In the field of in vitro diagnostics (IVD) experiments, cell culture is a crucial foundational technology. Due to their unique physiological characteristics, different types of cells require appropriate cultivation methods to ensure cell growth and provide reliable research subjects for experiments. Below, we will provide a detailed introduction to several common cell culture methods.

 

(HEPES biological buffer)

 

 

1、 Primary cell culture method

 

Primary culture is the initial cultivation of tissues or cells directly obtained from the body. Due to its short ex vivo time and close biological characteristics to its in vivo state, it can truly reflect the situation in the body to a certain extent. Taking obtaining liver cells from liver tissue as an example, the main methods for obtaining primary cells are tissue block method and enzyme digestion method. First, cut the liver into small pieces and further separate them with tweezers and scissors. Then, add trypsin and digest it in a 37 ℃ water bath to promote the separation of liver cells. The operation process needs to be strictly maintained in a sterile state. The instruments used, such as scissors and tweezers, need to be burned with an alcohol lamp before use, and the movements should be gentle to prevent damage to the cells. The digestion time should not be too long to avoid excessive digestion and damage to the cell structure. Primary cells have high requirements for the culture medium.

 

The commonly used basic culture media are DMEM and RPMI-1640. DMEM is nutrient rich and suitable for metabolically active cells, such as fibroblasts; RPMI-1640 is relatively mild and suitable for sensitive cells such as lymphocytes. In addition, it is necessary to add 10-20% fetal bovine serum, which is rich in growth factors and essential trace elements that can promote cell growth.

 

In terms of gas environment and temperature, the incubator generally maintains a gas environment of 5% CO ₂ and 95% air, with a constant temperature of 37 ℃. CO ₂ reacts with water in the culture medium to generate carbonic acid, thereby maintaining the pH of the culture medium at 7.2-7.4, which is the ideal pH range for cell growth. pH values that are too high or too low can inhibit cell growth.

 

2、 Passage cell culture method

 

Passage cells are cell lines formed by primary cells after passage culture, which have stable growth and are relatively easy to cultivate. The passaging methods are divided into suspended growth cell passaging, semi suspended growth cell passaging, and adherent growth cell passaging based on cell growth characteristics.

 

3、 Suspension growth cell passage

 

Suspension growth cells are often passaged by centrifugation. After centrifuging at a speed of 1000 revolutions per minute for 20-30 seconds, remove the supernatant and add the precipitated cells to the new culture medium. Mix well to complete passage. Direct passage method can also be used. When the suspended cells precipitate on the bottle wall, 1/2-1/3 of the supernatant culture medium is removed, and then the cell suspension is directly blown with a pipette for passage.

 

4、 Semi suspended growth cell passage

 

For semi suspended cells, such as Hela cells, some cells exhibit adherent growth, but the adhesion is not firm. The direct blowing method can be used to detach the cells from the bottle wall and proceed with passaging operations.

 

(Biological buffer PIPES)

 

5、 Cell passaging with adherent growth

 

Cells growing on the wall are usually passaged using enzymatic digestion. 0.25% trypsin solution is commonly used as digestive fluid. After adding trypsin EDTA solution, let it sit at 37 ° C for a few minutes and observe it under an inverted microscope. When the cells are about to separate and appear round, remove the trypsin EDTA solution. Then add an appropriate amount of fresh culture medium containing serum to terminate the action of trypsin, centrifuge and aspirate the supernatant. Gently tap the culture bottle to remove the cells from the bottle wall, then add an appropriate amount of fresh culture medium. Use a pipette to suck up and down several times to break up the cell clusters. After mixing evenly, transfer to a new culture bottle according to the dilution ratio and continue to culture under normal conditions.

 

6、 Stem cell culture methods

 

Stem cells are mainly divided into embryonic stem cells and adult stem cells. Embryonic stem cells can differentiate into various cells derived from the three germ layers, such as liver cells from the endoderm, myocardial cells from the mesoderm, and nerve cells from the ectoderm. Adult stem cells have the potential for multi-directional differentiation. Taking mesenchymal stem cells as an example, they can differentiate into various cell types such as osteoblasts, chondrocytes, and adipocytes. Maintaining the stemness of stem cells during the cultivation process is crucial. Once stemness is lost, stem cells cannot proliferate and differentiate normally. At the same time, the differentiation direction and degree of stem cells are controllable. By adding different inducing factors, stem cells can be guided to differentiate into specific cell types to meet different experimental needs.

 

Mastering different cell culture methods is the foundation for conducting research in IVD experiments. Whether it is primary cells, passaged cells, or stem cells, only by providing appropriate culture conditions can cells survive in an in vitro environment, providing favorable assistance for the development of medical research and in vitro diagnostic technology. To maintain the growth environment of cell culture medium, buffering agents are used to stabilize it. As a manufacturer of biological buffering agents, Hubei Xindesheng's supplied products have a purity of over 99% and excellent buffering performance. If you have any purchasing needs, please feel free to contact us at any time!