1. Culture environment requirements for different cells
The requirements of cells for culture environment vary depending on the cell type, which is reflected not only in the difference of culture medium, but also in the difference of growth space density.
Some cells grow more easily and perform better at higher density, and these cells usually grow slower, such as endothelial cells. Other cells perform better at low density, such as macrophages and some tumor cells.
Macrophages in particular grow and adhere very quickly, so fewer cells need to be retained during passaging to maintain good conditions.
Macrophages tend to grow in aggregates, but a certain amount of space needs to be retained between different cell piles. If the cells are too dense, the number of aging cells will increase, which will affect the results of subsequent experiments.
Therefore, when culturing cells, it is necessary to explore the optimal growth density of each cell type.
2. Strategies for dealing with poor cell morphology
For adherent cells, if you find that the cell morphology is poor or there seems to be foreign matter on the surface, you can try the following operations when passaging:
First, pour out the old culture medium, add 3 ml of new culture medium (with or without serum) to wash, and then aspirate it away.
Next, add 3 ml of culture medium for pre-blowing, gently blow along the bottom of the bottle once, and then suck it away.
After this, formal digestion and blowing operations are carried out. Macrophages only need to be blown, no digestion is required.
Next, transfer the blown cell suspension to a new culture bottle with culture medium added in advance.
Place the culture bottle in the incubator and regularly observe the cell adhesion, checking once every 10 minutes, 20 minutes, and 30 minutes.
When some cells are attached to the wall, quickly pour out the culture medium, add 3 ml of new culture medium, and gently wash again.
Then, add complete culture medium to continue culturing and observe the cell growth status and morphology.
This method is called "secondary passage".
If the effect is not ideal once, you can repeat this operation many times until you find the best morphology of the cells. You need to choose the appropriate passage time according to the growth habits of the cells. For cells that like to grow at high density, you can wait until more cells are attached before passage, and vice versa. This method is very effective.
3. The amount of culture medium
The amount of culture medium needs to be explored according to the specific cell type. It is not necessary to add culture medium according to the standard of 12 ml or 14 ml in all cases. Some cells perform better in a small amount of culture medium. For cells that grow rapidly and are easy to culture, a small amount of culture medium usually makes the cell morphology better, but special attention needs to be paid when changing the medium.
4. How to choose culture medium?
Classic or basal culture medium is usually a synthetic culture medium, which is based on natural culture medium components and simulated by using chemical substances. It contains amino acids, carbohydrates, inorganic salts, vitamins and other auxiliary components. The following are some commonly used culture media and their applicable cell types:
RPMI-1640: It is widely used in the culture of mammals, special hematopoietic cells, normal or malignant proliferating white blood cells, and hybridoma cells. It is currently a very widely used culture medium. It is mainly used for suspension cell culture. Others such as K-562, HL-60, Jurkat, Daudi, IM-9 and other lymphoblasts, T cell lymphoma cells and HCT-15 epithelial cells can be used for reference.
Genom Bio-RPMI1640 culture medium
MEM：It contains only 12 essential amino acids, glutamine and 8 vitamins. It has simple ingredients and can be widely adapted to the culture of various established cell lines and mammalian cell types from different places.
Genom Bio-MEM culture medium
DMEM-High sugar: It is a widely used culture medium that can be used for many mammalian cell cultures and is more suitable for high-density suspension cell culture. It is suitable for cloning cells with poor adhesion but do not want them to be separated from the original growth point. It can also be used for the culture of myeloma cells in hybridomas and transformed cells transfected with DNA.
Genom Bio-DMEM high glucose culture medium
DMEM-Low sugar: It is a widely used culture medium that can be used for many mammalian cell cultures. Low sugar is suitable for dependent adherent cell culture, especially for tumor cell culture with fast growth rate and poor adhesion.
Genom Bio-DMEM low-glucose culture medium
DMEM/F12（1:1）：DMEM/F12 The culture medium is suitable for clonal density culture. F12 culture medium is complex in composition and contains a variety of trace elements. It is combined with DMEM in a 1:1 ratio, called DMEM/F12 culture medium, as the basis for the development of serum-free formulas, taking advantage of the richer ingredients in F12 and the higher concentration of nutrients in DMEM. This culture medium is suitable for mammalian cell culture under conditions of low serum content. In order to enhance the buffering capacity of this culture medium, one of the improvements is to add 15mM HEPES buffer to DMEM/F12 (1:1).
Genom Bio-DMEM/F12 (1:1) culture medium
α-MEM：It is generally used to culture some difficult-to-culture cell types, while other cell lines without any special features can almost all be cultured using MEM.
Genom Bio-α-MEM culture medium
McCoy’s 5A：It is mainly designed for the culture of sarcoma cells and can support the growth of various primary transplants (such as bone marrow, skin, lung and spleen, etc.). In addition to being suitable for general primary cell culture, it is mainly used for tissue biopsy culture, some lymphocyte culture and the growth support of some difficult-to-culture cells, such as Jensen rat sarcoma fibroblasts, human lymphocytes, HT-29, BHL-100 and other epithelial cells.
Genom Bio-McCoy’s 5A culture medium
William’s Medium E：Suitable for long-term culture of rat liver epithelial cells.
Genom Bio-William’s Medium E culture medium
The above are some of the culture media products of Jinuo Biology. For more product details, please follow the "Jinuo Biology" official account~
5. Selection of culture bottles
According to personal experience, cells with faster growth rates tend to grow better in glass bottles than in disposable plastic bottles. The same type of cells also grow better in glass bottles during the vigorous growth period.
This may be because plastic bottles are easier to adhere to the wall than glass bottles, and cells with faster growth rates perform worse than glass bottles in the more comfortable environment of plastic bottles.
Therefore, if you want to obtain cells in better condition, you can choose plastic bottles for cells with slower growth rates, while glass bottles may be more suitable for cells with faster growth rates or cells in the vigorous growth period.
6. Digestion operation during subculturing
The digestion process during subculturing is crucial to the survival and state of cells. Many experimenters have found that cells grow well in the first few generations, but after several subculturings, the cell state begins to decline.
This is usually due to significant damage to the cells during the digestion process. During each digestion, all cells are acted upon by pancreatic enzymes, but the growth state and adhesion of each cell are different, so the same digestion time is unfair to all cells.
To solve this problem, you can try the "four-step digestion method":
Without adding pancreatic enzymes, pour out the old culture medium, add a small amount of new culture medium to wash 1-2 times to remove floating dead cells and other impurities in preparation for digestion.
Add a small amount of new culture medium to blow and blow down the cells that are not firmly attached to the wall, and then wash it again with culture medium. The resulting suspension can be mixed and subcultured to a new bottle for culture, and compared with the cells digested later.
After removing the remaining liquid in the bottle, add a small amount of pancreatic enzymes to rinse once, and then add a small amount of pancreatic enzymes for digestion, and observe under a microscope at the same time.
When there are obvious gaps between cells, suck out the pancreatic enzymes, add new culture medium to blow and blow, and then subculture to a new bottle for culture. This step can be performed multiple times according to actual conditions.
For cells that are particularly difficult to digest, they can be digested in batches multiple times to avoid excessive damage to cells by pancreatic enzymes and ensure the best state of cells.
For cells that are easy to digest, simple operations may be sufficient, but for cells that are difficult to digest, special attention should be paid to the details of the digestion process.
7. Precautions for changing medium, subculturing and washing
For cells using DMEM culture medium, if serum-free 1640 is used for washing when changing medium, the subsequent culture effect is usually better than that of direct washing with DMEM. Similarly, cells cultured with 1640 have similar performance.
If you don't mind the trouble, you can choose PBS for washing, which is equivalent to serum-free medium, and sometimes even better. The purpose of washing is to remove the residual serum in the culture bottle to prevent it from affecting the digestion effect of trypsin and ensure the optimal activity of trypsin.
After washing with PBS, you can try to let the cells stand for 10-15 minutes, wait for the cells to gradually separate, and then blow them. This method is suitable for some cells that are difficult to digest or when there is insufficient trypsin, but you need to test whether it is suitable for your cells first.
| Part Number | Product Name | Specification |
|---|---|---|
| GNM31800-5 | RPMI1640培养液 | 500ml |
| GNM41500-5 | MEM培养液 | 500ml |
| GNM12800-5 | DMEM高糖培养液 | 500ml |
| GNM31600-5 | DMEM低糖培养液 | 500ml |
| GNM12500-5 | DMEM/F12培养液(1:1) | 500ml |
| GNM11900-5 | α-MEM培养液 | 500ml |
| GNM16600-5 | McCoy's 5A培养液 | 500ml |
| GNM41250-5 | William's Medium E | 500ml |
| GNM15050-1 | 0.25%胰酶 含酚红 | 100ml |
| GNM10010-5 | PBS缓冲液 PH7.4 | 500ml |