Isolation and purification of primary epithelial cells and primary fibroblasts
PriCells- primary epithelial cells and primary fibroblast cultures into Purification of
Most of the primary cells and passage cells are mixed and grow, both epithelioid cells and fibroid cells, and fibroblasts include fibroblasts, muscle cells, bone cells, and synovial cells. Mixed cells directly affect the results of the experiment.
When culturing primary cells in vitro, in order to ensure the reliability, consistency, stability, and reproducibility of the experimental results, it is required to use a single type of cell to conduct experiments, so as to perform a change in the function and morphology of a certain cell. A series of studies, thus purification of cultured cells, has become an important step in experimental research, even requiring the isolation of individual cells from a mixed cell population for culture and experimental research.
First, the primary cell proliferation advantage purification method:
Natural purification is the use of a certain kind of cell proliferation advantage, in the long-term passaging process by natural elimination method, constantly crowding out other slow-growing cells, relying on the potential of natural proliferation, and finally leaving the cells with strong growth potential, to achieve the purpose of cell purification . However, this method often fails to select cells according to needs and experimental requirements and research purposes. This method takes a long time and often leaves fibroblasts. Only those malignant tumor cells or mutated cells can be retained by this method and continuously purified to establish a cell line.
Second, the primary cell commonly used purification methods:
Artificial purification is the use of artificial means to cause environmental conditions favorable for the growth of a certain cell, inhibiting the growth of other cells to achieve the purpose of purifying cells.
1 , cell time difference enzyme digestion method:
Enzymatic digestion is a relatively common purification method. It is not only feasible for adherent cells, but also has different tolerance to trypsin by epithelial cells and fibroblasts. It is separate for purification purposes; Separation and purification between semi-adherent cells and adherent cells is also very effective.
Under the action of trypsin, the fibroblasts first detached from the wall, and the epithelial cells have to be digested for a long time before they are detached. Especially in the primary passage and early passage of the primary cells, the two differences are particularly obvious. Separation of epithelial cells and fibroblasts by multiple differential digestion methods are as follows:
- 0.25% trypsin was injected into the culture flask twice by conventional digestion and passage, and 1 ml (25 ml culture flask) was added each time, and gently shaken 1-2 times to allow trypsin to flow through the surface of all cells, and then poured off.
- The stopper was capped, and the culture bottle was placed under an inverted microscope to observe that the fibroblasts were turned into gardens and partially detached, and the digestion was immediately terminated by adding 2 ml of serum-containing medium.
- Gently blow the fibroblast growth area with an elbow pipette (mark the pen on the flask with a marker beforehand). Do not use force when blowing, and do not blow the epithelial cell growth area. After the end of the pipetting, rinse with a small amount of medium, and then continue to culture by adding an appropriate amount of the medium, or repeat the above operation once more.
- After a few days or the next passage, the above operation is carried out, and after several treatments, the fibroblasts can be removed or separated.
2 , cell culture mechanical scraping method:
In primary culture, if epithelial cells and fibroblasts are divided into regions, each cell grows on the wall of the bottle in a small piece or in a regional distribution. Mechanical methods can be used to remove unwanted cell areas while retaining the desired cell area. The method steps are as follows:
- The culture flask to be purified is operated under the supervision of an inverted microscope in a clean room.
- Use a silicone rubber wiper to push the cells in areas where growth is not required, and suspend the cells in the medium, taking care not to damage the desired cells.
- After the deduction, the medium is added to the original bottle to continue the cultivation by shaking with the medium and shaking twice.
- If the undesired cells are found to grow again after a few days, the above operation can be performed, so that the cells can be purified repeatedly. Strict aseptic operation should be carried out during operation to prevent pollution.
3 , cell repeated adherence method:
Compared with epithelial cells, fibroblasts adhere quickly, most cells can complete the attachment process (but not necessarily fully stretched) in a short time (about 10-30min), while epithelial cells (mostly) are in short time. It is not attached or unstable, and it floats with a little oscillation. The difference can be used to purify the cells.
- The cell suspension was inoculated in a culture (preferably, the medium contained no serum, at which time the epithelial cells were attached more slowly) and allowed to stand for 20 min.
- Observed under an inverted microscope, see some cells adherent, and when the cells were shaken a little, the cell suspension was introduced into another flask.
- Continue to static culture for 20min, then repeat the above operation, the epithelial cells and fibroblasts can be separated, the first bottle and the second bottle are mainly fibroblasts, and the next few bottles are epithelial cells. The next time you pass the above method, you can achieve the goal of complete separation.
4 , cell electrophoresis screening method:
When the adherent cells are transformed, scattered foci will appear in the cell layer of the culture flask. The cells in the foci are densely packed, regularly arranged, and have obvious growth tendency, and have obvious regional boundaries with the cells that have not been transformed. Untransformed cells can be removed by mechanical scraping, and untransformed cells can be scalded by electroporation to retain the transformed cells.
- Pour the stock solution and use a marker to draw out the area of ​​the foci.
- The cells around the foci were all scalded with a heated micro-electrode (similar to a soldering iron for soldering), leaving only the foci cells. This method can also be used to kill cells surrounding a single cell during single cell cloning screening.
- The culture is then continued in an adaptive medium (50% stock solution) for purification purposes.
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