Basic cell culture guidelines

Preparation of medium

One can buy premade liquid medium but premade ones have much shorter shelf life.

In general a growth medium is composed of a chemically-defined base medium with necessary buffering salts and amino acids and carbohydrates and then supplemented with serum containing growth factors. For example, a Dulbecco's minimum essential medium (DMEM) with 10% fetal bovine serum is a common medium for many cancer cells.

One thing i'm not sure is buying premade liquid medium are in 1X and supplementation of serum inadverently dilute the base medium. This maybe of many people's approach.
However we usually like to compensate the base medium being diluted by the serum.
To do so, we also like to start with powered medium. One can purchase the powdered medium (made by freezedrying of the medium and fine grinding them).

On the package, you can see the name of the medium with different supplement.

Usually for medium to have good buffering capacity in a CO2 incubator, especially 5% CO2 nowadays, sodium bicarbonate are added in the medium. Usually sodium bicarbonate is not added in the medium. We take an approach of using fully saturated bicarbonate solution to ensure the stability of pH of cell culture medium in long term. Some labs use standard chemistry approach of using strong acid or base to adjust pH which will heavily affect the ionic strength of the medium and drive the CO2 gas out from the medium affecting the buffering capacity of the medium and the equilibrium pH.


We make medium from powder by the following guidelines.
1. Consider how much percentage of serum are to be added into the medium. For example, for a pack of powder medium usually is 1L. To make final 10% FBS supplement, we measure 900mL of Type I water (18.2MOhm)
2. put 870mL(don't need to be precise) of the water into a 1L beaker and put in a stir bar. Add all powder of from the package and use some water to wash out the left over powder in the package.
3. Measure the necessary sodium bicarbonate in a 50mL beaker and add the rest 30mL of water. mix to dissolve.
4. use CO2 gas to fully saturate the bicarbonate solution. This approach will ensure long term pH stability of the medium as evident of minimum phenol red color change in long term.


5. Mix with the base medium, you should see the right color now.



6. Filter the medium into sterile medium and add necessary serum before or after the filter. Start drawing vacuum from the port to filter all the medium.






7. Recep the solution through sterile operation. Label accordingly.

Sterile operation


Thawing frozen cells


Subculturing cell


Cryopreservation


Routine maintenance

Maintenance of Incubator

Modern incubators have well controlled temperature and humidity as well as CO2 concentration control to satisfy the buffering of bicaronate system in the solution. In some higher class models, sterilisation cycle of dry heat or steam heat is also available to enable seasonly or yearly cleaning to avoid contamination. Some incubators can be retrofitted with additional gas control to modulate the O2 gas concentration. Since the O2 in atmosphere is hardly ever the real tissue oxygen fraction, controllign the O2 is an essential parameters since the metabolism of cells changes. one can use N2 gas to displace atmospheric oxygen to create 1~21% O2, and use O2 gas to create 21~95% O2 atmosphere in the incubator.

Routine maintenance of incubator include
1. Disinfect incubator during daily operation
During opening and closing of the incubator, use 75% ethanol to disinfect the handle surface and nearby area of the door. 

2. Filling the water pan regularly to ensure the humidity in the chamber.
We used to use steam sterilized 18.2MOhm water added with Aqua Guard I in incubator water pan. But now it is not recommended as the ultra pure water is so pure it is corrosive to the water pan. It is recommended now to use only steam sterilized dH2O (15MOhm).
mixed with appropriate AquaGuard I or some disinfectant of choice (Copper ion). The disinfectant are meant to help with control of possible contamination it is optional.

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