Human retinal microvascular endothelial cells

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Separation of human retinal microvascular endothelium from retinal tissue; The retina is located in the inner layer of the eyeball wall and is a transparent thin film. The retina is composed of the pigment epithelium layer and the retinal sensory layer, which can be separated under pathological conditions, known as retinal detachment. The pigment epithelium layer is closely connected to the choroid and is composed of pigment epithelial cells. They have functions such as supporting and nourishing photoreceptor cells, shading, heat dissipation, regeneration, and repair. Histologically, the retina is divided into10 layers, from outside to inside: pigment epithelium layer, cone, rod cell layer, outer membrane, outer granular layer, outer plexiform layer, inner granular layer, inner plexiform layer, ganglion cell layer, nerve fiber layer, and inner limiting membrane. The inner layer of the retina is a thin film lining the inner surface of the vascular membrane, which has a photosensitive effect; There is a optic nerve nipple on the posterior nasal side. The sensory layer on the retina is composed of three neurons. Neurons are the layer of visual cells specialized in light sensing, which includes cone cells and rod cells. Rod cells are mainly located in the retina far from the fovea, while cone cells are most abundant in the fovea. The second layer is called double ganglion cells, which are responsible for communication between approximately 10 to hundreds of visual cells and a ganglion cell through double ganglion cells. The third layer is called the ganglion cell layer, which is responsible for conduction. The retina is a thin but very complex structure that is attached to the posterior wall of the eyeball. The nerve fibers that transmit impulses from retinal receptors cross the surface of the retina and reach the exit through the optic nerve. The resolution of the retina is uneven, and in the macular area, its resolution is strong. It is a single layer of flat epithelioid cells that make up the vascular lumen. The bioactive substances it produces and secretes play an important role in maintaining vascular tension, regulating blood pressure, and preventing thrombosis. They have significant pathophysiological significance in the pathogenesis of retinal vascular diseases. Due to the different characteristics of endothelial cells obtained from different tissues and organs, these endothelial cells have the function of an internal barrier in the brain and retina, playing an important role in regulating the physiological state of blood vessels, releasing vasoactive substances, and forming new blood vessels. In vitro isolation and culture of RCEC have important clinical significance for studying the pathological and physiological mechanisms of the occurrence and development of retinal vascular diseases, as well as early prevention and treatment of diseases.
Method Introduction:

The human retinal microvascular endothelium isolated in the company laboratory was treated with collagenase-The neutral dan white enzyme mixed digestion method is combined with density gradient centrifugation, and finally screened by endothelial cell specific culture medium. The total number of cells is about 5 × 10? Cells/bottle.
Quality inspection:

Human retinal microvascular endothelial cells isolated from the company laboratoryCD31 immunofluorescence identification, purity can reach over 90%, and does not contain HIV-1, HBV, HCV, mycoplasma, bacteria, yeast, fungi, etc.

Package conditions PLL（0.1mg/ml）， Gelatin (0.1%)

culture medium containFBS、 Growth additives, Penicillin, Streptomycin, etc

Fluid change frequency eachChange the fluid every 2-3 days

Growth characteristics wall sticking

cell morphology Endothelial cell like

Passage characteristics transmissible2-3 generations

digestive juice 0.25% protease

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

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.