1.8. Working with cell cultures

Sterility is a crucial issue during working with different cell cultures. Undesired microorganisms accidentally getting into a solution often start to grow by utilizing and degrading the available components. As these microorganisms synthesise the biomolecules of their cells, they release various metabolites into the solution. (Some algae can grow even in distilled water!) Cell cultures (e.g. the cultures of the E. coli bacterium used widely in recombinant DNA techniques) must be protected from such “invaders”. To achieve this, sterile laboratory tools, vessels and media must be used. A laminar flow cabinet (also known as tissue culture hood) is required for most operations performed with cell cultures (Figure 1.14, panel A). In the laminar flow cabinet, a constant air current is established. Air filtered through a HEPA (high-efficiency particulate air) filter is blown into the cabinet at the top, and leaves below the working bench. HEPA filters remove all microorganisms. The transparent door of the cabinet protects its inner contents from contamination. While the door is opened, work can be done through a narrow gap allowing only the hands of the operator to reach the equipment and the cell cultures inside. The operator must wear laboratory gloves. Ethanol and/or a Bunsen burner can be used to quickly sterilise equipment inside (e.g. inoculation loops, glass spreaders). Other accessories, including pipette tips and different tubes, are sterilised in an autoclave prior to use. It is very important to avoid situations in which the arm of the operator or any non-sterile equipment is directly placed above sterile cell cultures (or any sterile equipment), because dust particles or bacteria transferred by the air flow (pointing downwards) can easily contaminate the sterile components. Upon finishing the desired operations, the front door of the laminar flow cabinet must be closed. The inner contents can be sterilised by using a built-in UV lamp.

Incubators are used to maintain controlled conditions for bacterial or eukaryotic cell cultures (Figure 1.14, panels B-D). These devices can heat or cool their inner space in order to provide constant temperature conditions for cells. (Simpler and cheaper incubators can only heat; hence, these can only be used above ambient temperature.) Cells can be cultured on different surfaces. Bacteria are often grown in Petri dishes on the top of a layer of gelatinous nutrient agar medium (Figure 1.14, panel B), while eukaryotic (e.g. human) cells are grown at the bottom of cell culture flasks, covered by liquid medium at an appropriate depth (Figure 1.14, panel C). Most of the commonly used eukaryotic cells are adherent: they settle and grow while attached to the surface of the cell culture dish.

Besides temperature, the most important environmental factor is the gas mixture in which the cells are being cultured (for instance, obligate anaerobic bacteria are killed by oxygen, and eukaryotic cells often require elevated levels of carbon dioxide). If necessary, a controlled atmosphere can be maintained by using appropriate incubators.

In general, cells grown in liquid cultures (suspended in liquid medium) can reach higher densities than adherent cultures, because in liquid cultures the entire culture volume can be utilised for growth. Liquid cultures are used if cells are required in large numbers (e.g. during recombinant protein expression in E. coli bacteria) (Figure 1.14, panel D). As mentioned above, Erlenmeyer flasks are the most widely used vessels for this purpose. Incubators equipped with a shaking platform are necessary to ensure uniform cell densities and growth conditions within the medium. Intensive shaking of the flasks by circular motion facilitates effective gas exchange. The attained cell density can be further increased by the use of fermentors.

Laboratory equipment for culturing cells

Figure 1.14. A, Laminar flow cabinet. B, Incubator (37°C) with bacterial cultures on agar plates. C, Incubator (37°C) with cultures of eukaryotic cells. D, Incubator (37°C) with a shaking platform for liquid cultures of E. coli bacteria (in Erlenmeyer flasks).