Brand of human type II alveolar epithelial cells

Human airway epithelial cells

1、 Basic cellular properties

Culture medium: containing FBS, growth additives, Penicillin, Streptomycin, etc

Fluid change frequency: Change the fluid every 2-3 days

Passage characteristics: Can be passed down for 2-3 generations

Digestive fluid: 0.25% pancreatic amylase

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

Cell profile: Human tracheal epithelial cells are isolated from tracheal tissue; Trachea, a part of the respiratory organ; A cylindrical tube with a slightly flat rear wall. The lower edge of the sixth cervical vertebra is flat at the upper end and connected to the cricoid cartilage; Going down to the junction of the fourth and fifth thoracic vertebrae (equivalent to the sternal angle plane), it divides into left and right main bronchi, and the bifurcation is called the tracheal branch. The trachea is mainly composed of 14-16 semi-circular cartilages, which are elastic and have a "C" - shaped cartilage ring. The gap is backward, and each cartilage ring is connected by ligaments. The gap behind the ring is connected by smooth muscle and dense connective tissue, maintaining a continuous open state. The lumen is lined with mucosa, and the surface is covered with ciliated epithelium. The mucus secreted by the mucosa can adhere to dust particles inhaled into the air. The cilia constantly swing towards the throat to expel the mucus and dust, purifying the inhaled gas. The tracheal epithelium is the first line of defense between the airway and the external environment. It is not only a target cell for various pathogens and inflammatory mediators, but also an effector cell that synthesizes and releases various inflammatory mediators and cytokines to participate in airway inflammation and immune responses. The primary tracheal epithelial cells cultured in vitro are extremely important in basic and clinical research due to their significant similarity in morphology, structure, and functional activity with tissues in vivo.

The idea of primary cell isolation and culture:

Sampling - Separation - Cultivation - Identification

Firstly, the tissue is extracted from the body and treated with pancreatic amylase/collagenase to disperse into single cells. Then, it is cultured in a suitable medium to allow the cells to multiply to a certain coefficient before cell identification.

Experimental procedure:

1、 Male SD rats aged 7-10 days were euthanized by cervical dislocation and soaked in 75% ethanol for 3-5 minutes for disinfection.

2、 In the ultra clean workbench, decapitate the rat and place it in a glass culture dish. After opening the cranial cavity, take out the whole brain and place it in a PBS glass culture dish with pre cooling. Remove the cerebellum and diencephalon.

3、 Slowly roll the cerebral hemisphere on dry filter paper to remove the pia mater and meningeal blood vessels, and then place it in a new PBS glass culture dish with pre cooling.

4、 Use fine dissection forceps to remove white matter, residual large blood vessels, and pia mater of the brain, while preserving the cerebral cortex.

5、 The cerebral cortex was placed in DMEM, cut into approximately 1mm3 pieces, and placed in a 50ml centrifuge tube. Mixed enzyme solution I (0.05-0.1% type II collagenase, 0.025-0.05% type IV collagenase, 200-400U DNaseI) was added, and after mixing, digested in a 37 ℃ water bath for 1-1.5 hours.

6、 Centrifuge at room temperature of 1000 × g for 8 minutes and remove the supernatant.

7、 Add 20% BSA to the precipitate, resuspend thoroughly, mix well, 1000 × g for 20 minutes, centrifuge at 4 ℃, and remove the supernatant.

8、 Add 4ml of mixed enzyme solution II (0.05-0.1% collagenase/dispersant, 0.05-0.1% type I collagenase, 100-300U DNaseI) to the precipitate, resuspend, mix well, digest in a 37 ℃ water bath for 0.5-1 hour, centrifuge at 700 × g room temperature for 6 minutes, and remove the supernatant.

9、 Add 2ml of DMEM culture medium to the precipitate, suspend and mix well. Spread it on 12ml of 50% Percoll (25000 × g, 60 min, 4 ℃), 1000 × g, and centrifuge at 4 ℃ for 10 min to form a continuous gradient.

10、 The white yellow layer above the red blood cell layer near the bottom is the purified microvascular segment. After aspiration, rinse twice with DMEM (centrifuge at 1000 × rpm, 6 minutes, room temperature) to remove the supernatant.

Add DMEM culture medium (20% FBS, 4ug/ml) and resuspend. Inoculate in a cell culture dish prepared by overnight incubation at 37 ℃ with 5% rat serum. Place in a 37 ℃, 5% CO2 incubator and let it stand for 24 hours before changing to a non nutrient mixed medium. Then change the medium the next day.

Notes:

1. The cultivation can be stored for 3-6 months under 4 ℃ conditions.

During the cell culture process, please pay attention to maintaining aseptic operation.

During the process of subculture, the digestion time of enzymes should not be too long, otherwise it will affect the cell adhesion and growth status.

4. It is recommended that customers take several cell photos for each magnification within the first 3 days after receiving the cells, recording the cell status for communication with the technical department; Due to transportation reasons, some sensitive cells may experience instability. Please contact us promptly and provide detailed information about the specific situation of the cells, so that our technical personnel can track and follow up until the problem is resolved.

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