Human neuroglial cells

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Separation of human microglia from brain tissue; The brain is divided into two hemispheres, the cerebral cortexGray matter covers most of each hemisphere of the brain and is the place where neuronal cells are concentrated. Internally, it is white matter composed of nerve fibers or myelin sheaths. Each hemisphere has three faces, namely the lateral surface (approximately one-third of the entire cortical area), the medial surface, and the basal surface (two-thirds of the area); There are many grooves or fissures of varying depths on the surface of the hemisphere, and the elevations between the grooves or fissures are called "folds", which greatly increase the surface area of the brain; The important grooves and fissures on the lateral surface of the brain include the lateral fissure, occipital fissure, and central fissure. Due to the boundary between the three fissures, the cerebral cortex is divided into four major parts: frontal lobe, parietal lobe, temporal lobe, and occipital lobe. Microglia are a type of glial cell, equivalent to macrophages in the brain and spinal cord, and are the main immune defense line in the central nervous system (CNS). Microglia account for approximately 20% of the glial cells in the brain, constantly clearing damaged nerves, plaques, and infectious substances in the central nervous system. Numerous clinical and theological studies have shown that activated microglia play a crucial role in the pathogenesis of neurodegenerative diseases such as Parkinson's disease, multiple sclerosis, and Alzheimer's disease. However, excessive activation or loss of control of microglia can cause neurotoxicity. They are important sources of pro-inflammatory factors and oxidative stress, such as tumor necrosis factor (TNF), nitric oxide, interleukin, and other neurotoxic substances. The main functions of microglia are as follows: ① Microglia appear during the transition from early brain development to mature stage; ② When programmed cell death occurs, or when the central nervous system is damaged or subjected to pathological damage during brain development, glial cells can act as macrophages in the brain; ③ Glial cells can express antigens when CD-4 positive T cells are expressed in class II histocompatibility complexes, perform Fc mediated phagocytosis, and share antigens with hematopoietic cells and macrophage tissues.
Method Introduction:

The human microglia isolated in the company's laboratory were prepared by enzymatic digestion combined with differential adhesion method. After several days of nutrient deficiency in the culture medium, the exfoliated cells were collected by shaking on a shaker. The total number of cells was approximately5×10? Cells/bottle.
Quality inspection:

Human neuroglia isolated in the company laboratoryCD11b immunofluorescence identification, purity can reach over 90%, and does not contain HIV-1, HBV, HCV, mycoplasma, bacteria, yeast, fungi, etc.

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

Fluid change frequency eachChange the fluid every 2-3 days

Growth characteristics wall sticking

cell morphology Shuttle shaped, polygonal shaped

Passage characteristics Belonging to terminally differentiated cells; Belonging to non proliferating cell population

digestive juice Lido Ka Yin（12mM）

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.