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5. Add glutamine.
6. QS to final volume with purified water (See Glossary for definition of QS).
7. pH the medium (7.2).
8. Remove 15 ml of the medium.
9. Add 15 ml of a 1 M HEPES buffer.
10. Stir for an additional 10 min.
Filter Sterilization Materials
1-liter bottletop filter unit, 0.22 ^m filter (Corning) for Schott bottles, or 1-liter filter flasks. If more than 1 liter at a time is routinely made, the following apparatus may be more practical for filtration:
1. Variable flow peristaltic pump, 1 liter/min
2. Pump head (polycarbonate, with stainless steel rotor), to accommodate tubing
3. Thick-wall silicon tubing (4.8 mm inner diameter)
4. Millipore 20-liter Millipak filters
Clamp the tubing in the pump head and attach the pump head, tightening just enough to keep it in place. Adjust the tubing so that there is sufficient length in the Erlenmeyer reservoir and sufficient length in the outbound side of the pump to reach the receiving bottles at the filter end. Tighten the pump head. Attach the tubing to the filter (clamping is usually not necessary). Adjust the flow rate slowly at first and vent the filter by loosening the filter vent cap until there are no air bubbles in the filter unit. The flow rate can now be increased to maximum, but care should be taken that any back pressure does not force the tubing off the filter's hose barb.
The smallest filter that will handle the volume of medium to be sterilized without plugging should be used. Different lots of serum can vary considerably in their filterability. Horse serum and adult bovine serum have a higher lipid and protein content than fetal bovine serum and can be more difficult to filter. A prefilter can be used to extend the filter life and increase filtration rate. A much larger volume of serum-free medium than serum-containing medium can be filtered through a filter of a given area.
Serum can be added to medium when it is made up and the mixture filtered. However, since the serum has a longer shelf life than the medium and different cells may require different levels or kinds of serum, we find it more convenient to make up the medium without serum, store the sterile serum separately, and add it to the medium as needed for each experiment. Whether using disposable filters or a filtration setup, the first 30 to 100 ml of medium through the filter should be discarded to avoid contaminating the medium with chemicals washed out of the filter. The media bottles should be labeled, dated, and stored at 4jaC.
Serum is frequently spoken of as if it were a defined single substance. This is very far from the truth. Cell culture media can be supplemented with sera from any species of animal; bovine (fetal, newborn, or adult), horse, or human sera are the most frequently used. These are quite different in many ways and can have very different effects on the properties of cells grown in them. Additionally, serum varies from animal to animal, with changes in diet, and seasonally. Therefore, there is considerable variability from lot to lot of the commercially available sera. In addition to whole sera, which is allowed to clot and the clot removed, the blood can be collected with an anticlotting agent and the cellular portion spun out, resulting in plasma. Serum and plasma, even from the same animal, are quite different in composition and their effect on cells.
Sera can be treated before use in one or more ways: filtration, dialysis, diafiltration, heat treatment, or fractionation. These treatments can act as an added insurance against contamination, can remove or inactivate toxic components of the serum, can remove or inactivate growth-promoting or differentiating components of the serum, and specifically can remove low- or high-molecular-weight components of the serum or particular serum fractions. Clearly, this complex and undefined addition to medium must be treated with some care to insure consistent results. The only way to insure good results is to thoroughly test several lots of serum for their ability to support the desired cell characteristics (e.g., growth, differentiation, or lack of differentiation; specific biochemical markers; protein production, etc.) and then buy a large quantity of the best lot, store it at "C20 to "C80jaC, and use it for the next several years.
Most commercial sera come sterilely packaged. It is best to purchase serum that has been sterilely collected as well, as an added insurance against viruses or mycoplasma, which can go through some filters. When adding human serum to cultures, the entire culture and all waste should be treated as a biohazard. Human sera should be collected from known donors or blood banks that test for the common viruses such as human immunodeficiency virus (HIV) and hepatitis.
Testing Media and Components-Quality Control
We have stated that it is best to prepare media from commercially available powdered nutrient mixtures as outlined above. It is important to keep good records and do quality control testing of reagents used in making the medium. We generally keep one set of glassware exclusively for medium making. This is rinsed well with distilled water but not washed with detergent between each use. This avoids the possibility of any detergent residue getting into the medium. The sodium bicarbonate and other reagents that are used in media should be used only for media. When weighing out these reagents, a disposable tongue depressor and weigh boat should be used. This avoids contaminating these reagents with other, potentially toxic chemicals that may be in use in the laboratory.