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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 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 (<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% ± 1.2%, 94.4% ± 1.1%, and 92.4% ± 1.7% (mean ± 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.
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 endo....
The use of animals was approved by the Ethics Review Committee of Shenzhen Second People's Hospital, in accordance with the principles of animal welfare.
1. Preparation of animals, medium, and buffers
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
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 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&D Program of China, Grant/Award Number: 2017YFC1103704; Sanming Project of Medicine in Shenzhen, Grant/Award Number: SZSM201412020; Special ....
Name | Company | Catalog Number | Comments |
BD FACSAria II | BD Bioscience | ||
Boneforceps | Beijing HeLi KeChuang Technology Development CO.,Ltd. China | HL-YGQ | |
BOON Disposable Syringe (10ml) | Jiangsu Yile medical Article Co., Ltd. China | ||
CD31-FITC antibody | Bio-Rad | MCA1746F | |
Cell scraper | Corning | 3010# | |
Collagenase IV | Sigma-Aldrich | C5138#-1G | |
Compound ketamine injection | Veterinary Pharmaceutical Factory of Shenyang, China | Ketamine Hydrochloride:0.3g/2ml,Xylazine hydrochloride:0.3g/2ml, Phenacetin hydrochloride:1mg/2ml | |
DAPI (4',6-Diamidino-2-Phenylindole, Dihydrochloride) | Life Technologies | D1306# | |
DMEM | Life Technologies | 11965118# | |
ECM | Sciencell | 1001# | |
ECGS | Sciencell | 1052# | |
Eppendorf Snap-Cap Microtube(1.5mL) | AXYGEN | MCT-150-C# | |
Falcon 100mm Cell Culture Dish | Corning | 353003# | |
Fetal Bovine Serum | GIBCO | 10270-106# | |
Flowjo v10.0 | |||
Forceps | ShangHai medical instruments Co.,Ltd.China | ||
Heparin sodium | Jiangsu WanBang biopharmaceutical Co.,Ltd.China | ||
Iodine tincture | Guilin LiFeng Medical Supplies Co.,Ltd.China | ||
Miniature Pig (Bama or Wuzhishan) | Kang Yi Ecological Agriculture Co., Ltd, China | ||
Mshot microscope | Guangzhou Micro-shot Technology Co., Ltd. | M152 | |
Petri Dishes (150 x 15 mm) | Biologixgroup | 66-1515# | |
Penicillin/Streptomycin | Life Technologies | 15070063# | |
Rectangular Canted Neck Cell Culture Flask with Vent Cap (T25) | Corning | 3289# | |
Scissors | ShangHai medical instruments Co.,Ltd.China | ||
Serological Transfer Pipettes (10ml) | JET Biofil | GSP010010# | |
Sterile Pasteur Pipette | GeneBrick | GY0025# | |
Sterile Syringe Filter (0.22µm) | Millipore | SLGV033RS# | |
Surgical scalpel | ShangHai medical instruments Co.,Ltd.China | 22# | |
Surgical suture | Shanghai Pudong Jinhuan Medical Supplies Co., Ltd | 5-0# | |
Syringe(5mL) | Shengguang Medical Instrument Co., Ltd.China | ||
Trypsin-EDTA (0.25%), phenol red | GIBCO | 25200056# | |
75% Medical alcohol | Guilin LiFeng Medical Supplies Co.,Ltd.China | ||
20 x PBS solution (pH 7.4,Nuclease free) | Sangon Biotech | B540627# | |
Medical disinfectant 84 liquid | Sichuan Province Yijieshi Medical Technology Co., Ltd | 450ml/bottle |
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