In vitro studies

Cell-based in vitro research

When considering supplement research on cells in vitro the following questions are useful:

Cell species: Human or which animal? (e.g. in biogerontology, mice may be an unsuitable model because mice express telomerase whereas most human cell types do not)

Primary or cell line:
- Cell lines are cells that keep on proliferating in culture mainly without attention to the Hayflick Limit. These 'immortalised' cells are very easy to obtain for laboratory researchers but for obvious reasons they may not be a good model for biogerontology studies.
- Primary cells are isolated from fresh tissue. They may not be a close match to 'real cells', not just because they are no longer part of tissue specific interactions, but also because the 'stress' of isolating the cells and getting them to grow in a dish may change their behaviour and parameters.

Administration: It is also important to consider how the cells were exposed to the supplement in question:
- Pre-isolation exposure encompasses e.g. feeding or injecting the supplements to the cell donor prior to isolating the cells.
- Culture supplementation: exposing the cells to concentrations of the supplement as part of their metabolic intake (potentially aided by molecules that facilitate uptake by the cell). It is useful to consider the amount of supplement that cells were exposed to. Often, it is very difficult to emulate this exposure by oral supplementation.

Effect: There are a number of ways in which effects can be recorded. Examples include:
- Proliferation: Cells grow better than the control group. This is arguably a rather weak measure for life extension purposes. Indeed, increased proliferation in certain cell types could be considered potentially hazardous due to implications for neoplasia.
- Cell survival: Cells are exposed to a certain environmental stress (e.g. H202 exposure, increased oxygen levels or irradiation) and seen if supplemented cells are more resilient than the control.
- DNA damage: A common method is the 'comet assay' where cells undergo suspension in agarose, lysis and electrophoresis. The DNA is then stained with a fluorescent dye which allows visualization of DNA damage.
- ROS: ROS reacting with a dye causes light to be emitted. Flow cytometry is superior to luminometer-based techniques as it distinguishes between cell sub-populations and allows for analyzing intracellular events on a single-cell level.