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 medi...

<ol>
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V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+cells:+role+in+infection+and+inflammation.">Endothelial cells: role in infection and inflammation.</a> World Journal of Surgery. 22 (2), 171-178 (1998).</li><li>Ekser, B., Cooper, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overcoming+the+barriers+to+xenotransplantation:+prospects+for+the+future.">Overcoming the barriers to xenotransplantation: prospects for the future.</a> Expert Review of Clinical Immunology. 6 (2), 219-230 (2010).</li><li>Yeom, H. 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Chapter 2, 2.1.1-2.1.12 (2001).</li></ol>

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 border="0" cellpadding="0" cellspacing="0" style="width:1163px;" width="1164">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:280px;">BD FACSAria II</td>
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			<td style="width:424px;">Beijing HeLi KeChuang Technology Development CO.,Ltd. China</td>
			<td style="width:142px;">HL-YGQ&#160;&#160;</td>
			<td></td>
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			<td height="21" style="height:21px;width:280px;">BOON Disposable Syringe (10ml)</td>
			<td style="width:424px;">Jiangsu Yile medical Article Co., Ltd. China</td>
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			<td></td>
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			<td height="21" style="height:21px;width:280px;">Cell scraper</td>
			<td style="width:424px;">Corning&#160;</td>
			<td style="width:142px;">3010#</td>
			<td></td>
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			<td height="21" style="height:21px;width:280px;">Collagenase IV</td>
			<td style="width:424px;">Sigma-Aldrich</td>
			<td style="width:142px;">C5138#-1G</td>
			<td></td>
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			<td height="64" style="height:64px;width:280px;">Compound ketamine injection&#160;</td>
			<td style="width:424px;">Veterinary Pharmaceutical Factory of Shenyang, China</td>
			<td style="width:142px;"></td>
			<td style="width:318px;">Ketamine Hydrochloride&#65306;0.3g/2ml,Xylazine hydrochloride:0.3g/2ml, Phenacetin hydrochloride&#65306;1mg/2ml</td>
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			<td style="width:424px;">Life Technologies</td>
			<td style="width:142px;">D1306#</td>
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			<td style="width:142px;">11965118#</td>
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			<td height="21" style="height:21px;width:280px;">ECM</td>
			<td style="width:424px;">Sciencell</td>
			<td style="width:142px;">1001#</td>
			<td></td>
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			<td height="21" style="height:21px;width:280px;">ECGS</td>
			<td style="width:424px;">Sciencell</td>
			<td style="width:142px;">1052#</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:280px;">Eppendorf Snap-Cap Microtube(1.5mL)&#160;</td>
			<td style="width:424px;">AXYGEN</td>
			<td style="width:142px;">MCT-150-C#</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:280px;">Falcon 100mm Cell Culture Dish</td>
			<td style="width:424px;">Corning</td>
			<td style="width:142px;">353003#</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:280px;">Fetal Bovine Serum</td>
			<td style="width:424px;">GIBCO</td>
			<td style="width:142px;">10270-106#</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:280px;">Flowjo v10.0</td>
			<td style="width:424px;"></td>
			<td style="width:142px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:280px;">Forceps&#160;</td>
			<td style="width:424px;">ShangHai medical instruments Co.,Ltd.China</td>
			<td style="width:142px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:280px;">Heparin sodium</td>
			<td style="width:424px;">Jiangsu WanBang biopharmaceutical Co.,Ltd.China</td>
			<td style="width:142px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:280px;">Iodine tincture</td>
			<td style="width:424px;">Guilin LiFeng Medical Supplies Co.,Ltd.China</td>
			<td style="width:142px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:280px;">Miniature Pig (Bama or Wuzhishan)</td>
			<td style="width:424px;">Kang Yi Ecological Agriculture Co., Ltd, China</td>
			<td style="width:142px;"></td>
			<td style="width:318px;"></td>
		</tr>
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			<td height="21" style="height:21px;width:280px;">Mshot microscope&#160;</td>
			<td style="width:424px;">Guangzhou Micro-shot Technology Co., Ltd.</td>
			<td style="width:142px;">M152</td>
			<td></td>
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			<td height="21" style="height:21px;width:280px;">Petri Dishes (150 x 15 mm)</td>
			<td style="width:424px;">Biologixgroup</td>
			<td style="width:142px;">66-1515#</td>
			<td></td>
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			<td style="width:424px;">Life Technologies</td>
			<td style="width:142px;">15070063#</td>
			<td></td>
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			<td style="width:424px;">Corning&#160;</td>
			<td style="width:142px;">3289#</td>
			<td></td>
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			<td height="21" style="height:21px;width:280px;">Scissors</td>
			<td style="width:424px;">ShangHai medical instruments Co.,Ltd.China</td>
			<td style="width:142px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:280px;">Serological Transfer Pipettes (10ml)</td>
			<td style="width:424px;">JET Biofil</td>
			<td style="width:142px;">GSP010010#&#160;</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:280px;">Sterile Pasteur Pipette</td>
			<td style="width:424px;">GeneBrick</td>
			<td style="width:142px;">GY0025#</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:280px;">Sterile Syringe Filter (0.22&#181;m)</td>
			<td style="width:424px;">Millipore</td>
			<td style="width:142px;">SLGV033RS#</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:280px;">Surgical scalpel</td>
			<td style="width:424px;">ShangHai medical instruments Co.,Ltd.China</td>
			<td style="width:142px;">22#</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:280px;">Surgical suture</td>
			<td style="width:424px;">Shanghai Pudong Jinhuan Medical Supplies Co., Ltd</td>
			<td style="width:142px;">5-0#</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:280px;">Syringe&#65288;5mL&#65289;</td>
			<td style="width:424px;">Shengguang Medical Instrument Co., Ltd.China</td>
			<td style="width:142px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:280px;">Trypsin-EDTA (0.25%), phenol red</td>
			<td style="width:424px;">GIBCO</td>
			<td style="width:142px;">25200056#</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:280px;">75% Medical alcohol</td>
			<td style="width:424px;">Guilin LiFeng Medical Supplies Co.,Ltd.China</td>
			<td style="width:142px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:280px;">20 x PBS solution (pH 7.4,Nuclease free)</td>
			<td style="width:424px;">Sangon Biotech</td>
			<td style="width:142px;">B540627#</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:280px;">Medical disinfectant 84 liquid</td>
			<td style="width:424px;">Sichuan Province Yijieshi Medical Technology Co., Ltd</td>
			<td style="width:142px;">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 ...

The method is a highly effective method for isolating endothelial cells from miniature pig. And the cells can be expanded too, investigate the immune and coagulation response in xenotransplantation. The main advantage of technique is that it is not only easy, highly effective to isolate highly purified endothelium cells from pig, but also better for keeping other pAECs when passaged.After confirming a lack of response to pain reflex in the anesthetized pig, wash the porcine abdominal wall three times with 75%medical alcohol, and one tincture of iodine. After the last wash, use a scalpel to make an abdominal incision to expose the inferior vena cava, and use a five milliliter syringe to inject 5000 units of heparin sodium into the inferior vena cava. After five minutes, insert a catheter into the abdominal aorta, and use a scalpel to incise the diaphragm.With the help of a second investigator, remove the left ventricle and use bone forceps to excise the ribs to expose the heart and lungs. Locate the aorta posterior to the heart and lungs, and clamp the ends. Use scissors to excise the aorta, and wash it one time with pre-cooled washing buffer.Remove any excess tissue around the aorta with sterile forceps and scissors, and place the vessel into a 50 milliliter centrifuge tube containing fresh pre-cooled washing buffer for transfer to the laboratory. For porcine aortic endothelial cell isolation, transfer the pig aorta into a 150 millimeter petri dish under aseptic conditions, and gently remove both ends of the vessel. Trim any additional excess tissue where the vessel had been clamped, with sterile forceps and scissors, and use 20 to 50 milliliters of fresh washing buffer to rinse the outside and inside of the aorta.Next, pass a surgical suture through the aorta, and loosely tie one end of the vessel, keeping the suture within the lumen. 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. Wash the endothelial surface of the aorta three times with 10 milliliters of fresh washing buffer per wash, and place the aorta into a 15 milliliter centrifuge tube.Cover the sample with 10 milliliters of 37 degrees Celsius warmed 0.005%collagenase IV digestive solution, and incubate the tissue at 37 degrees Celsius for 15 minutes. At the end of the incubation, transfer the entire tube contents into a 100 millimeter culture dish, and arrest the digestion with 10 to 15 milliliters of pre-cooled stopping buffer. Using a cell scraper, gently remove the aortic endothelial cells from the inside surface of the vessel, and wash the vessel three times with five milliliters of washing buffer per wash.After the last wash, transfer the supernatant into a 50 milliliter centrifuge tube, and wash the bottom of the culture dish two times with five milliliters of fresh washing buffer, per wash, pulling the washes in the 50 milliliter tube. Collect the cells by centrifugation, and discard all but the last 10 milliliters of supernatant. Add 20 milliliters of washing buffer to resuspend the pellet, taking care to avoid bubbles, and collect the cells with another centrifugation.Resuspend the pellet in one milliliter of cell culture medium for counting, and plate the cells at a one times ten to the six cells per 25 centimeters squared flask concentration. Then place the cells in the cell culture incubator at 37 degrees Celsius in 5%carbon dioxide, refreshing the medium every two to three days. After isolation, the endothelial cells are inspected on day zero, one, and two, and after passages one and two for contamination and their morphological evaluation.Flow cytometric analysis using an anti-CD31-FITC antibody indicates a typically greater than 97%endothelial cell marker positive population on day three of culture, that is maintained at an over 90%purity even after two passages. The endothelial surface of the aorta was scraped in only one direction, with a cell scraper, for less than 10 times, and it was not compressed during the process of scraping. Best answer, optimized method of pAECs isolation, we can use purified pAECs to perform individual experiments and investigate the immune rejection and coagulation response in xenotransplantation.

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

Research

JoVE Journal

Biology

8.4K visualizações.  Shenzhen Longhua District Central Hospital, Affiliated Central Hospital of Shenzhen Longhua District, Guangdong Medical University. O método é um método altamente eficaz para isolar células endoteliais de suínos em miniatura. E as células também podem ser expandidas, investigar a resposta imune e coagulação na xenotransplante. A principal vantagem da técnica é que não só é fácil, altamente eficaz isolar células de endotélio altamente purificadas do porco, mas também melhor para manter outros pAECs quando passagem.Depois de confirmar a falta de resposta ao reflexo da dor no porco anestesiado, lave ...