Evaluation of Biocompatibility Using In Vitro Methods: Interpretation and Limitations - Tissue Engineering: Cell-Surface Interactions for Tissue Culture - page 127

A further variable portrays the surface to extract fluid volume ratio. It is
suggested that the surface to volume ratio should be 1.25-6 cm 2 /ml depending
on the thickness, or 0.1-0.2 g/ml or 6 cm 2 /ml in the case of materials with
indeterminate surface irregularities like foams [ 1 ]. Starting from the 100% stock
extract, dilution series may be prepared with culture medium. The standard is,
however, very inaccurate since it does not define the minimum surface to volume
ratio.
2.2.2 Limitations of Direct Contact Test
As discussed briefly above, the sample material is placed on top of a subcon-
fluent cell layer. That tight contact between material surface and cells is thought
to reflect the material-cell interaction. Such tests have to be carried out with
great care to avoid artefacts and misinterpretation of the results. The first
problems may be encountered if samples are placed on the cell layer. Most often,
the sample weight is either too high or too low. For instance, e.g. a sample made
of a CoCrMo alloy has a density of 8.3 g/cm 3 and might crush easily the cells,
while a dense polyurethane sample is slightly below 1 g/cm 3 and will float on the
cell culture medium. The contact regime will be completely different for these
two samples. Another problem that might occur is that the cells adhere to the
material surface instead of to the tissue culture plastic. Rupture of the underlying
cell layer due to sample removal or apparent ''reduction'' of the number of cells
below the sample may be the result. In this case, it will be difficult to judge the
contact test, although preferential adherence as compared to tissue culture plastic
is a good sign.
Another drawback of contact tests is that released toxic components may be
diluted in the culture media to such an extent that they no longer affect the
cells. In this situation the released components are extracted during the tests
instead of before testing as is the case in the extract tests. The difference
between the extract test and direct contact test is the ratio between the material
surface and medium and is well illustrated by the following situation. A circular
film sample with a thickness of less than 0.5 mm is evaluated with extract and
direct contact tests both using 3.5-cm diameter culture dishes with 3 ml of
culture medium. In order to obtain an extract according to the ISO 10993-12,
the surface to volume ratio must be 6 cm 2 /ml. In the present example, this is
the case if the diameter of the sample is 3.4 cm. However, that contradicts the
requirement of the direct contact test where the sample should cover only 10%
of the culture dish surface. The latter corresponds to 0.96 cm 2 and thus the
circular sample should not exceed 1.1 cm in diameter. Furthermore, in the case
of the extract test, cells are already treated from the beginning with the con-
centration of the constituents that is released over a period of 24 h. In the direct
contact test this maximal concentration occurs only at the end of the treatment
period, normally 24 h.