Two methods of cell purification

Purification of cells Two methods of purification of cells are generally divided into two types, namely, natural purification and manual purification. Commonly used purification methods include enzymatic digestion, cytokine-dependent purification, mechanical scraping, repeated adherence, and ironing. Screening method.  
The cells cultured in vitro are derived from human or animal or embryonic tissues, and the cells in the body are mixed and grown. Each tissue has blood vessels and mesenchymal tissues. Therefore, the primary cells and passage cells of the culture materials derived from the above tissues are absolutely Most of them are mixed and grow, both epithelial-like cells and fibroid-like cells. Fibro-like cells include fibroblasts, myocytes, bone cells, synovial cells, etc., and mixed cells directly affect the experimental results. When culturing cells for experimental research, in order to ensure the reliability, consistency, stability, and reproducibility of the experimental results, it is required to use a single type of cells to conduct experiments, so as to perform a series of changes on the function and morphology of a certain cell. Research, thus purification of cultured cells has become an important step in experimental research, even the need to separate individual cells from the mixed cell population for culture and experimental research.
Purification of cells:
Purification of cells is generally divided into two types, namely, natural purification and manual purification. It can be selected according to different cell types, sources, experimental requirements and purposes. Two methods for purification of cells <br> 1. Natural purification:
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, often leaving fibroblasts. Only those malignant tumor cells or mutant cells can be used by this method. Retained, continuously purified to establish cell lines.
Second, manual purification:
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. There are five main methods.
1. Cytokine dependent purification method:
It is the cell line that depends on the growth of this cytokine by adding certain special cytokines. Some cells in human and animal tissues need an environment with special cytokines to survive and grow for a long time, such as white blood cells. Interleukin-2 (IL-2) SHI The cytokine necessary for cell growth. B cell growth factor is a growth factor of B cells. If lymphocytes are added to IL-2 in vitro, T cells can grow and multiply to form IL. -2 dependent T cells, such as the CTLL-2 cell line, while other cells are naturally eliminated, using this method also established IL-6-dependent cell lines, such as B9, CTD7 cell lines.
2. Enzymatic 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.
(1) Separation and purification of epithelial cells and fibroblasts:
Under the action of trypsin, the fibroblasts are first detached, and the epithelial cells are 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. Multiple differential digestion methods separate epithelial cells from fibroblasts. The method steps are as follows.
0.25% trypsin was injected into the culture flask twice by conventional digestion and passage method, 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 pour off.
Cover the stopper and place the flask under an inverted microscope. It is found that the fibroblasts are turned into gardens and partially shed. Immediately add 2ml of serum-containing medium to stop digestion.
Use a curved pipette to gently blow the fibroblast growth area (you can use a marker to mark the flask on the microscope beforehand). Do not use force when blowing, and do not blow the epithelial cell growth area. After the end of the blow, use it again. Rinse a small amount of medium, then add the appropriate amount of medium to continue the culture in the bottle, or repeat the above operation.
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) Purification of bone marrow stromal cells and muscle-like cells:
When blood cells and bone marrow cells are cultured in a static state, many muscle-like cells and bone marrow stromal cells often adhere to the wall, but many lymphocytes, monocytes, and granulocytes adhere to them, and the types are mixed, in view of the adhesion of adherent cells. The enzymatic digestion method can also be used to separate the adherent cells to achieve the purpose of separating and purifying the muscle-like cells and lymphocytes in the bone marrow stromal cells or blood.
When the stromal cells or muscle-like cells form a monolayer, pour off the old solution, add PBS without calcium and magnesium ions, rinse it vigorously, then pour it off. After repeated washing for 2-3 times, wash serum and Calcium and magnesium ions are digested by enzymes, and on the other hand, most of the adherent cells are washed away, but there are still many adhering cells.
Inject 0.25% trypsin into the culture flask, 1ml (25ml culture flask) per bottle, gently shake the trypsin to flow through the cell surface, apply 1-2min, then gently shake 1-2 times and then pour.
Capsules were capped and observed under a normal microscope. It was found that stromal cells or muscle-like cells were not firmly detached due to adherence, and the cells that had previously adhered had floated. At this time, 2 ml of PBS was added and washed once, and the adherent cells were removed as much as possible. .
Add the serum-containing medium to terminate the digestion, continue to shake vigorously, then pour off, add the medium to continue the culture. After a few days or before the next passage, the above operation is carried out. After several treatments and subculture, the adherent cells can be adhered. Remove, separate the two, to achieve the purpose of purifying bone marrow stromal cells or muscle-like cells.
3. 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 regional distribution. Mechanical methods can be used to remove unwanted cell areas and retain The required cell area is as follows. The culture flask of the cell to be purified is operated under the supervision of an inverted microscope in a clean room.
Use a silicone rubber scraper to delineate areas of the cells that do not need to grow, so that the cells are suspended in the medium, taking care not to damage the desired cells.
After the deduction, rinse with the medium and shake it twice, then the medium can be added to the original bottle to continue the cultivation.
After a few days, if it is found that the unwanted cells grow again, the above operation can be carried out, so that the cells can be purified repeatedly several times. The operation should be strictly aseptically operated to prevent pollution.
4, 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 can not be attached or unstable, and it can be floated with a little oscillation. The difference can be used to purify 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.
Under the inverted microscope, see some cells attached to the wall, and when the shake is not raised, the cell suspension is 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.
5. Ironing 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 transforming cells.
Pour off the old solution and use the marker to draw out the area of ​​the foci.
Using a heated micro-electrode (similar to a soldering iron for soldering), the cells around the foci are all scalded, leaving only the foci cells. In the single-cell cloning screening, cells around the individual cells can also be killed by this method. .
Then continue to culture in an adaptive medium (50% is old) to achieve purification

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