Cytotoxicity
The cell or tissue culture including cytotoxicity methods is a fair predictor of biocompatibility when used together with other appropriate tests. 8, 9 Several highly specialized cell culture methods are available to monitor the biocompatibility of the raw materials used in manufacturing the device or auditing the manufacturing process. Cell or tissue culture testing offers several advantages, including the following:
It is simple, rather inexpensive, and easy to perform.
It allows testing of a biomaterial on human tissue.
It is sensitive to toxic material. It is easy to manipulate and allow more than one end-point investigation.
It can be used to construct a dose-response curve.
It can give quick and quantitative results and allows direct access or direct observation or measurement.
The objective of cell cytotoxicity testing is to screen the biocompatibility of the polymeric and elastomeric portions of medical devices using mammalian cell cultures. Cytotoxicity is a useful method for screening material. It can also serve as a quality control mechanism for batch testing programs, and is a basic part of all device biocompatibility evaluation.10-12 It is one of the oldest assays designed specifically to screen plastics for toxicity.13 Given the extreme sensitivity of this test, materials found to be cytotoxic must be assessed along with the results of in vivo and otherstudies to evaluate the risk to human health. Unlike the other studies utilized in biocompatibility testing, cytotoxicity is not a pass or fail test. Failure in cytotoxicity is generally grounds for performing a confirmatory test such as an implantation or intracutaneous reactivity.
14 The great majority of toxic compounds are chemically stable and produce their characteristic effects by interference with biochemical or physiological homeostatic mechanisms.
Cytotoxicity assays measure loss of some cellular or intercellular structure and/or functions, including cell death. They are generally simple to perform, are reproducible, and have a clearly defined endpoint. A variety of cell lines have been used, including corneal epithelial cells, lung fibroblasts, Chinese hamster ovary (CHO) cells, canine renal cells, HeLa (human tumor cell line) cells, and microorganisms. Most cells in culture are fibroblasts.
Primary cells that are taken directly from an animal often are difficult to establish in culture and become fibroblasts, losing the normal functions of growing differentiated cells. Numerous conditions have to be optimized for obtaining good growth of differentiated cells. Most cultured cells have a fibroblastic appearance, although they may not be true fibroblasts. For example, cells grown under non optimum conditions can temporarily take an appearance of fibroblasts. The fibroblasts in culture can take overcultures because they grow readily on plastic surfaces. The recent success ingrowing differentiated cells was partially due to techniques that have been developed to remove and limit the growth of fibroblasts to allow other cells to grow. The possible cytotoxicity end points are15-18 as follows:
Microscopic examination of cell morphology, membrane integrity, and fragility, Cell population and density, Cell adhesiveness, Cytopathic effect, Total protein content, Rate of growth, Rate of protein synthesis, Total DNA content, Rate of DNA synthesis, Colony-forming efficiency, Trypan blue uptake and other dye uptake, Biochemical assays of enzymes.
Much of the initial work on the range of cytotoxicity assays that have been developed (and their end point measurement methods) was done with the goal.
19-25 Early cell culture methods merely estimated the numbers of living or dead cells, but now morphological analysis by electron microscopy reveals a spectrum of microcellular changes, and cell function tests measure biochemical parameters, indicating the nature of cell stress. Although many modifications have been made, cell culture tests are of four main types: gel diffusion, direct contact, extracts dilution, and cell function tests.
As discussed above, several tissue culture methods are available for testing biomaterials. These are divided into two major groups: one tests the toxicity of a soluble extract of the material, and the other tests the toxicity by the direct contact of cells with the material or components of the device. The text extracts is incorporated into the culture medium, which is usually double strength minimum essential medium supplemented with serum and other essential nutrients at the maintenance level. The toxic effect on the monolayer, such as cell lysis and microscopic observation of cell morphology changes, is usually checked after 24 and 48 hr. Cell lysis can be scored by direct microscopic observation or with the use of radiolabels or tryphan blue dye uptake. The inhibition of cell growth is a more informative test requiring more time and skill. Distilled water extract is incorporated into the tissue culture medium and inoculated with the cells in the tissue culture tubes. After 72 hr, the extent of cell growth is determined by total protein assay, such as the Lowery photometric method, on the removed cells from the individual tubes. Most cells in culture are fibroblasts.
Primary cells that are taken directly from an animal often are difficult to establish in culture and become fibroblasts, losing the normal functions of growing differentiated cells. Numerous conditions have to be optimized for obtaining good growth of differentiated cells.
