Practical Mammal Cell Culture Manual (below)

Cleaning and disinfection of glass and plastic products used in cell culture

Cleaning

In tissue cell culture, cells in vitro are very sensitive to any harmful substances. Microbial products are accompanied by sundries, and the last cell residues and non-nutrient chemicals can affect the growth of cultured cells. Therefore, the new glassware and the reused culture vessels should be thoroughly and thoroughly cleaned, and different cleaning methods should be selected according to the composition of the vessels.

Cleaning of glassware

In tissue culture, the largest amount is glassware, so the most important thing is the cleaning of glassware. Cleaning of general glassware includes four steps of soaking, brushing, pickling and rinsing. The cleaned glassware not only requires clean, clear, oil-free, but also does not leave any material.

(1) Soaking: The glassware after initial use and culture should be soaked with water first to soften the deposit or be dissolved by the solution. The new glassware used for the first time has a large amount of dry dust on the surface of the glass during production and transportation, and the surface of the glass is often alkaline and has some harmful substances to the cells. The new bottle should be simply brushed with tap water before use and then soaked in dilute hydrochloric acid overnight to neutralize the alkaline material. Reusable glassware is often accompanied by a large amount of freshly used protein. It is not easy to be washed off after drying. Therefore, it should be immersed in water immediately after use, and it is required to be completely immersed, and no air bubbles or floating on the liquid surface.

(2) Brushing: The glassware after soaking should be brushed with a brush and detergent to remove the relatively tight impurities on the surface of the vessel. Brushing should be moderate and excessive will damage the surface gloss of the vessel.

(3) Pickling: The cleaning solution is prepared by a certain proportion of potassium dichromate, concentrated sulfuric acid and distilled water, and the treatment process is called pickling. The cleaning solution has no corrosive effect on the glassware, and its strong oxidation can remove trace impurities that cannot be washed away. The cleaning solution has a strong decontamination ability. It is a key part of the cleaning process. When soaking, the vessel should be filled with cleaning liquid. Do not leave air bubbles or utensils to expose the cleaning liquid. The soaking time is generally overnight and should not be less than 6 hours. The cleaning solution can be formulated into different strengths according to requirements. The following three types are commonly used: potassium dichromate (g) concentrated sulfuric acid (ml) distilled water (ml) (A) strong cleaning liquid 63:1000:200000 (B) strong Washing solution 120:200:1000 (C) weak cleaning solution 100:100:100.

Care should be taken when preparing the cleaning solution. Wear acid-resistant gloves and aprons, and protect the exposed parts of the face and body. Potassium dichromate can be dissolved in water during the preparation process, and then concentrated sulfuric acid is slowly added. Stirring with a glass rod is carried out to volatilize the generated heat. Potassium dichromate can be dissolved in water during the preparation process, and then concentrated sulfuric acid is slowly added. Stirring with a glass rod, the generated heat is volatilized, and the preparation solution should be selected from plastic products. After the preparation, the cleaning liquid is generally brownish red.

(4) Flushing: After use, the glassware must be thoroughly rinsed with water after brushing and soaking. Keep it as free as possible from any contamination or cleanliness. It is preferred to use a washing device for rinsing. That is to save effort and good results. If it is operated by hand, it needs to be rinsed more than ten times. The water should be filled and cleaned every day. It is best to wash it 3-5 times with distilled water and dry it for later use.

Rubber plug cleaning

The rubber products used in cell culture are mainly stoppers. The newly purchased cork has a large amount of talcum powder and impurities. It should be washed first with tap water and then routinely. The conventional cleaning method is: soak it in water immediately after each use, then boil it with 2% NaOH or washing powder 10- 20 minutes to remove the protein from the culture. Rinse with tap water, soak it in 1% dilute hydrochloric acid for 30 minutes or rinse with distilled water, then boil for 10-20 minutes, and let it dry for use.

Cleaning of plastic products Plastic products are mostly non-toxic and specially processed packaging, which can be used to open the package, mostly disposable items. If necessary, soak overnight with 2% NaOH, rinse thoroughly with tap water, soak for 30 minutes with 5% hydrochloric acid solution, rinse with tap water and distilled water, and dry for use.

disinfection

The greatest risk of cell culture is the contamination of the culture with bacteria, fungi and viruses. The pollution is mainly caused by the operator's negligence. Common causes are uncleanness in the operation room or surrounding space, and the culture vessels and culture fluids are not qualified or incompletely disinfected. Since every mistake in the culture can lead to failure of culture, every step of cell culture should strictly abide by the operating rules to prevent pollution. There are three types of disinfection methods: physical sterilization (UV, damp heat, dry roasting, filtration, etc.), chemical sterilization (various chemical disinfectants) and antibiotics.

(1) UV disinfection: used for air, surface of the console and cannot be disinfected and cultured using other methods. Direct ultraviolet radiation is convenient and effective. After a certain period of time, most of the bacteria in the air can be eliminated. The ultraviolet light in the culture room should not exceed 2.5 meters from the ground, and the disinfected items should not be shielded from each other. Less than disinfection. Ultraviolet rays can produce ozone, pollute the air, and the reagents and culture fluids have adverse effects. They are also harmful to human skin and should not be irradiated.

(2) Warm disinfection: high pressure steam disinfection is the most widely used and best disinfection method. When warming and disinfecting, the disinfectant items should not be overfilled to prevent gas clogging in the sterilizer and millions of dangers, ensuring the circulation of gas inside. Before heating and boosting, first open the exhaust air to discharge the cold air in the sterilizer. After the cold air is exhausted, close the exhaust valve and check that the safety valve is free to move, then start to increase the pressure. When the required pressure is reached, Start to calculate the disinfection time. During the disinfection process, the operator cannot leave the job, and should regularly check the pressure and safety to prevent disinfection and epidermal accidents.

Disinfection pressure and time of common items:

Culture medium, balanced salt solution and other liquids requiring sterilization: 121 ° C, 15 lbs, 20 minutes;

Cloth, glassware, metal equipment, etc.: 121 ° C, 15 lbs, 20 minutes, then dried in an oven;

Glass bottles: dry heat sterilization 170 ° C, 4 hours.

(3) Chemical disinfection method: The most common is 70% alcohol and 1 ‰ clean and dry, the former is mainly used for the operator's skin, the surface of the console and the wall of the sterile room. The latter is mainly disinfected by soaking of the instrument and wiping the skin of the skin and the operating room. The chemical disinfection method is simple, convenient and effective.

(4) Antibiotic disinfection: It is mainly used for sterilizing culture liquid or preventing culture pollution.

Common items for cell culture

Cell culture medium

Currently, dry powder media and liquid media are available on the market. The dry powder medium needs to be prepared and sterilized by the user, which has the advantage of being inexpensive. The disadvantage is that the preparation process is cumbersome and the quality is not easy to control. The liquid medium is produced by professional producers according to the standard scale, not only the quality is guaranteed, but also the use is very convenient.

Serum (omitted)

Balanced salt liquid

PBS (Phosphate-Buffered Sallines)

DPBS (Dulbecco's Phosphate-Buffered Sallines) standard type

Hanks' balanced salt solution ( Hanks'   B alanced Salt Solutions , HBSS )

D-Hanks' balanced salt solution (D-Hanks Balanced Salt Solutions, D-HBSS)

Digestive juice:

Isolation of tissue and dispersion of cells. Commonly used are trypsin and disodium edetate (EDTA) solutions. They can be used alone or in combination.

(1) Trypsin solution

Trypsin (referred to as pancreatic enzyme) is a yellow-white powder that is easily deliquescent and should be stored in a cool dark dry place. The currently applied trypsin is mainly derived from the pancreas of cattle or pigs. The main function of trypsin is to hydrolyze proteins between cells to make cells separate from each other. The separation of pancreatic enzymes from cells is closely related to the type of cells and the characteristics of the cells. In general, the concentration of pancreatic enzyme is large, the action temperature is high, the action time is long, and the ability to separate cells is also large, but the cells are damaged beyond a certain limit. The trypsin solution has the strongest action at pH 8.0 and a temperature of 37 °C. Note: The presence of calcium, magnesium and serum proteins reduces trypsin activity. Therefore, the trypsin solution is usually prepared as a 0.25% solution by using a Ca-free, Mg2+-free D-Hanks balanced salt solution. When digesting cells, adding some serum or serum-containing medium, or trypsin inhibitor can stop the digestion of trypsin.

Preparation of trypsin solution:

(1) Weigh the trypsin powder into a beaker, first adjust the salt solution with a little D-Hanks balanced salt solution (about pH 7.2), then make up the D-Hanks balanced salt solution, stir and mix, and set at room temperature for 4 hours. Or overnight in the refrigerator and stirring constantly;

(2) The next day, the filter paper is firstly filtered, then filtered and sterilized, and placed in a bottle, and stored in a low-temperature refrigerator for use. The commonly used concentration is 0.25% or 0.125%. The trypsin solution is slightly acidic, and the sodium bicarbonate solution can be called to adjust the pH to about 7.2 before use.

Storage conditions: The prepared trypsin solution must be stored in a -20 ° C refrigerator to avoid decomposition failure.

EDTA•4Na solution

EDTA is a chemical chelating agent that has a certain dissociation effect on cells, and is low in toxicity, low in price and convenient to use. The common working fluid concentration is 0.02%. Usually used in a 1:1 mixture with a 0.25% trypsin solution (mixture).

Note: After using EDTA to treat cells, be sure to rinse them off with Hanks solution, as residual EDTA will affect

Cell growth.

Preparation of EDTA solution: Dissolve in D-HBSS balanced salt solution without calcium and magnesium, autoclave in a high pressure steam, dispense into small vials, and store at room temperature or 4 °C in the refrigerator.

Trypsin-EDTA•4Na solution (0.05% trypsin, 0.53 mM EDTA•4Na)

0.5 g trypsin + 0.2 g EDTA•4Na+1L D-HBSS. Store at -20 ° C in the refrigerator.

pH adjustment solution

(1) NaHCO3 solution

The usual concentration is 7.5%. Dissolve in three steamed waters after preparation, filter and sterilize, dispense, store at 4 ° C refrigerator or at room temperature. When the pH value is exceeded, it can be adjusted by autoclaving 10% acetic acid solution or by introducing CO2 gas.

(2) HEPES (molecular weight 238.31) solution

HEPES is usually used at a final concentration of 10-50 mM, but is usually formulated as a 1M stock solution: 47.6 grams of HEPES is dissolved in 200 ml of double distilled water, and the pH is adjusted to 7.5-8.0 with 1N NaOH; then the bacteria are removed by filtration, and the vials are dispensed at room temperature. Or save at 4 °C. Antibiotic solution

Phenol red:

Phenol red is used as a pH indicator in most cultures. Red indicates neutral pH, yellow indicates acidic pH, and purple indicates alkaline pH. However, phenol red is not contained in some special culture solutions because some studies have shown that phenol red can mimic the effects of steroid hormones (especially estrogens).

Sodium pyruvate:

It is another carbon source for cells in cell fluid. D-glucose is a preferred carbon source for cells, but when D-glucose is depleted in the culture, the cells can metabolize sodium pyruvate to obtain a carbon source.

antibiotic

Mammalian cell cryopreservation

The main purpose: (1) Preservation of seed cells for easy access. This is the main purpose of preserving cells.

(2) Reduce the risk of cells being contaminated by microorganisms.

(3) Reduce the risk of cross-contamination between cells.

(4) Reduce genetic variation and morphological changes caused by subculture.

(5) Avoid aging or malignant transformation of finite cell lines.

(6) Reduce manpower and material resources.

Cryopreservation points: (1) The freezing process should be slow. Cells that need to be cryopreserved are placed in a 4 ° C freezer for 30-60 minutes; then transferred to -20 ° C for 30 minutes; then transferred to -80 ° C for 16-18 hours (or overnight); finally placed in liquid nitrogen Medium and long term preservation. Where conditions permit, a programmable temperature-lowering instrument can be used to cool down at a rate of -1 to -3 ° C per minute until it is below -80 ° C, and then directly placed in liquid nitrogen for long-term storage.

(2) The cryopreserved cells must be in the logarithmic growth phase, with a viability greater than 90% and no microbial contamination.

(3) The cell concentration is controlled at: 1 x 107 - 5 x 107 / ml.

(4) Use high concentration serum or protein protectant. In general, serum concentrations in cryopreserved complete cell growth media are greater than 20%.

(5) Use a suitable cell cryoprotectant to protect cells from ice crystal damage during freezing. Currently, the most commonly used cryoprotectant is DMSO (dimethyl sulfoxide) at a final concentration of 5-10%. However, some cell lines cannot use DMSO as a cryoprotectant, such as the human leukemia cell line HL-60. Because DMSO can induce HL-60 cell differentiation. In this case, other cryoprotectants such as glycerin, hydroxyethyl starch and the like may be used.

Special attention: (1) The cells should not be placed in the refrigerator at -20 °C for more than 1 hour during the freezing process to prevent the ice crystals from being too large to destroy the cells. You can also skip the -20 °C step and put it directly into the -80 ° C refrigerator, but the cell survival rate is

Lower.

(2) A large amount of heat is released when DMSO is diluted. Therefore, DMSO cannot be directly added to the cell fluid and must be prepared in advance.

(3) DMSO must be of cell culture grade. The newly purchased DMSO itself is sterile and should be dispensed in small quantities in sterile vials or vials immediately after opening the bottle, and stored at 4 °C. Avoid repeated freezing and thawing to cause DMSO cracking to produce harmful substances. And reduce pollution opportunities. To filter DMSO, a DMSO resistant nylon filter must be used.

The main components of the cell cryopreservation solution: cryoprotectant, basal medium, serum or protein.

Common cell cryopreservation solution: (1) 10% DMSO + complete cell growth medium (20% serum + basal medium)

(2) 10% glycerol + complete cell growth medium (20% serum + basal medium) suspension cell cryopreservation procedure (liquid nitrogen storage method):

(1) Take a logarithmic growth phase cell culture and centrifuge at 200-400 g for 5 minutes. Cryopreservation procedure using adherent (adherent) cells (liquid nitrogen storage method):

Thawing and resuscitation points of cryopreserved cells: (1) rapid thaw. Once the cryopreserved cells have been removed from the liquid nitrogen, they should be placed in a 37 ° C water bath and gently shake the cryotubes to melt them in 1 minute (not more than 3 minutes).

(2) The action of the thawing operation should be light. Since the cryopreserved cells become very fragile, not only is the thawing speed fast, but the action is lighter. In general, the thawed cells can be directly inoculated directly into a cell culture flask containing the fully grown culture medium (1 ml of cryopreserved cell solution is added to 25-30 ml of fresh complete medium) and reused after 24 hours. Fresh complete culture replaces the old medium to remove DMSO. If the cells are particularly sensitive to cryoprotectants, the thawed cells should first be centrifuged to remove the cryoprotectant and then inoculated into a culture flask containing the fully grown broth.

Special attention: (1) Be sure to pay attention to safety when thawing, and prevent the freezing tube from bursting. To bring gloves, use a forceps to remove the cell cryotube from the liquid nitrogen and place in a 37 ° C water bath. Do not use your hands directly to avoid frostbite.

(2) When the freezing tube is thawed in the water bath, the liquid level shall not exceed the surface of the frozen tube, otherwise it may be contaminated.

Centrifugation method for thawing cryopreserved cells:

(1) The frozen cells were taken out from the liquid nitrogen and immediately thawed in a 37 ° C water bath.

(2) Add 1-2 ml of cryopreserved cell solution to 25 ml of fresh complete cell growth medium and mix gently.

(3) Centrifuge at 80 g for 2-3 minutes and discard the supernatant.

(4) Gently suspend the cell pellet with the complete growth medium and count the cells.

(5) The cells were seeded into a culture flask at a starting density of 3 x 105 / ml.