Isolation and Culture of Primary Aortic Endothelial Cells from Miniature Pigs

Summary

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.

Abstract

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.

Introduction

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 similarities^2,3. The failure of a pig xenograft is largely related to the primate immune rejection and coagulation responses. Porcine endo....

Protocol

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

1. Prepare the miniature pig.
   NOTE: All miniature pigs were Chinese Wuzhishan or Bama pigs (male). The age and weight of pigs were 100 days ± 8 days and 5.7 kg ± 1.0 kg (mean ± SD, n = 3), respectively.
2. Prepare the culture medium: endothelial cell medi.......

Discussion

Endothelial cells are commonly used in research of vascular dysfunction, diabetes, tissue regeneration, transplantation, and cancers^{14,15,16,17,18}. 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 reported^8,<.......

Materials

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