Cultivation and passaging of cell lines

For a successful cell culture, cultivation under optimal conditions as well as regular checking and maintenance of the culture is mandatory.

Most cell lines require a constant temperature of 37 °C, a relative humidity of 95%, and an atmospheric CO2 content of 5%. A CO2 incubator ensures these conditions (Figure 1, left).

The culture is checked by means of an inverted microscope. The special construction of the microscope allows the cultures to be viewed in the original culture flask. If normal microscopes were used, the cultures would have to be placed on slides. Also, multiwell plates could not be viewed with assays with a normal microscope. (Figure 2)

If the culture flask is densely overgrown or the medium is used up, the cells must be resupplied. This is done under sterile conditions in the sterile bench, which is exclusively available for the cell culture (Figure 1, right).

Transfection of cells – fluorescence microscopy

Various methods can be used to introduce foreign DNA into animal cells transiently (temporarily). Commonly used methods are calcium phosphate precipitation, transfection via cationic polymers and lipofection. The effects and the efficiency of transfection of the different methods are tested in the laboratory course “Chemistry of Active Substances.”

If a plasmid encoding the GFP (green fluorescent protein) is used for transfection, the living cells and the efficiency of the transfection (proportion of successfully transfected cells) can be observed on the existing Zeiss fluorescence microscope.

Furthermore, transfections are used for current research projects as well as when carrying out a reporter gene assay (practical course in the chemistry of active substances).

Figure 1: Zeiss Phomi III fluorescence microscope

Figure 2: View into the microscope, with CHO cells transfected with GFP plasmid
           (liposome-mediated transfection)

Cryopreservation of cells

Cryopreservation – what for mankind is still a dream that is the stuff of science fiction films, has long been reality in cell culture. Living cells can be deep-frozen, stored and then cultivated again after thawing. The cells can be stored at -80 °C in the freezer (short-term storage) but also in a nitrogen tank at -196 °C (long-term storage, nitrogen tank see figure).

In the laboratory course “Cell Culture Technology” students can try out this technique themselves and determine the survival rate of “their” cell culture.