Sign In

A subscription to JoVE is required to view this content. Sign in or start your free trial.

In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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 similarities2,3. 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.......

Representative Results

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

Discussion

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,<.......

Acknowledgements

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 ....

Materials

NameCompanyCatalog NumberComments
BD FACSAria IIBD Bioscience
BoneforcepsBeijing HeLi KeChuang Technology Development CO.,Ltd. ChinaHL-YGQ  
BOON Disposable Syringe (10ml)Jiangsu Yile medical Article Co., Ltd. China
CD31-FITC antibodyBio-RadMCA1746F
Cell scraperCorning 3010#
Collagenase IVSigma-AldrichC5138#-1G
Compound ketamine injection Veterinary Pharmaceutical Factory of Shenyang, ChinaKetamine Hydrochloride:0.3g/2ml,Xylazine hydrochloride:0.3g/2ml, Phenacetin hydrochloride:1mg/2ml
DAPI (4',6-Diamidino-2-Phenylindole, Dihydrochloride)Life TechnologiesD1306#
DMEMLife Technologies11965118#
ECMSciencell1001#
ECGSSciencell1052#
Eppendorf Snap-Cap Microtube(1.5mL) AXYGENMCT-150-C#
Falcon 100mm Cell Culture DishCorning353003#
Fetal Bovine SerumGIBCO10270-106#
Flowjo v10.0
Forceps ShangHai medical instruments Co.,Ltd.China
Heparin sodiumJiangsu WanBang biopharmaceutical Co.,Ltd.China
Iodine tinctureGuilin 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)Biologixgroup66-1515#
Penicillin/StreptomycinLife Technologies15070063#
Rectangular Canted Neck Cell Culture Flask with Vent Cap (T25)Corning 3289#
ScissorsShangHai medical instruments Co.,Ltd.China
Serological Transfer Pipettes (10ml)JET BiofilGSP010010# 
Sterile Pasteur PipetteGeneBrickGY0025#
Sterile Syringe Filter (0.22µm)MilliporeSLGV033RS#
Surgical scalpelShangHai medical instruments Co.,Ltd.China22#
Surgical sutureShanghai Pudong Jinhuan Medical Supplies Co., Ltd5-0#
Syringe(5mL)Shengguang Medical Instrument Co., Ltd.China
Trypsin-EDTA (0.25%), phenol redGIBCO25200056#
75% Medical alcoholGuilin LiFeng Medical Supplies Co.,Ltd.China
20 x PBS solution (pH 7.4,Nuclease free)Sangon BiotechB540627#
Medical disinfectant 84 liquidSichuan Province Yijieshi Medical Technology Co., Ltd450ml/bottle

References

  1. Zhang, G. Y., Liao, T., Fu, X. B., Li, Q. F. Organ transplantation in China: concerns remain. Lancet. 385 (9971), 854-855 (2015).
  2. Ekser, B., et al. Clinical xenotransplantation: the next medical revolution?. Lan....

Explore More Articles

Primary Aortic Endothelial CellsMiniature PigsIsolationCultureEndotheliumXenotransplantationCollagenase IVAortaAbdominal IncisionHeparinCatheterHeartLungsCentrifugeWashing BufferDigestive SolutionIncubation

This article has been published

Video Coming Soon

JoVE Logo

Privacy

Terms of Use

Policies

Research

Education

ABOUT JoVE

Copyright © 2024 MyJoVE Corporation. All rights reserved