Evaluation of Biocompatibility Using In Vitro Methods: Interpretation and Limitations - Tissue Engineering: Cell-Surface Interactions for Tissue Culture - page 130

living cells. It accumulates linearly in the lysosomes of living cells as a function of
time [ 162 ]. There is one limitation to this test, due to the lipophilic characteristics of
NR. It may bind to certain test samples or adsorb on components such as carbon
black. The resulting high background levels depend on sample surface or concen-
tration of components and have to be subtracted in analysis [ 98 ].
2.3.3 Example: MTS
The MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulf-
ophenyl)-2H-tetrazolium, inner salt] assay can be used as an alternative to the
MTT test. As in the MTT test, the conversion of MTS to its formazan product is
measured. The advantage over the MTT test is that the MTS formazan product is
water-soluble and by that does not need to be solubilized. In analogy to the
MTT, the MTS assay cannot be used for measuring antiproliferative activity
since it also underestimates the antiproliferative effect [ 146 , 148 ]. It may be
assumed that the MTS conversion cannot be taken as an index of cell viability,
like the MTT conversion. In toxicological studies comparing the results of MTS
and neutral red assay, MTS systematically underestimated toxicity [ 11 , 110 ].
Since cell number and cell activity both play a role in total culture MTT con-
version activity level, this test may be taken as an index for total culture cell
activity, like the MTT test.
2.3.4 Example: DCF Test for Measuring ROS
Reactive oxygen species (ROS) are important in several metabolic pathways, act
as intracellular signalling molecules and are important in homeostasis. ROS are
formed as metabolic products and exhibit a very high reactivity due to their
unpaired oxygen electron. Below a certain concentration range, their adverse
effects are reduced by numerous scavenger molecules that are produced by the cell
for this purpose. If cells are stressed, e.g. due to drugs, particles, UV light or
endotoxins, the production of ROS is increased, reaching concentrations which
may be harmful for the cells. One of the common tests for evaluating ROS for-
mation is the H 2 DCF-DA test [ 58 , 103 , 118 , 139 , 158 ]. In cell cultures, the
H 2 DCF-DA test is primarily used for the detection of a variety of ROS species
including peroxyl and hydroxyl radicals, the peroxynitrite anion and nitric oxide,
as well as hydrogen peroxide [ 44 , 104 ]. The 2 0 ,7 0 -dichlorodihydrofluorescein
diacetate (H 2 DCF-DA) test is based on the assumption that this non-fluorescent
dye is taken up by cells and deacetylated to its non-fluorescent congener 2 0 ,7 0 -
dichlorodihydrofluorescein (H 2 DCF), which is thereafter entrapped within cells.
ROS react with H 2 DCF, converting it to the highly fluorescent 2 0 ,7 0 -dichloroflu-
orescein (DCF) which is not membrane-permeable [ 13 , 80 , 137 ]. Recently, some
doubts have arisen as to how far the DCF test is adequate for measuring ROS [ 22 ].
Additionally against current opinion, H 2 DCF as well as DCF is found not to be