Porcine Aorta Isolation: A Surgical Procedure to Excise the Thoracic Aorta From Miniature Pigs for Downstream Analysis

Samenvatting

Source: Zhao, Y., et al. Isolation and Culture of Primary Aortic Endothelial Cells from Miniature Pigs J. Vis. Exp. (2019)

In this video, we demonstrate a procedure for the surgical removal of the aorta from miniature pigs. Endothelial cells harvested from these isolated aortae can be further cultured in in vitro conditions to study coagulation and immune responses.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. 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- 1x PBS solution (pH 7.4) with 1% (v/v) Penicillin/Streptomycin. (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).

Resultaten

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...

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
20 x PBS solution (pH 7.4, Nuclease free)	Sangon Biotech 	B540627#
75% Medical alcohol	Guilin LiFeng Medical Supplies Co., Ltd. China
Boneforceps	Beijing HeLi KeChuang Technology Development CO., Ltd. China	HL-YGQ
BOON Disposable Syringe (10ml)	Jiangsu Yile medical Article Co., Ltd. China
Compound ketamine injection	Veterinary Pharmaceutical Factory of Shenyang, China		Ketamine Hydrochloride: 0.3g/2ml, Xylazine hydrochloride:0.3g/2ml, Phenacetin hydrochloride: 1mg/2ml
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
Penicillin/Streptomycin 	Life Technologies 	15070063#
Scissors	ShangHai medical instruments Co., Ltd. China
Surgical scalpel	ShangHai medical instruments Co., Ltd. China	22#