from Miniature Pigs at JoVE.com

Isolation and Culture of Primary Aortic Endothelial Cells from Miniature Pigs

An effective enzymatic method for isolation of primary porcine aortic endothelial cells (pAECs) from miniature pigs is described. The isolated primary pAECs can be used to investigate the immune and coagulation response in xenotransplantation.

Xenotransplantation is a promising way to resolve the shortage of human organs for patients with end-stage organ failure, and the pig is considered as a ...

isolation-culture-primary-aortic-endothelial-cells-from-miniature

Research

JoVE Journal

Biology

8.5K Views.  Shenzhen Longhua District Central Hospital, Affiliated Central Hospital of Shenzhen Longhua District, Guangdong Medical University. De methode is een zeer effectieve methode voor het isoleren van endotheelcellen van miniatuurvarken. En de cellen kunnen ook worden uitgebreid, onderzoeken de immuun- en stollingsrespons bij xenotransplantatie. Het belangrijkste voordeel van techniek is dat het niet alleen gemakkelijk, zeer effectief is om zeer gezuiverde endotheelcellen te isoleren van varkens, maar ook beter om andere pAC's te houden wanneer deze worden doorgegangerd.Na bevestiging van een gebrek aan reactie op pijnr...

Video: Isolation and Culture of Primary Aortic Endothelial Cells from Miniature Pigs

Autologous Endothelial Progenitor Cell-Seeding Technology and Biocompatibility Testing For Cardiovascular Devices in Large Animal Model

Implantable cardiovascular devices are manufactured from artificial materials (e.g. titanium (Ti), expanded polytetrafluoroethylene), which pose the risk of thromboemboli formation1,2,3. We have developed a method to line the inside surface of Ti tubes with autologous blood-derived human or porcine endothelial progenitor cells (EPCs)4. By implanting Ti tubes containing a confluent layer of porcine EPCs in the inferior vena cava (IVC) of pigs, we tested the improved biocompatibility of the cell-seeded surface in the prothrombotic environment of a large animal model and compared it to unmodified bare metal surfaces5,6,7 (Figure 1). This method can be used to endothelialize devices within minutes of implantation and test their antithrombotic function in vivo. 
Peripheral blood was obtained from 50 kg Yorkshire swine and its mononuclear cell fraction cultured to isolate EPCs4,8. Ti tubes (9.4 mm ID) were pre-cut into three 4.5 cm longitudinal sections and reassembled with heat-shrink tubing. A seeding device was built, which allows for slow rotation of the Ti tubes. 
We performed a laparotomy on the pigs and externalized the intestine and urinary bladder. Sharp and blunt dissection was used to skeletonize the IVC from its bifurcation distal to the right renal artery proximal. The Ti tubes were then filled with fluorescently-labeled autologous EPC suspension and rotated at 10 RPH x 30 min to achieve uniform cell-coating9. After administration of 100 USP/ kg heparin, both ends of the IVC and a lumbar vein were clamped. A 4 cm veinotomy was performed and the device inserted and filled with phosphate-buffered saline. As the veinotomy was closed with a 4-0 Prolene running suture, one clamp was removed to de-air the IVC. At the end of the procedure, the fascia was approximated with 0-PDS (polydioxanone suture), the subcutaneous space closed with 2-0 Vicryl and the skin stapled closed. 
After 3 - 21 days, pigs were euthanized, the device explanted en-block and fixed. The Ti tubes were disassembled and the inner surfaces imaged with a fluorescent microscope.
We found that the bare metal Ti tubes fully occluded whereas the EPC-seeded tubes remained patent. Further, we were able to demonstrate a confluent layer of EPCs on the inside blood-contacting surface.
Concluding, our technology can be used to endothelialize Ti tubes within minutes of implantation with autologous EPCs to prevent thrombosis of the device. Our surgical method allows for testing the improved biocompatibility of such modified devices with minimal blood loss and EPC-seeded surface disruption.

A method for seeding titanium blood-contacting biomaterials with autologous cells and testing biocompatibility is described. This method uses endothelial progenitor cells and titanium tubes, seeded within minutes of surgical implantation into porcine venae cavae. This technique is adaptable to many other implantable biomedical devices.

Implantable cardiovascular devices are manufactured from artificial materials (e.g. titanium (Ti), expanded polytetrafluoroethylene), which pose the risk ...

Bioengineering

Isolation of Valvular Endothelial Cells

Heart valves are solely responsible for maintaining unidirectional blood flow through the cardiovascular system. These thin, fibrous tissues are subjected to significant mechanical stresses as they open and close several billion times over a lifespan. The incredible endurance of these tissues is due to the resident valvular endothelial (VEC) and interstitial cells (VIC) that constantly repair and remodel in response to local mechanical and biological signals. Only recently have we begun to understand the unique behaviors of these cells, for which in vitro experimentation has played a key role. Particularly challenging is the isolation and culture of VEC. Special care must be used from the moment the tissue is removed from the host through final plating. Here we present protocols for direct isolation, side specific isolation, culture, and verification of pure populations of VEC. We use enzymatic digestion followed by a gentle swab scraping technique to dislodge only surface cells. These cells are then collected into a tube and centrifuged into a pellet. The pellet is then resuspended and plated into culture flasks pre-coated with collagen I matrix. VEC phenotype is confirmed by contact inhibited growth and the expression of endothelial specific markers such as PECAM1 (CD31), Von Willebrand Factor (vWF), and negative expression of alpha-smooth muscle actin (&alpha;-SMA). The functional characteristics of VEC are associated with high levels of acetylated LDL. Unlike vascular endothelial cells, VEC have the unique capacity to transform into mesenchyme, which normally occurs during embryonic valve formation1. This can also occur during significantly prolonged post confluent in vitro culture, so care should be made to passage at or near confluence. After VEC isolation, pure populations of VIC can then be easily acquired.

We provide a method for isolating and culturing pure populations of heart valve endothelial cells (VEC). VEC can be isolated from either side of the cusp or leaflet and immediately following, underlying interstitial cell (VIC) isolation is straightforward.

Heart valves are solely responsible for maintaining unidirectional blood flow through the cardiovascular system.  These thin, fibrous tissues are ...

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 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.

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 ...

Medicine

Assessment of the Anticoagulant and Anti-inflammatory Properties of Endothelial Cells Using 3D Cell Culture and Non-anticoagulated Whole Blood

In vivo, endothelial cells are crucial for the natural anticoagulation of circulating blood. Consequently, endothelial cell activation leads to blood coagulation. This phenomenon is observed in many clinical situations, like organ transplantation in the presence of pre-formed anti-donor antibodies, including xenotransplantation, as well as in ischemia/reperfusion injury. In order to reduce animal experimentation according to the 3R standards (reduction, replacement and refinement), in vitro models to study the effect of endothelial cell activation on blood coagulation would be highly desirable. However, common flatbed systems of endothelial cell culture provide a surface-to-volume ratio of 1 - 5 cm2 of endothelium per mL of blood, which is not sufficient for natural, endothelial-mediated anticoagulation. Culturing endothelial cells on microcarrier beads may increase the surface-to-volume ratio to 40 - 160 cm2/mL. This increased ratio is sufficient to ensure the &#34;natural&#34; anticoagulation of whole blood, so that the use of anticoagulants can be avoided. Here an in vitro microcarrier-based system is described to study the effects of genetic modification of porcine endothelial cells on coagulation of whole, non-anticoagulated human blood. In the described assay, primary porcine aortic endothelial cells, either wild type (WT) or transgenic for human CD46 and thrombomodulin, were grown on microcarrier beads and then exposed to freshly drawn non-anticoagulated human blood. This model allows for the measurement and quantification of cytokine release as well as activation markers of complement and coagulation in the blood plasma. In addition, imaging of activated endothelial cell and deposition of immunoglobulins, complement- and coagulation proteins on the endothelialized beads were performed by confocal microscopy. This assay can also be used to test drugs which are supposed to prevent endothelial cell activation and, thus, coagulation. On top of its potential to reduce the number of animals used for such investigations, the described assay is easy to perform and consistently reproducible.

We present an in vitro model which allows the study and analysis of coagulation in whole, non-anticoagulated blood. Anticoagulation in the system depends on the natural anticoagulation effect of healthy endothelial cells and endothelial cell activation will result in clotting.

In vivo, endothelial cells are crucial for the natural anticoagulation of circulating blood. Consequently, endothelial cell activation leads to blood ...

Isolation of Primary Mouse Lung Endothelial Cells

Endothelial cells play critical roles in the regulation of vascular tone, immunity, coagulation, and permeability. Endothelial dysfunction occurs in medical conditions including diabetes, atherosclerosis, sepsis, and acute lung injury. A reliable and reproducible method to isolate pure endothelial cells from mice is needed to investigate the role of endothelial cells in the pathogenesis of these and other conditions. In this protocol, lung microvascular endothelial cells were prepared from 5-7 day old neonatal mouse pups. Lungs are harvested, minced, and enzymatically digested with collagenase I, and released cells are cultured overnight. Endothelial cells are then selected using anti-PECAM1 (CD-31) IgG conjugated to magnetic beads, and cells again are cultured to confluence. A secondary cell selection then occurs with anti-ICAM2 (CD-102) IgG conjugated to magnetic beads to increase the purity of the endothelial cells, and the cells again are cultured to confluence. The entire process takes approximately 7-10 days before the cells can be used for experimentation. This simple protocol yields highly pure (purity &#62;92%) endothelial cells that can be immediately used for in vitro studies, including the studies focused on endothelial cytokine and chemokine production, leukocyte-endothelial interactions, endothelial coagulation pathways, and endothelial permeability. With many knockouts and transgenic mouse lines available, this procedure also lends itself to understanding the function of specific genes expressed by endothelial cells in healthy and pathologic responses to injury, infection, and inflammation.

In this article, primary lung endothelial cells were isolated and cultured from neonatal mice.

Endothelial cells play critical roles in the regulation of vascular tone, immunity, coagulation, and permeability. Endothelial dysfunction occurs in ...

Immunology and Infection

Isolation of Murine Valve Endothelial Cells

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and surrounded by a monolayer of valve endothelial cells (VECs). VECs play essential roles in establishing the valve structures during embryonic development, and are important for maintaining life-long valve integrity and function. In contrast to a continuous endothelium over the surface of healthy valve leaflets, VEC disruption is commonly observed in malfunctioning valves and is associated with pathological processes that promote valve disease and dysfunction. Despite the clinical relevance, focused studies determining the contribution of VECs to development and disease processes are limited. The isolation of VECs from animal models would allow for cell-specific experimentation. VECs have been isolated from large animal adult models but due to their small population size, fragileness, and lack of specific markers, no reports of VEC isolations in embryos or adult small animal models have been reported. Here we describe a novel method that allows for the direct isolation of VECs from mice at embryonic and adult stages. Utilizing the Tie2-GFP reporter model that labels all endothelial cells with Green Fluorescent Protein (GFP), we have been successful in isolating GFP-positive (and negative) cells from the semilunar and atrioventricular valve regions using fluorescence activated cell sorting (FACS). Isolated GFP-positive VECs are enriched for endothelial markers, including CD31 and von Willebrand Factor (vWF), and retain endothelial cell expression when cultured; while, GFP-negative cells exhibit molecular profiles and cell shapes consistent with VIC phenotypes. The ability to isolate embryonic and adult murine VECs allows for previously unattainable molecular and functional studies to be carried out on a specific valve cell population, which will greatly improve our understanding of valve development and disease mechanisms.

The ability to isolate heart valve endothelial cells (VECs) is critical for understanding mechanisms of valve development, maintenance, and disease. Here we describe the isolation of VECs from embryonic and adult Tie2-GFP mice using FACS that will allow for studies determining the contribution of VECs in developmental and disease processes.

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and ...

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an important model for cardiovascular disease research. This study demonstrates a simple method to isolate and culture endothelial cells from the mouse aorta without any special equipment. To isolate endothelial cells, the thoracic aorta is quickly removed from the mouse body, and the attached adipose tissue and connective tissue are removed from the aorta. The aorta is cut into 1 mm rings. Each aortic ring is opened and seeded onto a growth factor reduced matrix with the endothelium facing down. The segments are cultured in endothelial cell growth medium for about 4 days. The endothelial sprouting starts as early as day 2. The segments are then removed and the cells are cultured continually until they reach confluence. The endothelial cells are harvested using neutral proteinase and cultured in endothelial cell growth medium for another two passages before being used for experiments. Immunofluorescence staining indicated that after the second passage the majority of cells were double positive for Dil-ac-LDL uptake, Lectin binding, and CD31 staining, the typical characteristics of endothelial cells. It is suggested that cells at the second to third passages are suitable for in vitro and in vivo experiments to study the endothelial biology. Our protocol provides an effective means of identifying specific cellular and molecular mechanisms in endothelial cell physiopathology.

The vascular endothelial cells play a significant role in many important cardiovascular disorders. This article describes a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Our protocol provides an effective means of identifying mechanisms in endothelial cell physiopathology.

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an ...

Isolation of Human Primary Valve Cells for In vitro Disease Modeling

Calcific aortic valve disease (CAVD) is present in nearly a third of the elderly population. Thickening, stiffening, and calcification of the aortic valve causes aortic stenosis and contributes to heart failure and stroke. Disease pathogenesis is multifactorial, and stresses such as inflammation, extracellular matrix remodeling, turbulent flow, and mechanical stress and strain contribute to the osteogenic differentiation of valve endothelial and valve interstitial cells. However, the precise initiating factors that drive the osteogenic transition of a healthy cell into a calcifying cell are not fully defined. Further, the only current therapy for CAVD-induced aortic stenosis is aortic valve replacement, whereby the native valve is removed (surgical aortic valve replacement, SAVR) or a fully collapsible replacement valve is inserted via a catheter (transcatheter aortic valve replacement, TAVR). These surgical procedures come at a high cost and with serious risks; thus, identifying novel therapeutic targets for drug discovery is imperative. To that end, the present study develops a workflow where surgically removed tissues from patients and donor cadaver tissues are used to create patient-specific primary lines of valvular cells for in vitro disease modeling. This protocol introduces the utilization of a cold storage solution, commonly utilized in organ transplant, to reduce the damage caused by the often-lengthy procurement time between tissue excision and laboratory processing with the benefit of greatly stabilizing cells of the excised tissue. The results of the present study demonstrate that isolated valve cells retain their proliferative capacity and endothelial and interstitial phenotypes in culture upwards of several days after valve removal from the donor. Using these materials allows for the collection of control and CAVD cells, from which both control and disease cell lines are established.

This protocol describes the collection of human aortic valves extracted during surgical aortic valve replacement procedures or from cadaveric tissue, and the subsequent isolation, expansion, and characterization of patient specific primary valve endothelial and interstitial cells. Included are important details regarding the processes needed to ensure cell viability and phenotype specificity.

Calcific aortic valve disease (CAVD) is present in nearly a third of the elderly population. Thickening, stiffening, and calcification of the aortic valve ...

Porcine Aorta Isolation: A Surgical Procedure to Excise the Thoracic Aorta From Miniature Pigs for Downstream Analysis

Source: Zhao, Y., et al. <a href="https://www.jove.com/v/59673">Isolation and Culture of Primary Aortic Endothelial Cells from Miniature Pigs</a> J. Vis. Exp. (2019)
In this video, we demonstrate a procedure for the surgical removal of the aorta from miniature pigs. Endothelial cells harvested from these isolated aortae can be further cultured in in vitro conditions to study coagulation and immune responses.

Source: Zhao, Y., et al. Isolation and Culture of Primary Aortic Endothelial Cells from Miniature Pigs J. Vis. Exp. (2019)
In this video, we demonstrate a ...

Isolation and Culture of Primary Aortic Endothelial Cells from Miniature Pigs

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