Epidermal cell culture experiment
Epidermal cell culture experiment
Epidermal cell culture experiments refer to the culture of epidermal cells under in vitro conditions.
Principle
The basic principle of the culture experiment of epidermal cells is to treat the skin tissue mass with specific digestive enzymes, its epidermis can be separated from the dermis at the basement membrane, when the epidermis is digested into cell clusters or single cells, and then cultured with the appropriate culture solution, a large number of epidermal cells can be proliferated.
Operation method
Experiments on Culturing Epidermal Cells (Experiments on Culturing Epidermal Cells)
Principle
The basic principle of the culture experiment of epidermal cells is to treat the skin tissue block with specific digestive enzymes, its epidermis can be separated from the dermis at the basement membrane, when the epidermis is digested into cell clusters or single cells, and then cultured with the appropriate culture solution, a large number of epidermal cells can be proliferated.
Materials and Instruments
Equipment: Move The basic procedure of the epidermal cell culture experiment can be divided into the following steps： （1） Take the materialA. Take a piece of skin from the back of a newborn 1~2 d mouse and remove the fat tissue under the skin.B. Wash the skin block with D-Hanks liquid for 3 times, cut the skin into 0.5 x 1.0 c㎡ pieces with ophthalmic scissors, and place them in 0.25% pancreatic enzyme.(2) Digestion and inoculationA. Digest at 4 ℃ for 24 h or 37 ℃ for 2~3 h.B. Separate the epidermis and dermis carefully with ophthalmic forceps.C. Place the separated epidermis in a centrifuge tube, add DMEM culture medium containing 10% fetal bovine serum, blow the epidermis and prepare cell suspension.D. Centrifuge at 2000r/min for 10 min and discard the supernatant.E. Add DMEM/F12 medium containing 10% fetal bovine serum, mix well, and then inoculate the cells into 25 ml culture flasks at a density of 1 x 106 cells/ml.F. Incubate at 37 ℃ for 4 h, gently aspirate the culture supernatant and add fresh medium to continue incubation.G. After 2 d, observe the cells under an inverted phase contrast microscope and take photos.H. Discard the original medium, add fresh medium and continue to incubate for 3 d and 7 d. Observe the cells under an inverted phase contrast microscope and take photographs. Caveat 1. After extraction, the fat tissue under the skin must be removed, otherwise the residual fat tissue will affect the enzyme digestion. 2. Before digestion, the size of the skin block should be controlled when the skin is cut with ophthalmic scissors. If the skin block is too large, the action of trypsin on the skin will be incomplete; if the skin block is too small, it is not easy to operate the process of separating the epidermis from the dermis after the action of trypsin. 3. Trypsin digestion of the epidermis for blowing, so that it is dispersed into single cells or cell clusters, often part of the residual epidermis is not easy to be blown scattered, the cells that make up the epidermis are mainly from the stratum corneum, the degree of differentiation of its cells is high, the connection is tight, the cells are generally not able to proliferate, therefore, it can not be necessary to continue to blow on these epidermis. 4. In the in vitro culture of epidermal cells, the cells of the basal layer are the main proliferating cells, and their number accounts for only a small part of the total number of epidermal cells, therefore, in order to improve the survival rate of the cultured cells, the number of inoculated cells should be increased appropriately. For more product details, please visit Aladdin Scientific website.
① newborn 1~2 d mice
② Culture bottle
② Culture bottle ③ Suction tube
③ Straw ④ Cap
⑤ Petri dish
⑥ Ophthalmic scissors
⑦ Ophthalmic forceps
⑧ Centrifuge
⑨ Inverted microscope
⑩ Ultra-clean bench
⑪ CO2 incubator, etc.
Gel cap
Reagents
① D-Hanks liquid
① D-Hanks liquid ② DMEM medium (containing 10% fetal bovine serum)
③ 0.25% trypsin solution