Cell culture assays

In biomaterials testing, a cell culture assay is any method which is used to assess the cytotoxicity of a material. This refers to the in vitro assessment of material to determine whether it releases toxic chemicals in sufficient quantities to kill cells either directly or indirectly through the inhibition of cell metabolic pathways. Cell culture evaluations are the precursor to whole animal studies and are a way to determine if significant cytotoxicity exists for the given material. Cell culture assays are standardized by ASTM, ISO, and BSI (British Standards Institution.)

Methods

Direct contact method

1. A near confluent layer of fibroblasts are prepared in a culture plate
2. Old cell culture media (agar generally) is removed
3. Fresh media is added
4. Material being tested is placed onto the cultures, which are incubated for 24 hours at 37 degrees Celsius
5. The material is removed
6. The culture media is removed
7. The remaining cells are fixed and stained, dead cells are lost during fixation and only the live cells are stained
8. The toxicity of the material is indicated by the absence of stained cells around the material.

Agar diffusion method

1. A near confluent layer of fibroblasts are prepared in a culture plate
2. Old cell culture media is removed
3. The cells are covered with a solution of 2% agar, which often contains red vital stain
4. When the agar solidifies the cells will have dispersed throughout its volume
5. The material is then placed on the surface of the agar and incubated for 24 hours at 37 degrees Celsius
6. Live cells take up the vital stain and retain it, dead cells do not
7. The toxicity of the material is evaluated by the loss of vital stain under and around the material
8. Surface microscopy is also needed to evaluate the material-cell interface

Elution method

1. A near confluent layer of fibroblasts are prepared in a culture plate.
2. An extract of the material which is being tested is prepared using physiological saline or serum free media (the latter is generally preferred).
3. Extraction conditions are used which are appropriate for the type of exposure which the cells would receive in the in vivo environment if the material were to be implanted.
4. The extract is placed on the cells and incubated for 48 hours at 37 degrees Celsius.
5. After 48 hours the toxicity is evaluated using either a histochemical or vital stain.

Each method has its own advantages and disadvantages, and some are more suitable for certain applications than others. For example the direct contact method offers conditions which are most similar to the physiological environment but the cells are susceptible to trauma if the material moves. The agar diffusion method is good for materials with high densities and offers an even concentration gradient for potential toxicants, but there is a serious risk of the cells going into thermal shock when they are overlaid with agar. The elution method is best for applications which might require extra incubation time, but additional time and steps are required for preparing such a test.

In vitro biomaterials testing yields fundamental information about the behaviour of materials in contact with living cells, but cannot qualify or even accurately predict the performance of a material in vivo.