Porcine aortic endothelial cells

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Product Name:Porcine aortic endothelial cells

Organizational source:Aortic tissue

Product Specifications5×105cells/T25cell culture flask

Training Information:

Package conditionsPLL（0.1mg/ml）Gelatin（0.1%）

The culture medium containsFBSGrowth additivesPenicillinTheStreptomycinwait

Fluid change frequency per2-3Change the fluid once every day

Growth characteristics: Wall adhesion

Cell morphology endothelial cell like

Passage characteristics can be passed down2-3generation

digestive juice0.25%

Cultivation conditions gas phase: air,95%；CO2，5%

Cell Introduction:

Isolation of autonomous arterial tissue from porcine aortic endothelium; The aorta is the main artery of the systemic circulation. Its operating path is: the ascending aorta starts from the left ventricle and ends at the right side2The height of the thoracic rib joint moves towards the aortic arch, and the arch moves to the left posterior to the th4The lower edge of the thoracic spine moves towards the descending aorta; at the12The aortic hiatus that penetrates the diaphragm at the height of the thoracic vertebrae moves to the abdominal aorta, above which is the thoracic aorta, until the th4The lower body of the lumbar spine is connected to the left and right common iliac arteries; The common iliac artery is divided into internal and external iliac arteries at the height of the sacroiliac joint. Aortic endothelial cells are a single layer of cells covering the inner surface of the aorta, which can secrete a series of vasoactive substances to maintain vascular homeostasis. When stimulated by inflammation or other factors, their homeostasis is disrupted, leading to the occurrence of some cardiovascular diseases. Therefore, aortic endothelial cells have become a tool for studying the pathogenesis and therapeutic drugs of cardiovascular diseases. Endothelial cells or vascular endothelium are a thin layer of specialized epithelial cells composed of a layer of flat cells. It forms the inner wall of blood vessels and serves as the interface between the blood within the vessel lumen and other vessel walls (monolayer squamous epithelium). Endothelial cells run along the entire circulatory system, from the heart to the smallest microvessels.

Method Introduction:

The porcine aortic endothelium isolated in the company laboratory was used-It is prepared by combining collagenase digestion method with differential adhesion method and screening through endothelial cell specific culture medium, with a total cell count of approximately5×10?cells/Bottle.

Quality inspection:

Porcine aortic endothelial cells isolated in the company laboratoryCD31Immunofluorescence identification, purity can reach90%Above, and not containingHIV-1TheHBVTheHCVMycoplasma, bacteria, yeast, fungi, etc.

Cultivation steps:

1、 Resuscitate cells: Quickly shake and thaw a cryovial containing 1mL of cell suspension in a 37 ℃ water bath, then add 5mL of culture medium and mix well. Centrifuge at 1000 RPM for 5 minutes, discard the supernatant, add 4-6 mL of culture medium, and blow evenly. Then add all cell suspensions to the culture bottle and culture overnight (or add the cell suspensions to a 6cm dish), and culture overnight. The next day, change the solution and check the cell density.

2、 Cell passage: If the cell density reaches 80% -90%, passage culture can be carried out.

a）、 For adherent cells, the following methods can be used for passaging:

1. Discard the culture supernatant and rinse the cells 1-2 times with PBS that does not contain calcium or magnesium ions.

2. Add 1-2ml of digestion solution (0.25% Trypsin 0.53mM EDTA) to a culture bottle, place it in a 37 ℃ incubator for digestion for 1-2 minutes, and then observe the cell digestion under a microscope. If most of the cells become round and fall off, quickly take them back to the operating table, lightly tap the culture bottle a few times, and add 5ml or more of culture medium containing 10% serum to terminate digestion.

3. Gently blow the cells, remove and aspirate, centrifuge at 1000RPM for 8-10 minutes, discard the supernatant, add 1-2mL of culture medium, and blow evenly.

4. Add 5-6ml/bottle of culture medium and divide the cell suspension into new dishes or bottles containing 5-6 ml of culture medium in a ratio of 1:2.

b）、 For suspended cells, the following methods can be used for passaging:

Method 1: Collect cells, centrifuge at 1000RPM for 8-10 minutes, discard the supernatant, add 1-2ml of culture medium and blow evenly. Divide the cell suspension into new dishes or bottles containing 8ml of culture medium in a ratio of 1:2 to 1:5.

Method 2: You can choose the method of half changing the medium. After discarding half of the medium, suspend the remaining cells and divide the cell suspension into new dishes or bottles containing 8ml of medium in a ratio of 1:2 to 1:3.

How to handle received cells?

1. Firstly, observe whether the cell culture bottle is intact and whether there is any leakage or turbidity in the culture medium. If there are any, please take photos and contact technical support in a timely manner (the photos taken will be used as the basis for subsequent services).

2. Wipe the surface of the cell culture bottle with 75% alcohol and observe the cell status under a microscope. Due to transportation issues, a small amount of adherent cells may detach from the bottle wall; Do not open the culture bottle cap yet, place the cells in the cell culture box and let them stand for 2-4 hours to stabilize the cell state.

3. Carefully read the cell manual to understand cell related information, such as adhesion characteristics (adhesion/suspension), cell morphology, basic culture medium used, serum ratio, required cytokines, passage ratio, fluid exchange frequency, etc.

4. After settling, take out the cell culture bottle, examine it under a microscope, take photos, and record the cell status (the photos taken will be used as the basis for subsequent services); It is recommended to regularly take photos and record the growth status of cells after subculture.

5. Adherent cells: If the cell growth density exceeds 80%, it can be passaged normally; If the cell density does not exceed 80%, remove the culture medium from the cell culture bottle and reserve about 5ml for further cultivation until the cell density reaches around 80% before proceeding with the passage operation. The bottle cap can be slightly loosened.

6. Suspended cells: Transfer all the liquid in the cell culture bottle to a 50ml sterile centrifuge tube and centrifuge at 1200rpm for 5 minutes. After centrifugation, collect the supernatant culture medium for later use. Add 5ml of culture medium to the bottom of the tube and resuspend the cell precipitate. During microscopic examination, if the cell density exceeds 80%, the cell suspension can be cultured in two cell culture bottles and supplemented with culture medium up to 5ml. If the cell density does not exceed 80%, the cell suspension can be transferred to the original bottle for further cultivation until the cell density reaches around 80% before passaging

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