Biochemical Techniques and Instrumentation (1+2)

6.3.3.3. Manipulation of cultured cells

Manipulation of cultured cells becomes important as the cells that grow generate several issues:

• Nutrient depletion in the growth media
• Accumulation of apoptotic / necrotic (dead) cells.
• Cell-to-cell contact that stimulate cell cycle arrest.
• Cell-to-cell contact that stimulate cellular differentiation.

The common manipulations that are carried out in cell culture are change of media, passaging and transfection of cells. Manipulations are carried out in a biosafety hood or laminar flow cabinet to exclude contamination by micro-organisms.

a. Change of media: As the cells grow, they undergo metabolic processes. Acid is produced which in turn decreases the pH. Addition of a pH indicator to the medium helps to identify the nutrient depletion. Then, the media can be removed directly by aspiration and replaced with the new media.

b. Passaging:

Passaging is also known as "subculture" or "splitting cells". This involves transfer of a small number of cells into a new vessel. Cells that are subcultured regularly can be grown for a longer time, as it avoids the senescence associated with prolonged high cell density. Suspension cultures are easily passaged with a small amount of culture containing a few cells diluted in a larger volume of fresh media. In adherent cultures, cells are first detached with a mixture of trypsin-EDTA and then a small number of detached cells are grown as a new culture.

c. Transfection and transduction:

Introduction of foreign DNA by transfection in another common method for cell manipulation. This helps the cells to express a protein of interest. The transfection of RNAi constructs is used to suppress the expression of a particular gene/protein. Foreign DNA can also be inserted into cells using viruses by the process called "transduction".