Experimental technique: cell culture medium technology

Cell culture

First, the operating procedures:

1. Before the experiment, sterile rooms and sterile console irradiated with UV light for 30-60 minutes sterilization, aseptic table wiping with 70% alcohol, and after 10 minutes sterile console operation of the fan is turned on, before starting the experiment operation of each operation processing only one cell, the cell to avoid cross-contamination. after the experiment, the test article with a stage. The need to continue such a test, then with 70% alcohol wipe table aseptic, sterile console let the operation of the fan 10 minutes, before the next test operation.

2. The aseptic work area should be kept clean and spacious, essential items, such as test tube racks, pipette or pipette can be placed temporarily top, other experimental articles should be removed after use, to facilitate gas flow. Lab Supplies use 70 After the alcohol is wiped, it can be taken into the aseptic table. The experimental procedure should be carried out in the sterile area in the center of the station , not in the non-sterile area at the edge.

3. Carefully remove the sterile laboratory supplies to avoid contamination. Do not touch the suction tube and the tip of the tip or the mouth of the container . Do not operate the experiment directly above the open container. After the container is opened , hold the bottle cap by hand and hold the bottle body , and use it at an angle of about 45° . Try not to put the cap on the table face up.

4. The staff should pay attention to their own safety . They must wear the lab coat and gloves before conducting the experiment. Care must be taken to the cell lines derived from human or viral infection, and select the appropriate level of sterile console (at least two). During operation, should avoid the generation of aerosol, carefully toxic reagents, such as DMSO and the like TPA, and to avoid sharp objects wounding.

The periodic inspection of the following items: CO ₂ in the CO ₂ pressure cylinder; CO CO ₂ concentration, temperature, and water in the pan ₂ incubator for contamination; sterile console stream pressure is normal, periodic replacement UV lamp And HEPA filter membrane , pre-filter ( 300 hours / pre-filter , 3000 small / HEPA) .

Second, the preparation of powder cell culture medium ( taking 1 liter as an example ) :

    The cell culture medium usually requires 10 % serum, so the powder medium has a formulation volume of 900 ml and a pH of 7.2 - 7.4 . NaHCO _{3 is} additionally added, and if the NaHCO ₃ powder is directly added to the liquid medium, pH error or local overbased may occur. Therefore, the powder medium and the NaHCO ₃ powder should be dissolved separately before mixing, and then the pH is adjusted with CO ₂ gas instead of using strong acid (HCl) or strong alkali (NaOH) because chloride ions may have an effect on cell growth and when stored. The pH of the medium is subject to change.

[Reagents and equipment]

1. Pure water (milli-Q water or two to three times distilled water, water quality is very important )

2. Powder medium ( 1640 , DMEM, etc.)
3. NaHCO ₃ (Sigma S-4019)
4. Electromagnetic stirrer
5. Sterile serum bottle
6. 0.22 Micron sterile filtration membrane
7. pH meter
8. Vacuum valve
9. CO ₂ gas
[ Preparation steps]
1. Dissolve the powder medium in 700 ml milli-Q water and stir to dissolve.
2. Weigh an appropriate amount of NaHCO ₃ powder (the amount varies depending on the type of the medium ), dissolve it in 200 ml of milli-Q water, stir to dissolve it, and then pass CO ₂ gas to saturation for about 3-5 minutes.
3. Mix the dissolved and saturated CO ₂ NaHCO ₃ solution into the dissolved liquid medium. The pH of the mixed solution should be 7.2-7.4 . Unless the pH deviation is too large, it is not necessary to adjust it with acid or alkali. If it is too alkaline, it can be adjusted to pH by introducing CO ₂ gas. When the medium is passed through the membrane with a vacuum, the pH will increase by 0.1-0.2 .
4. to 0.1 or 0.2 mm The sterile filter membrane is sterilized by filtration and dispensed into a sterile container, and the type, date, bottle number, etc. of the medium are indicated, and stored at 4 ° C (the serum can also be added to the medium for filtration ) .
5. The prepared medium is prepared for growth test and pollution test.
【Precautions】

1. The liquid medium was stored in a refrigerator at 4 °C to avoid light, and it was warmed in a 37 °C water tank before the experiment.

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