What Is the Epicranium?

Epithelial cells are cells located on the surface of the skin or cavity. Epithelial cells vary according to different organs: conventional epithelial cells in urine are declining and exfoliated skin cells, which do not have a specific specific meaning. People with non-SLE, including normal people, have any related inflammation, fall off, and routine urine tests are also available.

Epithelial cells in the outer layer of the skin are common

1) Epidermal cell culture

1. Materials: Small skin grafts from surgical skin grafts or surgical remnants are preferred. Those with thin keratinized layers are better. The skin of premature and aborted children is better. Cut into 0.5 to 1 cm 2 small pieces.

2. EDTA treatment: first put in 0.02% EDTA at room temperature for 5 minutes.

3 Cold digestion: change into 0.25% trypsin and set at 4 ° C overnight.

4 Separation: Remove the skin and separate the epidermis from the dermis with vascular forceps or forceps.

5. Warm digestion: take out the epidermis and treat it separately, cut it into smaller pieces with scissors, put it in a new 0.25% trypsin, and digest it at 37 ° C for another 30 to 60 minutes.

6. Gently blow repeatedly with a pipette to make a cell suspension.

7. Culture medium: After filtering through a 80 mesh stainless steel gauze, centrifuge at low speed, aspirate the supernatant, directly add Eagle's solution and 20% calf serum to make a cell suspension, inoculate it into a dish, and culture in a CO2 incubator.

2) Breast tissue culture

Direct culture method: (suitable for cultivating soft tissue with less fiber)

1. In a container containing a small amount of culture or Hanks' solution, use a sharp blade to repeatedly cut the tissue into pieces.

2. Inject the tissue fragments and liquid into the centrifuge tube, add a little culture solution, blow with a pipette for a while, and set the test tube rack for 3 to 5 minutes. Aspirate the supernatant to exclude non-breast cells. Repeat 2 to 3 times.

3 After the last treatment, the culture solution was replenished into the sedimentation tube and blown with a pipette to resuspend the sediment. Don't wait for the cell mass to drop and immediately filter it into another tube through 3 to 4 layers of sterile gauze.

4 Adjust the appropriate density and inoculate it into a culture flask.

Collagenase digestion method: (suitable for treating hard tissues with more fibers)

The process is the same as culturing other tissues.

3) Gastric epithelial cell culture

1. Source: Stomach ulcer or gastric cancer was surgically removed to remove a small amount of mucosa from the distal non-lesion area of the gastric specimen.

2. Cleaning: After rinsing with a solution containing gentamicin (400 g / ml) and amphotericin (2 g / ml Hanks), the mucous membrane was peeled off with a blunt instrument and cut to a size of 1 mm3.

3 Digestion: Digestion in collagenase I and hyaluronidase at 37 ° C for 80 minutes.

4 Centrifugation: Collect the cell suspension. After 800 rpm, the Hanks solution was rinsed twice.

5. Inoculation: After the last centrifugation, complete medium containing 1% to 2% fetal bovine serum is added, and inoculated into culture plates with different numbers of wells. The inoculation amount depends on different experimental purposes.

4) Liver cell culture

Primary tissue block culture: take fresh liver, first remove the fibrous components such as capsules and blood vessels, cut the liver into small pieces of about 1 mm ³ with a knife or scissors, and adopt the wall culture method.

5) Endothelial cell culture

1. Take fresh umbilical cord after delivery. If it is not cultured immediately, it can be stored at 4 ° C, but it should not exceed 12 hours. Aseptically cut a length of 10 to 15 cm. Other large blood vessels such as embryos and larvae can also be used for culture.

2. First, use a three-way syringe to inject warm PBS solution into the vein of the umbilical cord to wash away the residual blood. The injection port should be ligated with a string to prevent the liquid from flowing back.

3 Clamp one end of the umbilical cord with vascular forceps, and slowly inject a collagenase with a final concentration of 0.1% from the other end into the umbilical vein. After the liquid appears at the end, it will be ligated to fill the blood vessels. The injection port should also be ligated to prevent the liquid from flowing back. Digest for 3 to 10 minutes.

4 Aspirate the digestive juice containing endothelial cells and inject it into a centrifuge tube. In order to obtain more cells, rinse with warm PBS 2 to 3 times to completely remove the remaining cells, and then inject into the centrifuge tube and centrifuge.

5. Aspirate the supernatant, add 1640 culture solution to make a cell suspension, inoculate it into a bottle and culture, and when it is smooth, the cells can grow into a single layer within 2 to 3 days.

6) Capillary endothelial cell culture

Preparation of tumor conditioned media:

1. S-180 solid sarcoma tissue of C3H mice was taken.

2. After trypsinization, 15 ml of Dulbecco's modified Eagle's medium (containing 10% calf serum) was inoculated into T-75 Falcon culture flasks.

3 When the cell growth is near full confluence, the culture medium is collected to make a conditioned medium; and after a new culture medium containing 10% calf serum is used, it is also collected every two days to obtain a large amount of conditioned culture.

4. After 4,000 rpm / separation of the heart, it was filtered through a 0.22 m microporous filter and frozen at -20 ° C for use (thaw before use).