Human ovarian fibroblasts

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Human ovarian fibroblasts are isolated from ovarian tissue; The ovaries are the reproductive organs of female animals, and their function is to produce eggs and steroid hormones. The position of the ovary is the same as that of the testicle, with only the left side developing (the right side has degenerated), appearing grape shaped, and all follicles at different stages of development. The follicles are yellow in color, and the surface of the ovary is densely covered with blood vessels. The size of the ovaries is related to age and spawning period. Most vertebrates have two ovaries, but some fish have two ovaries fused into a single structure, while all birds only have one ovary on the left. The ovaries are a pair of oval shaped reproductive organs located on both sides of the uterus, with a layer of epithelial tissue on the surface and a thin layer of connective tissue below it. The internal structure of the ovary can be divided into cortex and medulla. The cortex is located around the ovary and is mainly composed of follicles and connective tissue; The medulla is located in the center and is composed of loose connective tissue, which contains many blood vessels, lymphatic vessels, and nerves. Fibroblast cells（Fibroblast is the main cellular component of loose connective tissue, differentiated from mesenchymal cells during embryonic development; Fibroblasts are relatively large, with clear contours and mostly flat structures resembling spindle or star shaped protrusions. Their nuclei are regular oval shaped, and their nucleoli are large and distinct. Fibroblasts have strong functional activity, weak alkaline cytoplasm, and significant protein synthesis and secretion activities. Under certain conditions, they can achieve interconversion with fibroblasts; Fibroblasts play a crucial role in the repair of varying degrees of cellular degeneration, necrosis, and tissue defects. The newly isolated ovarian fibroblasts are round in shape and have good refractive index, suspended in the culture medium. After 30 minutes, the cells adhered to the wall, and some of them began to extend pseudopodia, appearing as small protrusions; After 6 hours, the cells basically adhered to the wall, stretched into a spindle shape, had clear nuclei, distributed evenly, grew sporadically, and did not aggregate into clusters; The cells grow rapidly, reaching a fused state within 5-7 days. The cells are arranged tightly, with some overlapping and growing horizontally. They are flat, with large cell bodies, transparent cytoplasm, and large oval shaped nuclei with light colors. Cells fuse and connect with each other to form a network; The cells are distributed in a flat spindle or star shape with protrusions. Most ovarian fibroblasts are distributed on the surface of the ovary, and their main characteristics and functions are: ① Fibroblasts are easy to culture in vitro; ② After ovarian injury, fibroblasts can repair and reshape the ovary; ③ Can effectively control the spread of ovarian inflammation.
Method Introduction:

The company's laboratory separates human ovarian fibroblasts usingPrepared by a combination of protease collagenase mixed digestion method and differential adhesion method, with a total cell count of approximately 5 × 10? Cells/bottle.
Quality inspection:

Human ovarian fibroblasts isolated in the company laboratoryVimentin immunofluorescence identification shows a purity of 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 Fibroblast like

Passage characteristics transmissibleAround 5 generations; Status within 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.