Evaluation of Biocompatibility Using In Vitro Methods: Interpretation and Limitations - Tissue Engineering: Cell-Surface Interactions for Tissue Culture - page 124

and (3) indirect-contact tests (agar diffusion test, filter diffusion test, see Fig. 3 for
schemes). One or more tests may be used for the evaluation depending on the
nature of the sample material.
Cytotoxicity is assessed using different parameters based on cell and culture
morphology (qualitatively), quantitative measurement of cell impairment, such as
effects on cell growth (proliferation), and specific aspects of cell metabolism.
Qualitative and quantitative measurements are reported to correlate very well [ 20 ].
Extract tests are normally based on a so-called extract obtained by exposing cell
culture medium to the test material or compound of interest for 24 h at 37 C.
Subconfluent cell cultures are treated by measuring effects on cell functionality
typically after 24 h, or low-density cultures are revealed by measuring effects after
a prolonged time period of six days. In the latter case, effects are assessed by
counting the number of colonies consisting of, as proposed, at least 50 cells. In the
direct contact test, a test sample covering about 10% of the subconfluent cell layer
is placed on top of that layer, while in the agar diffusion test an agar layer covers
the cells instead of cell culture medium and the test samples are placed on top of
the agar layer. In both tests, the sample is removed after 24-72 h exposure time
and the cells are qualitatively and quantitatively assessed below and adjacent to the
test samples. Another suggested test method is the filter test. For this cells are
cultured until confluency on one side of the filter, which is then placed with
the cell side on top of an agar layer. Subsequently, the test material is placed on the
other side of the filter. Effects on cells are qualitatively assessed after 2 h exposure
time.
The most striking limitation of all suggested tests in ISO 10993-5 is the short
test period, i.e. 2 h (filter diffusion), 24 h (extract acute cytotoxicity), or 24-72 h
(agar diffusion). By defining a reduction of 30% as the threshold for an extract to
be toxic, only a cell lysing compound, a compound inducing apoptosis in a very
short term, or very strong inhibitor of cell proliferation will be able to give rise to
such a reduction after a treatment period of 24 h. For instance lidocaine (1 mM),
known to reduce cell proliferation of the suggested cell line (NIH 3T3), yields a
65% reduction in cell count after 120 h, but only 30% reduction is observed after
the required culture period of 72 h. No clear-cut effects on cell numbers are seen
even 48 h after treatment [ 49 ]. Clear results are only obtained if the colony-
forming test is chosen, having the prolonged incubation period of six days.
Therefore, the short test duration is very limiting regarding the informative value
and the kind of effects that can be assessed. In particular, effects based on accu-
mulation and delayed/progressive effects will not be detected.
A second general limitation is the use of cell lines, in particular of cell lines that
may not be relevant for the proposed use of the biomaterial. The standard asks
indirectly for a justification of the cell sources used in tests, but limits the use of
primary cells to applications that require specific sensitivity. The result is that
industry will hardly ever test with primary cells or organ cultures, in order to
reduce the risk of a cytotoxic outcome. Therefore, fibroblast L-929 or NIH 3T3
cells are used for most if not all medical devices that are in contact with muscle
and skeletal tissues.