In Biomaterials Testing, a cell culture assay is any method which is used to assess the cytotoxicity

Cytotoxicity

Cytotoxicity is the quality of being toxic to cells. Examples of toxic agents are a chemical substance, an immune cell or some types of venom .-Cell physiology:...

 of a material. This refers to the in vitro

In vitro

In vitro refers to studies in experimental biology that are conducted using components of an organism that have been isolated from their usual biological context in order to permit a more detailed or more convenient analysis than can be done with whole organisms. Colloquially, these experiments...

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.)

Direct contact method

1. A near confluent layer of fibroblasts are prepared in a culture plate
2. Old cell culture media (agar
   Agar
   Agar or agar-agar is a gelatinous substance derived from a polysaccharide that accumulates in the cell walls of agarophyte red algae. Throughout history into modern times, agar has been chiefly used as an ingredient in desserts throughout Asia and also as a solid substrate to contain culture medium...
   
    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
   Vital stain
   A vital stain is a stain that can be applied on living cells without killing them. Vital stains have been useful for diagnostic and surgical techniques in a variety of medical specialties...
   
   
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
   Vital stain
   A vital stain is a stain that can be applied on living cells without killing them. Vital stains have been useful for diagnostic and surgical techniques in a variety of medical specialties...
   
    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
   Microscopy
   Microscopy is the technical field of using microscopes to view samples and objects that cannot be seen with the unaided eye...
   
    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
   Vital stain
   A vital stain is a stain that can be applied on living cells without killing them. Vital stains have been useful for diagnostic and surgical techniques in a variety of medical specialties...
   
   

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

Agar

Agar or agar-agar is a gelatinous substance derived from a polysaccharide that accumulates in the cell walls of agarophyte red algae. Throughout history into modern times, agar has been chiefly used as an ingredient in desserts throughout Asia and also as a solid substrate to contain culture medium...

 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 overlayed with agar

Agar

Agar or agar-agar is a gelatinous substance derived from a polysaccharide that accumulates in the cell walls of agarophyte red algae. Throughout history into modern times, agar has been chiefly used as an ingredient in desserts throughout Asia and also as a solid substrate to contain culture medium...

. 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

In vitro

In vitro refers to studies in experimental biology that are conducted using components of an organism that have been isolated from their usual biological context in order to permit a more detailed or more convenient analysis than can be done with whole organisms. Colloquially, these experiments...

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

In vivo

In vivo is experimentation using a whole, living organism as opposed to a partial or dead organism, or an in vitro controlled environment. Animal testing and clinical trials are two forms of in vivo research...

.