Biomedical Engineering Reference
In-Depth Information
2.2.3 Limitations of Indirect Contact Test
(i) Agar diffusion test. This tests exhibits the same limitation regarding dilution of
the toxicant as the direct contact test, since again only 10% of the cell surface
should be covered. This test is even less sensitive because only one sample side
comes into contact with the agar layer separating the cells and sample. In addition,
the toxicants have to be able to diffuse freely through the agar and must not react
with the agar. In cases where the chemical properties of the released compounds
are not known, it may be very challenging to prove the latter. Furthermore, a good
contact between the sample and the agar must be ensured. The advantage of the
present test is that an incubation period of 24-72 h is suggested, of which 72 h is
certainly preferred, enabling the measurement of cytotoxic effects via other
pathways in addition to acute cytotoxicity. Another advantage of the agar diffusion
test is that the samples can be heavier, since the agar layer protects the cell to a
certain extent and the cell layer cannot be disrupted due to a sample removal.
(ii) Filter diffusion test. The advantage over the agar diffusion test is that the
distance between cells and material is much smaller and mimics the direct contact
test in this regard. The only limitation of the test is that a good contact between
sample and filter must be ensured, i.e. the fluid layer is in contact with the cells and
the sample surface over the whole filter surface. Furthermore, it must be dem-
onstrated that the released constituents do not bind to the filter. The advantage of
testing extracts instead of pure samples is that with extracts exposure to maximal
constituent concentration is ensured from the moment the extract is applied.
However, the exposure period is extremely short (2 h ± 10 min).
2.3 Limitations of Parameters for Cytotoxicity
Typically, only final values of the cytotoxicity parameters are considered after a
specified exposure period in cell cultures according to ISO 10993-5. The history,
i.e. what occurred in between, is not taken into account. For instance, the presence
of fewer cells in an exposed well relative to control may be evoked by cell death,
reduction of cell proliferation or a combination of both. The absence of an effect
may be the result of a real absence of adverse effects or of a too short exposure
period (for instance, inhibition of cell proliferation can certainly not be detected in
the filter diffusion test). Furthermore, the cells used for these tests will affect the
outcome. This is due to the variable sensitivity of cells and the activity of the cells
(e.g. the MTT conversion activity varies between different cell types and may also
differ between cell line and primary cells of the same cell type). As a rule of
thumb, it can presumed that the relevance of the chosen cell types and therefore
the test outcome are directly connected with the proposed application of the
biomaterial. Furthermore, it must be noted that false positive results may be
obtained if the biochemical assay is influenced by a cross-reaction between test
sample and/or the compound itself and the constituents of the biochemical assay.
Search WWH ::
Custom Search