What are fibroblasts cells?
Fibroblasts cells are the major cellular components of loose connective tissue and belong to terminal differentiation cells, which produce proteins such as collagen. The feature for Fibroblasts includes:largest amount, large cell body, regular oval nucleus, unclear cell contour with protrusions. The shape will diverse base on the function and the physical properties of the attachment. Rough endoplasmic reticulum with electron microscopy, free ribosome, and developed Golgi complex indicates that it has the function of synthesis and secretion of the protein. In the trauma and other factors, some fibroblasts can be re-transformed into naive fibroblasts, the functional activities can be restored, involved in tissue damage repair. In addition, there is a small amount of differentiation potential of mesenchymal cells in connective tissue, they can proliferate and differentiate into fibroblasts In the case of trauma repair.
How to culture Fibroblasts:
1. Cutting out the fresh skin, proliferative scar, and keloid tissue, and repairing the epidermis and subcutaneous tissue in the aseptic operation room.Then put into the PS containing DMEM culture medium after saline rinses repeatedly and bring back to the sterile work room.
2. Place tissue block in a Petri dish, rinsing Hank, s solution 3 times and then absorb Hank, s solution. Cut the tissue into 0.5-1mm3 size with the ophthalmic scissors repeatedly, and inoculating tissue block in 40 ml of the bottle wall of the culture bottle with the elbow pipette, making the space about 0.3-0.5 cm between the tissue piece
3. Plug the stopper into the thermostat incubator at 37°C for 3.5 hours to dry the tissue slightly
4. Get the culture medium out from the incubator, injecting 5 ml of DMEM medium that containing 20% CS and PS into the bottom of the flask. Then turn the culture flask to make the culture medium slowly cover the tissue pieces attached to the wall of the flask. Incubated again in a 37°C incubator
5. Take the culture flask out one week later, discard the culture medium and rinse with Hank, s solution for 2 times. Then adding the 5ml same culture medium to continue for culturing. While cell eruption, replacing the medium with the same method
Subculture can be processed while the primary cells fusion into slices:
1. Aspirate the culture medium and rinse twice with Hank, s solution. Add 0.5% trypsin solution to the flask for covering the bottom of the flask
2.Check microscopic observation of cell cytoplasm retraction one minute later, aspirating the trypsin solution and adding DMEM culture medium to stop digestion once cell gap increased
3. Pipetting the medium with straw and blew the cell wall to make cells fall off to form cell suspensions. Knock the subculture 1:3 ratio down and incubated in a 37 ℃ incubator with efficient amount of DMEM medium added
4. Replacing the medium twice a week, processing the subculture again until the Cells are pooled into monolayers
The above-mentioned content is the brief introduction for fibroblasts cells and methods to culture fibroblasts.