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

Method Article

Isolation and Culture of Primary Aortic Endothelial Cells from Miniature Pigs

8.5K views

⸱

DOI:

10.3791/59673

⸱

August 14th, 2019

 ,  ,  ,  ,  ,  ,  ,  ,  ,  , 
1Department of Nephrology, Shenzhen Longhua District Central Hospital, Affiliated Central Hospital of Shenzhen Longhua District, Guangdong Medical University, 2Shenzhen Xenotransplantation Medical Engineering Research and Development Center, Shenzhen Second People's Hospital, First Affiliated Hospital of Shenzhen University, Shenzhen University School of Medicine, 3Department of Medical Laboratory, Shenzhen Longhua District Central Hospital, Affiliated Central Hospital of Shenzhen Longhua District, Guangdong Medical University, 4Department of Endocrinology, Shenzhen Longhua District Central Hospital, Affiliated Central Hospital of Shenzhen Longhua District, Guangdong Medical University, 5Xenotransplantation Program, Department of Surgery, University of Alabama at Birmingham, 6People's Hospital of Longhua

In This Article

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 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.

Access restricted. Please log in or start a trial to view this content.

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 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).
  3. Prepare the washing buffer: 1x PBS solution (pH 7.4) with 1% (v/v) P/S.
  4. 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 °C prior to the digestion.
  5. Prepare the stopping buffer: Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated FBS and 1% P/S.

2. Surgery and preparation for porcine aortic endothelial cell isolation

  1. 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.
  2. 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: 100μL/kg, Table of Materials) (Figure 1A), confirm the animal’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. 
  3. 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.
  4. 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. 
  5. 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).
  6. 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).

3. Isolation of porcine aortic endothelial cells

  1. Under aseptic conditions, remove the pig aorta from the washing buffer, and place it on a 150 mm Petri dish (Figure 2A).
  2. 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 20−50 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.
  3. 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).
  4. 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.
  5. 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 °C before digestion.
  6. Incubate at 37 °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 10−15 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.
  7. 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 5−8x is recommended.
  8. Centrifuge the tube at 600 x g for 6 min at 4 °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.
  9. Centrifuge at 600 x g for 6 min at 4 °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 ± 1.4 x 104 (mean ± SD, n = 3).
  10. 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 °C with 5% CO2 and replace the medium every 2−3 days.
    NOTE: Some cells are damaged by the cell scraper. A cell viability assay found the percentage of live cells to be 95.7% ± 1.7% (mean ± SD, n = 3). The doubling time of the isolated cells is about 1−2 days.

4. Harvest and characterization of pure endothelial cells

  1. Leave the cells grow for about 4−5 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).
  2. 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 µL antibody for per 100 µL cells, stained for 30 min at 4 °C) for flow cytometry analysis.
  3. 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% ± 1.2% (Day 3), 94.4% ± 1.1% (P1) and 92.4% ± 1.7% (P2) (mean ± SD), respectively (Figure 3B).

Access restricted. Please log in or start a trial to view this content.

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

Access restricted. Please log in or start a trial to view this content.

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,<...

Access restricted. Please log in or start a trial to view this content.

Disclosures

The authors have nothing to disclose.

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 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.

Access restricted. Please log in or start a trial to view this content.

Materials

List of materials used in this article
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? Lancet. 379 (9816), 672-683 (2012).
  3. Cooper, D. K., Ekser, B., Ramsoondar, J., Phelps, C., Ayares, D. The role of genetically engineered pigs in xenotransplantation research. The Journal of Pathology. 238 (2), 288-299 (2016).
  4. Pober, J. S., Sessa, W. C. Evolving functions of endothelial cells in inflammation. Nature Reviews Immunology. 7 (10), 803-815 (2007).
  5. McGill, S. N., Ahmed, N. A., Christou, N. V. Endothelial cells: role in infection and inflammation. World Journal of Surgery. 22 (2), 171-178 (1998).
  6. Ekser, B., Cooper, D. K. Overcoming the barriers to xenotransplantation: prospects for the future. Expert Review of Clinical Immunology. 6 (2), 219-230 (2010).
  7. Yeom, H. J., et al. Porcine aortic endothelial cell genes responsive to selected inflammatory stimulators. The Journal of Veterinary Medical Science. 71 (11), 1499-1508 (2009).
  8. Beigi, F., et al. Optimized method for isolating highly purified and functional porcine aortic endothelial and smooth muscle cells. Journal of Cellular Physiology. 232 (11), 3139-3145 (2017).
  9. Jansen of Lorkeers, S. J., et al. Xenotransplantation of Human Cardiomyocyte Progenitor Cells Does Not Improve Cardiac Function in a Porcine Model of Chronic Ischemic Heart Failure. Results from a Randomized, Blinded, Placebo Controlled Trial. PLOS One. 10 (12), e0143953(2015).
  10. Zhang, J., et al. Potential Antigens Involved in Delayed Xenograft Rejection in a Ggta1/Cmah Dko Pig-to-Monkey Model. Scientific Reports. 7 (1), 10024(2017).
  11. Paris, L. L., et al. ASGR1 expressed by porcine enriched liver sinusoidal endothelial cells mediates human platelet phagocytosis in vitro. Xenotransplantation. 18 (4), 245-251 (2011).
  12. Paris, L. L., Chihara, R. K., Sidner, R. A., Tector, A. J., Burlak, C. Differences in human and porcine platelet oligosaccharides may influence phagocytosis by liver sinusoidal cells in vitro. Xenotransplantation. 19 (1), 31-39 (2012).
  13. Bernardini, C., et al. Heat shock protein 70, heat shock protein 32, and vascular endothelial growth factor production and their effects on lipopolysaccharide-induced apoptosis in porcine aortic endothelial cells. Cell Stress & Chaperones. 10 (4), 340-348 (2005).
  14. Endemann, D. H., Schiffrin, E. L. Endothelial dysfunction. Journal of the American Society of Nephrology: JASN. 15 (8), 1983-1992 (2004).
  15. Graupera, M., Claret, M. Endothelial Cells: New Players in Obesity and Related Metabolic Disorders. Trends in Endocrinology and Metabolism: TEM. 29 (11), 781-794 (2018).
  16. Kawamoto, A., Asahara, T., Losordo, D. W. Transplantation of endothelial progenitor cells for therapeutic neovascularization. Cardiovascular Radiation Medicine. 3 (3-4), 221-225 (2002).
  17. Rafii, S., Lyden, D. Therapeutic stem and progenitor cell transplantation for organ vascularization and regeneration. Nature Medicine. 9 (6), 702-712 (2003).
  18. Jain, R. K., et al. Endothelial cell death, angiogenesis, and microvascular function after castration in an androgen-dependent tumor: role of vascular endothelial growth factor. Proceedings of the National Academy of Sciences of the United States of America. 95 (18), 10820-10825 (1998).
  19. Pratumvinit, B., Reesukumal, K., Janebodin, K., Ieronimakis, N., Reyes, M. Isolation, characterization, and transplantation of cardiac endothelial cells. BioMed Research International. 2013, 359412(2013).
  20. Crouch, E. E., Doetsch, F. FACS isolation of endothelial cells and pericytes from mouse brain microregions. Nature Protocols. 13 (4), 738-751 (2018).
  21. Nakano, H., Nakano, K., Cook, D. N. Isolation and Purification of Epithelial and Endothelial Cells from Mouse Lung. Methods in Molecular Biology. 1799, 59-69 (2018).
  22. Naschberger, E., et al. Isolation of Human Endothelial Cells from Normal Colon and Colorectal Carcinoma - An Improved Protocol. Journal of Visualized Experiments. (134), e57400(2018).
  23. Yu, S., et al. Isolation and characterization of endothelial colony-forming cells from mononuclear cells of rat bone marrow. Experimental Cell Research. 370 (1), 116-126 (2018).
  24. Chi, L., Delgado-Olguin, P. Isolation and Culture of Mouse Placental Endothelial Cells. Methods in Molecular Biology. 1752, 101-109 (2018).
  25. Hawthorne, W. J., Lew, A. M., Thomas, H. E. Genetic strategies to bring islet xenotransplantation to the clinic. Current Opinion in Organ Transplantation. 21 (5), 476-483 (2016).
  26. Yi, E., et al. Mechanical Forces Accelerate Collagen Digestion by Bacterial Collagenase in Lung Tissue Strips. Frontiers in Physiology. 7, 287(2016).
  27. Masson-Pevet, M., Jongsma, H. J., De Bruijne, J. Collagenase- and trypsin-dissociated heart cells: a comparative ultrastructural study. Journal of Molecular and Cellular Cardiology. 8 (10), 747-757 (1976).
  28. Yonenaga, K., et al. Optimal conditions of collagenase treatment for isolation of articular chondrocytes from aged human tissues. Regenerative Therapy. 6, 9-14 (2017).
  29. French, M. F., Mookhtiar, K. A., Van Wart, H. E. Limited proteolysis of type I collagen at hyperreactive sites by class I and II Clostridium histolyticum collagenases: complementary digestion patterns. Biochemistry. 26 (3), 681-687 (1987).
  30. Hara, H., et al. In vitro investigation of pig cells for resistance to human antibody-mediated rejection. Transplant International: Official Journal of the European Society for Organ Transplantation. 21 (12), 1163-1174 (2008).
  31. Takashima, A. Establishment of fibroblast cultures. Current Protocols in Cell Biology. Chapter 2, 2.1.1-2.1.12 (2001).

Access restricted. Please log in or start a trial to view this content.

Reprints and Permissions

Explore More Articles

Miniature Pig AortaCollagenase IV DigestionEndothelial Cell IsolationFlow Cytometry AnalysisCD31 Antibody StainingCell Scraping TechniqueXenotransplantation ResearchVascular Endothelial CellsCell Culture Expansion
Isolation and Culture of Primary Aortic Endothelial Cells from Miniature Pigs
Contact Us
Recommend to Library

Privacy

Terms of Use

Policies

Copyright © 2026 MyJoVE Corporation. All rights reserved.