Tubular type of renal epithelial cell Epithelial cells can be divided into monolayer and multilayer according to the number of cell layers, and can be divided into columnar and squamous according to the shape.
Columnar epithelial cells: mainly distributed in nasal cavity , nasopharynx, organs, lungs, stomach, intestines, cervix endometrium and Fallopian tube Etc. Squamous epithelial cells: covering the whole body skin, mouth, throat, part of nasopharynx, esophagus vagina All of and cervix 。 Squamous epithelial cells are divided into Basement layer Cell, middle layer cell and surface layer cell. Epithelial cells in the outer layer of the skin are common Keratinization , has the function of protection and absorption. The epithelial cells in the lumen are multi differentiated and have the functions of secretion, drainage and absorption.
Epithelial cells on the surface of digestive organs and lungs play an important role in digestion, absorption and exchange of oxygen [1] 。
1) Epidermal cell culture
1. Materials: Take small pieces of surgical skin graft or residual skin, preferably with thin keratoderma, and cut the skin of premature abortions into small pieces of 0.5~1 square centimeter.
2． EDTA Treatment: Put 0.02% EDTA in room temperature for 5 minutes. 3. Cold digestion: put it into 0.25% trypsin and put it at 4 ℃ overnight.
4. Separation: Take out the skin and separate the epidermis from the dermis with vascular forceps or forceps.
5. Warm digestion: take out the epidermis for separate treatment, cut it into smaller pieces with scissors, put it into a new 0.25% trypsin, and digest it at 37 ℃ for 30~60 minutes.
6. Blow gently and repeatedly with a straw to make the cell suspension.
7. Culture medium: filtered through 80 mesh stainless steel gauze, centrifuged at low speed, aspirated the supernatant, directly added Eagle solution and 20% calf serum to make cell suspension, inoculated into dish, and cultured in CO2 incubator.
2) Breast tissue culture
Direct culture method: (suitable for cultivating soft tissue with less fiber)
1. In the container containing a small amount of culture medium or Hanks solution, use a sharp blade to cut the tissue into pieces repeatedly.
2. Inject the tissue fragments together with the liquid into the centrifuge tube, add a little culture liquid, blow with a straw for a while, and place in the test tube rack for 3-5 minutes. The non mammary gland cells can be excluded by aspirating the upper fluid. Repeat 2-3 times.
3. After the end of the last treatment, add culture solution to the settling tube and blow it slightly with a straw to resuscitate the sediment. Filter the cells into another tube through 3~4 layers of sterile gauze immediately without dropping the cell mass.
4. Adjust the appropriate density and inoculate it into the culture bottle for cultivation.
Collagenase digestion method: (suitable for handling hard tissues with more fibers)
The process is the same as that of cultivating other tissues.
3) Gastric epithelial cell culture
1. Sampling: Take a small amount of mucosa in the non pathological area at the distal end of the gastric ulcer or gastric cancer surgical resection specimen.
2. Cleaning: rinse with gentamicin (400 μ g/ml) and amphotericin (2 μ g/ml Hanks) solution, peel off the lower mucosa with a blunt instrument, and cut it into 1mm3.
3. Digestion: Digest at 37 ℃ for 80 minutes in type I collagenase and hyaluronidase.
4. Centrifuge: collect cell suspension, and rinse twice with Hanks solution after heart separation at 800 rpm.
5. Inoculation: after the last centrifugation, add complete culture medium containing 1%~2% fetal bovine serum, and inoculate it into culture plates with different number of holes. The amount of inoculation depends on different experimental purposes.
4) Hepatocyte culture
Primary tissue block culture: take fresh liver, peel off fibrous components such as capsule and blood vessel, and cut the liver into 1mm with knife or scissors three The left and right small pieces were cultured by adherent culture method.
5) Endothelial cell culture
1. Take the fresh umbilical cord after delivery. If not cultured immediately, it can be stored at 4 ℃, but should not exceed 12 hours. Sterile cutting is 10~15cm long. Other large blood vessels such as embryos and young animals can also be used for culture.
2. Use a three-way syringe to absorb PBS solution and inject it into the vein of the umbilical cord to wash the residual blood. The injection port should be tied with a cord to prevent the liquid from flowing back.
3. Clamp one end of the umbilical cord with a vascular clamp, from the other end to the Umbilical vein Slowly inject collagenase with the final concentration of 0.1% into the middle, and ligate it after the liquid appears at the end to fill the blood vessels. The injection port should also be ligated to prevent the liquid from flowing back and digest for 3 to 10 minutes. 4. Suck out the digestive fluid containing endothelial cells and inject it into the centrifuge tube. In order to obtain more cells, inject warm PBS to wash for 2-3 times to completely remove the residual cells, and then inject it into the centrifuge tube for centrifugation.
5. Suck out the supernatant, add 1640 culture medium, make cell suspension, inoculate it into a bottle dish for culture, and the cells can grow into monolayer 。 6) Capillary endothelial cell culture
Preparation of tumor conditioned medium:
1. Take S-180 solid sarcoma tissue from C3H mice.
2. Trypsin digestion, 15ml Dulbecco modified Eagle's culture solution (containing 10% calf serum), inoculated into T-75 Falcon production culture bottle for culture.
3. When the cells grow close to complete confluence, collect the culture medium to make the conditioned medium; After using the new culture medium containing 10% calf serum, it is also collected every two days to obtain a large amount of conditional culture.
After separation at 4.4000 rpm, filter it through 0.22 μ m microporous membrane, and freeze it at - 20 ℃ for standby (thaw it before use).
