How many scientific researchers have been frustrated because of the poor state of cell culture! How many laboratories have ever had to terminate their carefully developed long-term experiments due to microbial contamination! So how should cells be cultured to keep them in a normal state? How can we avoid contamination of cultured cells?
Cell culture is also a basic technology for virus research and vaccine development. Therefore, cell culture technology has been widely used in genetics, immunology, oncology, virology, molecular biology and other fields. Nuclear transfer, cell hybridization, DNA-mediated gene transfer and the establishment of some physical maps developed in recent years also need to be closely integrated with cell culture.
Below, Abbkine cell culture method gives you the answer.
The general process of cell culture mainly includes the following points:
1. Preparation: The preparation includes the cleaning, drying and disinfection of utensils, the preparation, distribution and sterilization of culture media and other reagents, and the preparation of sterile rooms or ultra-clean benches. Cleaning and disinfection, inspection and debugging of incubators and other instruments.
2. Extracting materials: Take out a certain tissue cell from the body in a sterile environment (depending on the purpose of the experiment), and put it into a culture vessel after a certain treatment (such as digestion and dispersion of cells, separation, etc.). This process is called sampling . In the case of cell line expansion, there is no such process as obtaining materials. The first culture of tissue cells taken out of the body is called primary culture.
3. Cultivation: The process of putting the obtained tissue cells into a culture flask or culture plate is called culture. For tissue block culture, connect the tissue block directly to the bottom of the culture vessel. After a few hours, the tissue block can be attached to the bottom, and then add the culture medium.
4. Cryopreservation and resuscitation: In order to preserve cells, especially mutant cells or cell strains that are not easily available, the cells should be frozen. The freezing temperature is generally -196℃ of liquid nitrogen. Collect the cells into the cryopreservation tube and add a medium containing a protective agent (usually dimethyl sulfoxide or glycerol), freeze at a certain cooling rate, and finally save In liquid nitrogen. At extremely low temperatures, the storage time of cells is almost unlimited. The resuscitation generally adopts a quick-thaw method, that is, after removing the cryotube from the liquid nitrogen, immediately put it in 37°C water to make it melt quickly within one minute. Then transfer the cells to a culture vessel for culture.
The basic steps of cell primary culture:
Primary separation cell culture refers to the separation of tissues from the donor body into single cells or monotype cell groups by mechanical and digestion, so that they can imitate the physiological environment of the human body in vitro. Under temperature and certain nutritional conditions, it can survive, grow and reproduce. Primary cultured cells often have different cell components and grow slowly, but they are more representative of the tissue cell type from which they are derived and the specific characteristics of the expression tissue. Using primary cell culture to do various experiments, such as drug testing, cell differentiation and virology, has a good effect. The operation steps are as follows:
1. Cut the tissue: first clean the obtained tissue with D-Hanks or Hanks solution to remove blood stains on the surface, and use surgical tweezers to remove attached connective tissue and other non-cultivating tissues. After cleaning again, use a scalpel to cut the tissue into several small pieces, transfer them into a penicillin vial or a small beaker, add an appropriate amount of buffer, and use elbow ophthalmic scissors to repeatedly cut the tissue until the tissue becomes a paste, about 1mm3 in size. After standing for a while, use a straw to suck off the upper layer of liquid, add appropriate buffer solution and wash again.
2. Digestion and separation: The purpose of digestion and separation is to digest and separate small tissue pieces into cell clusters or scattered single cells to facilitate further culture. Commonly used digestive enzymes are trypsin and collagenase.
3. Cultivation: Cell suspension is counted with a counting plate. Adjust the cell number to (2～5)×105 cells/ml with the culture medium, or the density required for the experiment, and divide it into the culture flask, so that the amount of cell suspension is slightly higher than the bottom of the culture flask after covering. Place in a CO2 incubator, 5% CO2, 37°C static culture. Generally 3~5d, the primary cultured cells can adhere to the wall of the bottle and stretch and start to grow. You can add 1/2 of the original culture medium to the new medium, continue to culture for 2~3d and then change the medium, generally 7~14d can be longer Fill the wall of the bottle and pass it down.
Specific steps of subculture:
Note
1. Digestion time: Master the time for cell digestion. When the digestion time is too short, the cells should not fall off the bottle wall. Excessive digestion will cause the cells to fall off and damage.
2. Digestion concentration: Master the digestion concentration. When the digestion solution concentration is too high, the digestion time should be shortened, and when the digestion solution concentration is too low, the cell digestion time will be relatively prolonged.
The meaning
of cell cryopreservation is to store cells in a low temperature environment (liquid nitrogen) to reduce cell metabolism for long-term storage. Cryopreservation of cells is one of the main methods of cell preservation, which plays a role in cell preservation.
Significance of
cell resuscitation Cell resuscitation, a term in biology, refers to the process of re-cultivating cells frozen in liquid nitrogen or in a refrigerator at -70°C to restore growth.
Cell culture link:
http://www.abbkine.cn/avoid-contamination-in-cell-culture