Isolation and Culture of Primary Aortic Endothelial Cells from Miniature Pigs

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 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 xenotransplantation^6,7. 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 setting^8,9,10,11,12. 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 studies^8,10,13. 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.

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).

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 1B-D). Another critical step to maintain cell viability is to minimize the period of time between obtaining the surgical specimen and the isolation procedure.

The processes involved in the purification of pAECs are shown in Figure 2. Our method is different from others since one end of the aorta is tied off and the endothelial surface is exposed (Figure 2C-G). Subsequently, the endothelial surface of the entire aorta is digested with pre-warmed collagenase digestive solution (5-10 mL) in a 15 mL centrifuge tube (Figure 2H). Compared to other methods, the endothelial surface is more completely, and preferentially, exposed to the digestive solution because of the inversion of the aorta and submersion into the digestive solution (without bubbles). After the digestion is stopped with stopping buffer, the endothelial surface of the aorta must be gently scraped in only one direction with a cell scraper (Figure 2I). The direction of scraping is important to avoid damage to the ECs. Do not touch the holes and cut edges of the digested aorta.

After isolation, the ECs are inspected on Days 0, 1, and 2, and after passage 1 (P1) and passage 2 (P2) (Figure 3A). The isolated ECs are identified by flow cytometry using an anti-CD31-FITC antibody. Flow cytometry analysis has indicated that the percentages of CD31-positive cells are 97.4% ± 1.2% (Day3), 94.4% ± 1.1% (P1) and 92.4% ± 1.7% (P2) (mean ± SD), respectively (Figure 3B). Thus, the isolation of pAECs can be successfully achieved with this protocol.

Figure 1: The surgical procedure and excision of the aorta from a miniature pig.
(A) Photograph of a miniature pig (Bama). (B) The aorta is exposed after laparotomy and thoracotomy. (C) The aorta is clamped at each end. (D) The aorta is placed in a 50 mL tube with cold washing buffer. Please click here to view a larger version of this figure.

Figure 2: Porcine aorta digestion and pAECs isolation.
(A) The aorta is placed in a 150 mm Petri dish with cold washing buffer. (B) Photograph of porcine aorta after removing connective tissues. (C) The glass bar tied with a sterile surgical suture to pass through the porcine aorta. (D) One end of the aorta is tied with a sterile surgical suture, which is kept on the inside of the aorta. (E,F) The aorta is reversed by pulling the surgical suture. (G) The endothelial surface of porcine aorta is exposed. (H) The digested aorta. (I) Scraping the endothelial surface of the aorta with a cell scraper. Please click here to view a larger version of this figure.

Figure 3: Identification of highly-purified pAECs by flow cytometry with an anti-CD31 monoclonal antibody.
(A) Representative images of isolated pAECs on Days 0, 1, and 2, and after passage 1 and passage 2 (200x magnification). (B) Flow cytometry analysis of pAECs (Day 3, passage 1 and 2) with anti-CD31-FITC antibody. Statistical data are presented in the bottom right. Data are representative of three independent experiments (mean ± SD). Please click here to view a larger version of this figure.

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^{8,19,20,21,22,23,24}. Recently, an increasing number of reports have demonstrated that porcine ECs have various functions in immune rejection and coagulation of xenotransplantation^6,25. However, the isolation of highly-purified aortic endothelial cells from miniature pigs has been reported less frequently. Here, we describe a stable and easy method for the isolation of aortic endothelial cells from miniature pigs.

Collagenase can degrade different tissues, and is superior to trypsin or other digestive solutions^26,27,28,29. We used 0.005% collagenase IV to dissociate pAECs from a small pig aorta. Importantly, the concentration of the digestive solution should be optimized to different pigs. Collagenase digestive solutions at 0.025%, 0.05% and 0.2% have been used respectively in different pigs, according to age and breed^10,13,30. Here, we recommend the optimal concentration of collagenase IV to be 0.005%, and the optimal digestive time to be 15 min. The time should not be <10 min or >20 min, which is consistent with previous studies^8,30. The concentration of collagenase IV and the digestive time are critical for obtaining high-purity and large numbers of ECs. Lower concentrations of collagenase IV or shorter digestive times will result in fewer ECs. Higher concentrations of collagenase IV or longer digestive times will lead to more fibroblast contamination.

Cell damage and fibroblast contamination are two problems in the isolation of pAECs. In order to reduce cell damage and fibroblast contamination, we adopted a method in which the ECs were scraped gently with a cell scraper. The direction, frequency, and intensity of scraping are critical to prevent cell damage and fibroblast contamination. First, we scraped the endothelial surface of the aorta in one direction only. Second, we recommend that the scraping frequency be less than 10 times (5 to 8 times is recommended), and the intensity must be gentle. Finally, the areas surrounding the holes of arterial side branches and the cut edges of the aorta (which might lead to fibroblast cells and more damage cells) should not be scraped.

A higher collagenase concentration, longer digestion time, and increased frequency and intensity of scraping may increase fibroblast contamination. Fibroblasts may rapidly outgrow the CD31-positive cells, thus reducing the purity of the isolated ECs³¹. Comparing to existing protocols, although the protocol has been carefully designed to prevent fibroblast contamination, and the CD31-positive cells on Day 3 reached 97.4% ± 1.2% (mean ± SD), the percentage of CD31 positive cells slowly decreased following culture. If the isolated ECs are used to carry out experiments after 5 passages, we suggest that the cells should be sorted with a flow cytometry cell sorter¹⁰.

Usually, we not only isolate the endothelial cells, but also get other organs for xenotransplantation research, such as the pancreas and kidney. According to our experience, it would better to isolate the pancreas and lung first, and then perform the procurement of liver and kidney. Even after that it is not too late to isolate the heart and aorta, if the surgeon is quick enough to finish all of the isolation in less than half an hour. During the process, it is very critical to keep the surgical area sterile and cold. The cold PBS with antibiotics was poured to the target organ to clean the possible contamination and to keep the tissues in low temperature. It is also necessary to prevent the coagulation by injecting heparin after anesthesia of animal. The clot in the micro vascular vessel would induce cell death and inflammation of organs with more capillaries, such as the pancreas, the lung and the liver. We always inject heparin to the inferior vena cava and insert a catheter to the abdominal aorta to blood. This will effectively prevent coagulation related cell death.

In conclusion, we provide an effective method to isolate highly-purified pAECs from miniature pigs. The isolated pAECs are beneficial for xenotransplantation research. Although we have not isolated the pAECs from a large pig using the protocol, we believe we will obtain highly purified ECs from a large pig with the protocol by modifying some critical steps.