The culture conditions of cell culture, the basic operation of cell culture

There is a special accurate detection equipment for detecting oxygen and carbon dioxide in the cell culture process, refer to ** - Shanghai Yongzhou Experimental Equipment****.

Recently, the author has been culturing human umbilical vein endothelial cells (HUVECs) to record some basic operations of Zhiqiao in cell culture.

Take out the cell culture flask from the incubator with 5% CO2 at 37°C, first observe whether the liquid in the flask is cloudy (turbidity indicates that the cells may be contaminated), then observe the growth of the cells under the microscope, and when the cells are more than 70% long, they can be passaged.

Add 3ml PBS to the 25ml culture flask and wash it twice, then add trypsin (cactus pancreatic enzyme used in the laboratory is more warm and silvery), put it in the incubator and digest for 3-5min (to ensure that the cells are digested), and add 2ml of culture medium (ECM) to stop the digestion. Collect the liquid into a centrifuge tube, centrifuge at 350g for 5min (so that the cells can be precipitated), then remove the supernatant, add 6ml ECM to the centrifuge tube, blow the cells well (blow into single cells), add 2ml to each of the three 25ml flasks, and then add the medium to 6ml in the flask.

After the cells in the 25 ml flask are full, wash twice with 3 ml PBS, centrifuge after trypsinization, discard the supernatant, add medium and 500 L DMSO (10%), blow the cells well, and aliquot into 5 cryovils, 1 ml each, and put in -80 cryopreservation.

After the cryopreserved cells are taken out from -80 or liquid nitrogen, immediately put them in warm water at 37 °C (pay attention to whether liquid nitrogen leaks into the cryopreservation tube), after thawing, centrifuge at 350g for 5min, discard the supernatant, add 1ml ECM, blow the cells well, add 25ml culture flask, and make up ECM to 6ml.

Note: a. The lid of the culture flask should be passed through the alcohol lamp, and the cap of the culture flask should not be tightened;

Two. The 75ml flask is a 10ml culture system, plus trypsinization, and 4 times the amount of medium to terminate digestion; The 6-well plate is a culture system (trypsinized), the 12-well plate is a 2 ml culture system (trypsinized), the 24-well plate is a 1 ml culture system (trypsinized), and the 96-well plate is a 100 L culture system (1 drop trypsinized). After adding trypsin, shake the flask or culture plate slightly so that the trypsin can cover the adherent cells;

Three. The amount of PBS used for cleaning is half the amount of medium;

Four. The 25 ml flask is full of cells and there are about one cell;

Five. Huvec is no longer available after the 4th generation.

Cell culture aims and uses:

1. Scientific research.

Drug research and development, such as new drug screening, vaccines, genetic engineering drugs, cell engineering drugs, research and development, monoclonal antibody production, etc. Basic research, such as the mechanism of drug action, gene function, disease pathogenesis, etc.

2. Biopharmaceutical.

Vaccine production: such as viral vaccines (hepatitis virus vaccines, AIDS vaccines, etc.), peptide vaccines (tumor vaccines), etc. Production of genetically engineered drugs: such as EPO, etc. Production of antibody drugs and gene drugs.

Cell engineering drug production: some bioactive polypeptides, bioactive substances, etc. in biological cells. In vitro determination of the activity of bioactive substances by cell method; And its efficacy in vivo and alternative in vivo methods to detect the biological activity of its finished products.

Cell culture purpose and use, mainly for:

Related experiments, taking primary cells as an example, are not only widely used in basic research on molecular, cell biology and biological, such as proteomics, genomics, cell line (line) research, DNA, RNA and genetics research, but also can be applied to today's popular biomedical industries such as drug screening, drug metabolism and toxicology research, cancer drug research, etc.

Hope it helps!

Cell culture is first divided into primary culture and subculture.

Primary culture requires digestion, isolation, and purification of single cells from tissues, and then proceeding with passage and cryopreservation.

Subculture first:

Cell recovery:1The principle of slow freezing and fast thawing should be observed. First, adjust the water temperature to 37-38 degrees, take out the cryopreserved cells, quickly put them into the posterior cell surface, immerse them below the water surface, and shake continuously until they melt.

2.Add the complete medium to the culture flask in the sterile table, then take out the cells from the cryopreservation tube with a sterile pipette, shake gently in the culture flask, and make the cells evenly cultured in the incubator, and change the liquid after 24 hours; It can also be revived at the time of centrifugation (about 5min at 1000 rpm), complete medium resuspension culture, timely change.

Cell passaging:1Adherent cells:

For adherent cells, the medium should be aspirated (poured down) first, and the cleaner the sucking, the better, so as not to neutralize the digestive juice added after it, so as to weaken the strength. PBS wash 2-3 times, remove serum and dead cells, add 50ml flask to the digestive solution about, digest according to this ratio, (according to the preparation strength experience), shake to make the digestive solution evenly spread in the 37 degree incubator for about 2 minutes, after the cells shrink and become round or a few fall off, gently vibrate the bottom of the bottle to make the cells all deswim and fall, add 2-3ml complete medium, gently pipette to make the cells basically into a single suspension, and then collect centrifugation (1000rpm for about 5min), Resuspend the pellet with fresh medium, add complete medium and continue the culture or experiment.

2.Suspension cells:

If you want high concentration or need to change the new medium, you can centrifuge (1000rpm, about 5min) and then add the complete medium, gently blow well, and then add complete medium to other culture flasks to continue the culture.

For more details, you should read the old grinding pants and read the professional books on cell culture!

The basic methods of cell culture are adherent culture, suspension culture and immobilized culture. Adherent culture is mainly suitable for adherent-dependent cells. These need to grow, survive, and maintain on the surface of chemically inactive substances (inactive substances such as glass or plastic) that are interdependent, and eventually grow to a single layer on the adhesion surface, where contact inhibition occurs when the surface is covered with a flat surface.

**Cells in blood, lymphoid tissues, and many tumor cells (including hybridoma cells) and some transformed cells belong to suspension culture cells, cells are non-adherent-dependent cells, no support surface, cells or cell aggregates are suspended in liquid medium for proliferation. Suspension culture is ideal for large-scale cell culture and can be performed in a microbial-like manner. Animal cells are more fragile than microorganisms and are susceptible to shear forces, while cell immobilization technology can better protect cells from mechanical forces and environmental changes, improve cell tolerance, promote high-density cell culture, and improve the yield of target products.

Phyto-finch cell culture needs ().

a.Serozoar.

b.Inorganic salts and trace elements.

Carbohydrate sock shaft.

Correct answer: B