Cytotoxicity assay

In vitro cytotoxicity assays using isolated cells have been applied intermittently to cyanobacterial toxicity testing over several years." Cells investigated for suitability in cyanobacterial toxin assays include primary liver cells (hepatocytes) isolated from rodents and fish, established permanent mammalian cell lines, including hepatocytes, fibroblasts and cancerous cells, and erythrocytes. Earlier work suggested that extracts from toxic cyanobacteria disrupted cells of established lines and erythrocytes," but studies with purified microcystins revealed no alterations in structure or ion transport in fibroblasts or erythrocytes,... 

The authors are grateful to Professor Dr. Lucymara Fassarela Agnez Lima and Acarizia Eduardo da Silva for the contribution with the cytotoxicity assay. 

ICso is defined as the concentration of drug required to inhibit cell growth by 50% compared to a control. The IC50 values were calculated from the graphs obtained from the in vitro cytotoxicity assays and are the average of three independent experiments, each performed in triplicate. 

We decided to apply the elimination-based dendritic system to the synthesis of an anticancer prodrug and to evaluate it in a tumor cell cytotoxicity assay. Dendritic prodrugs 20 and 21 were synthesized with the chemotherapeutic drug melphalan as a tail unit and a trigger that is activated by PGA (Fig. 5.14). 

H. Gazzano-Santoro, P. Ralph, T. Ryskamp, A. Chen, and V. Mukku, A non-radioactive complement-depen-dent cytotoxicity assay for anti-CD20 monoclonal antibody, J. Imunol. Methods, 202, 163 (1997). 

Cytotoxicity assay to evaluate the PFCs toxicity and to select the concentrations for the transformation assay... 

Fig. 7 BALB/c 3T3 cytotoxicity assay red bar (NT) is untreated cells while green bar (DMSO) is the vehicle control...

Data was reported as transformation frequency (TF), calculated on the cells that survived after chemical exposure. TF is expressed as a function of the total number of foci per treatment divided by the number of surviving cells estimated from the clonal efficiency observed in the cytotoxicity assay performed in parallel with the transformation test [52],... 

Several factors are associated with the difficulty in assessing CNT toxicity. Among them, CNT interactions with components of the dispersing medium, the cytotoxicity assay employed, impurities, CNT surface chemistry, and dispersion state are the... 

Table 7.1 S ummary of cytotoxicity assays used in in vitro toxicity studies on CNTs. 

The calcein-AM assay [82-84] and cytotoxicity assays (e.g., performed with doxorubicin) [77, 78] are both basically competition assays. The accumulation of a primary substrate (e.g., calcein-AM or doxorubicin) in the cytosol of living cells is measured after addition of a second substrate (also called modifier or reverser) that reduces the efflux of the primary substrate. In the case of the calcein-AM assay, the primary substrate, calcein-AM, is hydrolyzed as soon as it reaches the cytosol, and the highly fluorescent hydrolysis product (calcein) can be determined using fluorescence spectroscopy. The more effective the reversal agent, the stronger is the increase in calcein fluorescence. Data can be quantified in terms of inhibitory constants, IQ, of the reversal agent. 

In cytotoxicity assays the concentration of the cytotoxic compound reaching the cytosol is estimated via its apparent toxicity. Cytotoxicity is expressed as the concentration that inhibits the growth of the MDR-expressing cells by 50% or 20% (known as IC50 or IC2o values, respectively), and indicates whether the secondary, co-administered compound is a substrate or a modulator of P-gp. The activity of the reversal agent is generally expressed as a fold reversion called the MDR ratio [36] ... 

Cell cytotoxicity assays with the encapsulated L929 cells were performed. Methanol and CoCl2 solutions were used as toxins. Whereas methanol is a strong... 

Fig. 8.8 Macrophage cytotoxic assays of plant extracts. Supernatant samples were tested from Trgeneration of chloroplast transgenic line pLD-JWl (proteins were extracted in buffer containing no detergent and MTTwas added after 5 hours). pLD-JWl (extract stored for 2 days) pLD-JWl (extract stored for 7 days) -> PA 5 pg ml-1 —x— Control wild type (extract stored for 2 days) ...

Cell-mediated immunity Mitogen-induced T-cell proliferation, NK cell activity Spontaneous cytotoxicity assays... 

Figure 29) was tested in several cell lines in a one-dose in vitro primary cytotoxicity assay, and passed the criteria for activity (20-29% growth percentages) next, it was scheduled for evaluation against the full panel of 60 human tumor cell lines at minimum of five concentrations at 10-fold dilution, showing very favorable cytotoxicity <2003EJM781>. 

The trypsinized cultures are counted and a sample is assessed for survival as for the cytotoxicity assay. In addition, an appropriate number of cells are reseeded for estimation of mutation frequency at the day 8 expression time. The cells are transferred to roller bottles (usually 490 cm2) for this stage. The bottles are gassed with pure CO2, the tops are tightened and the bottles are incubated at 37°C on a roller machine (approximate speed 0.5-1.0 rev min-1). Usually 106 7 8 9 viable cells are reseeded in 50 ml of Eagle s medium containing serum, but more cells are required at the toxic dose levels. 

For estimation of cloning efficiency and mutant induction, cells are plated out in 96-well microtiter plates. Flasks and microtitre plates are incubated at 37°C in a C02 incubator as in the cytotoxicity assays. 

Cell suspensions of exponentially growing cells are prepared as in the cytotoxicity assay, except that 6 ml of media required for each treatment culture contains 107 cells (3-h treatment) or 3 x 106 cells (more than 3 h treatment). The number of cells per treatment may be increased if marked cytotoxicity is expected, to allow enough cells to survive (e.g., if 20% survival or less is expected, 2 x 107 cells may be treated). 

For survival estimation, cells are placed into 96-well microtitre trays at a cell density of 1 cell per well as per the cytotoxicity assay. 

The calculation for cloning efficiency is made as for the cytotoxicity assay. 

The test material, test cells, used, method of treatment, harvesting of cells, cytotoxicity assay, and so on, should be clearly stated as well as the statistical methods used. Richardson et al. (1989) recommend that comparison be made between the frequencies in control cells and at each dose level using Fisher s Exact Test. 

Thus, cytotoxicity assays are unlikely to provide an adequate predictor of in vivo irritation in every case. There is no doubt that some type of compounds (such as surfactants) will give (and have given) acceptable correlations with in vivo data. However, for the diversity of compounds common in the pharmaceutical industry, cytotoxicity assays alone are inadequate predictors of ocular irritation, though they may have a place in a battery of tests. For instance, if one is interested in a very quick assessment that will be corroborated later, cytotoxicity assays may be indicated. But each laboratory needs to make its own evaluation of the utility and value of these methods. 

Additionally, the test materials used in the validation process should be as closely related as possible to the characteristics of the unknowns to be tested. It is clear from the literature, for instance, that many cytotoxicity assays give good correlations with the in vivo ocular irritancy data for surfactants, but the correlations fail when compounds from other chemical classes are tested. Since any particular assay may be used differently by individual safety assessment programs, users must evaluate potential methods under conditions likely to be encountered in their own situations. 

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