Human pulmonary microvascular endothelial cells

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Separation of human pulmonary microvascular endothelium from lung tissue; The lungs are the respiratory organs of the body, located in the chest cavity, one on each side, covering the heart. The lungs have lobes, two on the left and three on the right, totaling five lobes. The lung meridian system (referring to the trachea, bronchi, etc.) is connected to the larynx and nose, hence the larynx is called the gateway to the lungs, and the nose is the extrapulmonary orifice. Microvascular endothelial cells are closely involved in a series of physiological and inflammatory responses, including regeneration, development, and wound healing. The cells are spindle shaped or polygonal, and after forming a single layer, they are arranged in a cobblestone like or cobblestone like pattern. Pulmonary microvascular endothelial cells form a semi selective barrier, which is of great significance for lung gas exchange, regulating the flow of fluids and soluble substances between blood and lung interstitium.

Method Introduction:

The human lung microvascular endothelium isolated in the laboratory was prepared using tissue patch method combined with endothelial cell specific culture medium for screening, with a total cell count of approximately 5 × 10 ⁵ cells/bottle.

Quality inspection:

The laboratory isolated human lung microvascular endothelium was identified by CD31 immunofluorescence, with a purity of over 90%, and did not contain HIV-1, HBV, HCV, mycoplasma, bacteria, yeast, fungi, etc.

Package conditionsPLL (0.1mg/ml) or gelatin (0.1%)

culture mediumContaining FBS, growth additives, Penicillin, Streptomycin, etc

Fluid change frequencyChange the fluid every 2-3 days

Growth characteristicswall sticking

cell morphologyEndothelial cell like

Passage characteristicsCan be passed down to 2-3 generations

digestive juice0.25%

preparation

1. Preparation of experimental equipment: Prepare sterile culture dishes, culture bottles, pipettes, centrifuge tubes, surgical instruments, etc., and perform high-pressure sterilization or other suitable disinfection treatments.

2. Reagent preparation: Prepare or purchase suitable culture media, digestive enzymes (such as collagenase, etc.), fetal bovine serum, bispecific antibodies, and other reagents to ensure their sterility and expiration date.

3. Preparation of experimental animals or tissue sources: Select suitable animals according to experimental needs and perform corresponding anesthesia or execution to obtain the required tissue; Or obtain the organization from an existing organizational sample library.

Material selection and processing

1. Sampling: Under sterile conditions, quickly remove the target tissue, minimize damage to the tissue, and remove excess non target tissues such as fat and connective tissue.

2. Cleaning: Rinse the extracted tissue several times with pre cooled sterile PBS to remove blood and impurities.

3. Cutting: Cut the tissue into small pieces of about 1-2mm ³ for subsequent digestion.

cell separation

1. Digestion: Put the cut tissue pieces into centrifuge tubes containing an appropriate amount of digestive enzymes, and digest them for a period of time in a 37 ℃ constant temperature shaker or incubator. During this period, gently shake the centrifuge tube to make digestion more uniform.

2. Termination of digestion: When most of the tissue block is digested into single-cell suspension or small cell clusters, add serum containing culture medium to terminate digestion.

3. Filtration and centrifugation: Use a cell sieve to filter the cell suspension, remove undigested tissue fragments, and then centrifuge the filtrate at an appropriate speed to collect cell sediment.

Cell observation and detection

1. Daily observation: Use an inverted microscope to observe the morphology, growth status, density, etc. of cells every day, record the changes in cells, and take corresponding measures in a timely manner if cell contamination or abnormalities are found.
2. Cell counting and vitality detection: When necessary, methods such as trypan blue staining can be used to count and detect the vitality of cells, in order to understand their growth and health status.

1、 Material selection and separation

1. Quick operation

-After tissue sampling, it is necessary to handle it immediately and avoid prolonged exposure to room temperature or non nutrient environments.

-Rinse the tissue with sterile PBS or physiological saline to remove blood and impurities.

2. Selection of digestive enzymes

-Choose digestive enzymes based on the type of organization to avoid excessive digestion that can cause cell damage.

-The digestion time needs to be strictly controlled (usually 10-30 minutes), and the state of cell dissociation can be observed under a microscope.

2、 Optimization of cultivation conditions

1. Selection of culture medium

-Using culture media containing serum or specific growth factors (such as DMEM, RPMI 1640, etc.), some cells require the addition of insulin, EGF, etc.

-Avoid frequent changes in culture medium brands or batches to reduce cell adaptation pressure.

2. Wall sticking and passage

-Primary cells have weak adhesion ability and may need to be wrapped in culture dishes (such as collagen, polylysine).

-It is recommended to control the density between 70% and 80% during passage, as excessive convergence can lead to contact inhibition and differentiation.

3、 Pollution control

1. Aseptic operation

-Operate the entire process on a clean bench, using disposable consumables to avoid cross contamination.

-Double antibodies can be added to the culture medium, but long-term use may affect cell activity.

2. Mycoplasma testing

-Regularly detect mycoplasma contamination (such as PCR method), and promptly discard cells after contamination.

4、 Status monitoring

1. Daily observation

-Check the cell morphology, density, and color of the culture medium daily, and replace the medium promptly (usually every 2-3 days).

-Abnormal morphology (such as cells becoming round or fragments increasing) may indicate contamination or malnutrition.

2. Passage and cryopreservation

-The number of primary cell divisions is limited (usually 5-10 passages), and early passage cells need to be frozen in a timely manner.

-It is recommended to use DMSO+serum (or specialized cryopreservation medium) as the cryopreservation solution, and store it in liquid nitrogen after gradient cooling.