What Are Some of the Problems Faced by Cultured Cells?
Avoiding Contamination
Cell culture contamination is of two main types: chemical and biological. Chemical contamination is the most difficult to detect since it is caused by agents, such as endotoxins, plasticizers, metal ions or traces of chemical disinfectants, that are invisible. The cell culture effects associated with endotoxins are covered in detail in the Technical Bulletin: Endotoxins and Cell Culture (Ref. 10). Biological contaminants in the form of fast growing yeast, bacteria and fungi usually have visible effects on the culture (changes in medium turbidity or pH) and thus are easier to detect (especially if antibiotics are omitted from the culture medium). However, two other forms of biological contamination, mycoplasmas and viruses, are not easy to detect visually and usually require special detection methods.
There are two major requirements to avoiding contamination. First, proper training in and use of good aseptic technique on the part of the cell culturist. Second, properly designed, maintained and sterilized equipment, plasticware, glassware, and media. The careful and selective (limited) use of antibiotics designed for use in tissue culture can also help avoid culture loss due to biological contamination. These concepts are covered in detail in a Corning Technical Bulletin: Understanding and Managing Cell Culture Contamination (Ref. 7).
Finding A “Happy”En vironment
To cell culturists, a “happy” environment is one that does more than just allow cells to survive in culture. Usually, it means an environment that, at the very least, allows cells to increase in number by undergoing cell division (mitosis). Even better, when conditions are just right, some cultured cells will express their “happiness” with their environment by carrying out important in vivo physiological or biochemical functions, such as muscle contraction or the secretion of hormones and enzymes. To provide this environment, it is important to provide the cells with the appropriate temperature, a good substrate for attachment, and the proper culture medium. Many of the issues and problems associated with keeping cells “happy” are covered in the Corning Technical Bulletin: General Guide for Identifying and Correcting Common Cell Culture Growth and Attachment Problems (Ref. 8).
Temperature is usually set at the same point as the body temperature of the host from which the cells were obtained. With cold-blooded vertebrates, a temperature range of 18° to 25°C is suitable; most mammalian cells require 36° to 37°C. This temperature range is usually maintained by use of carefully calibrated, and frequently checked, incubators.
Anchorage-dependent cells also require a good substrate for attachment and growth. Glass and specially treated plastics (to make the normally hydrophobic plastic surface hydrophilic or wettable) are the most commonly used substrates. However, Attachment Factors, such as collagen, gelatin, fibronectin and laminin, can be used as substrate coatings to improve growth and function of normal cells derived from brain, blood vessels, kidney, liver, skin, etc. Often normal anchoragedependent cells will also function better if they are grown on a permeable or porous surface. This allows them to polarize (have a top and bottom through which things can enter and leave the cell) as they do in the body. Transwell® inserts are Corning vessels with membrane-based permeable supports that allow these cells to develop polarity and acquire the ability to exhibit special functions such as transport. Many specialized cells can only be truly “happy” (function normally) when grown on a porous substrate in serum-free medium with the appropriate mixture of growth and attachment factors.
Cells can also be grown in suspension on beads made from glass, plastic, polyacrylamide and cross-linked dextran molecules. This technique has been used to enable anchorage-dependent cells to be grown in suspension culture systems and is increasingly important for the manufacture of cell-based biologicals.
