Biomedical Engineering Reference
In-Depth Information
In the following examples, the use and limitations of some common tests will be
discussed.
2.3.1 Example: MTT-Test for Measuring Viability or Cell Number
One of the most common tests used in cytotoxicity evaluation is the test based on
the conversion of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide
(MTT) to its formazan product. MTT is membrane-impermeable and the cellular
uptake of MTT is endocytosis-dependent [
83
]. The reduction is associated with the
cytoplasm and non-mitochondrial membranes including the endosome/lysosome
compartment [
83
]. How far mitochondria participate in formazan formation, as
first suggested [
127
], remains unclear [
18
,
83
,
86
]. As reducing enzymes,
NAD(P)H-oxidoreductases [
19
,
83
] and glutathione S-transferase [
161
] are pro-
posed, among others. The final formazan product is transported to the cell surface
through exocytosis [
83
]. Some research teams use the test to enumerate cells [
86
].
This might be correct under normal conditions but it has been shown that in
several situations where adverse effects of compounds and particles are assessed
this is not the case [
30
,
72
,
148
,
152
]. Another application is to evaluate cell
viability. Here also cases have been reported where results obtained with the MTT
test do not reflect viability measures using other kinds of assays [
123
,
146
,
148
].
Since both aspects, cell number and cell activity, will affect the level of MTT
conversion, this test may be taken as an index of cell activity of the culture as a
whole. Special attention has to be paid to the interaction between test sample and
assay. For instance, evidence was found that the MTT formazan product is able to
bind to the carbon nanotubes (CNT), reducing the dissolved MTT formazan yield.
A pseudo-reduction of MTT formazan levels is therefore observed [
16
,
98
].
In addition, CNT interferes with the assay by a second pathway. In a control
experiment, under cell- and serum-free conditions it could be shown that CNT by
themselves are able to reduce MTT [
16
]. Similar effects are observed in systems
containing high salt levels, such as in combination with absorbable calcium
phosphate scaffolds (Luginbuehl et al., unpublished data). One other disadvantage
of the test is that the MTT by itself may affect cell functionality, as shown for
astrocytes. As a result, some discrepancies of cell functionality data using other
assays may occur [
71
]. This test should therefore be used with care; i.e. its limi-
tations and interference potentials must be addressed before use.
2.3.2 Example: Neutral Red Uptake Test
Neutral red (3-amino-7-dimethylamino-2-methylphenazine hydrochloride, Basic
Red 5, Toluylene red, NR) is a lipophilic free base used for staining in histology,
as a pH indicator in the range of 6.8-8.0 and for measuring cell viability [
10
,
162
,
26
].
It can be measured by light absorbance (at 540 nm) but also by fluorescence
(488 nm extinction, 590-600 nm emission). After addition, NR is taken up by
Search WWH ::
Custom Search