2.3. Disadvantages of cytotoxicity tests

the in vivo cells. Also, they are very sensitive to any effect during cultivation and they have a determined lifespan, meaning that they can only endure a limited number of passages. Secondary cell lines are immortalized by some method, which means that they have a hypothetically infinite lifespan. However, we can say from our own experience that for example Caco-2 cells are best suited for transport experiments (where they have to create a monolayer on an insert) between the 20th and the 30th passages. After 50 passages, the cells are hardly able to

Also, it is crucial to choose the right cell line for the given experiment. If the question is the biocompatibility of a chemical compound, which is about to be used on humans, then a human cell line must be chosen for the given experiment and even, an appropriate organ should be

As it can be seen, there are multiple available cell lines with the same origin. The selection and the test system must be based on the later application of the device/compound, as each cell line has a different medium requirement and cultivation method as they can act differently in cell

Also, because anti-proliferative drugs main attribute is their cytotoxicity, the following methods are ideal for testing these substances on cell lines. Because the secondary, immortalized cells can be seen as cancer cells (because their apoptotic or growth stop signals are suppressed by mutations), they are capable to react to certain promising anti-cancer molecules like their in vivo

As scientific and medical studies are getting more expensive over the recent decades, most of the universities, research institutes are underfinanced, the importance of a certain method's price is greater than ever before. Animal experiments are expensive and the administrative burden is overwhelming, so they are only used when no other test is suitable. Also, every year new plants and their respective metabolites are described and through the various methods of

HeLa, A431 Epithelial cervical cancer Very rapid growth, cells commonly used in cancer research Caco-2, HT29, HCT-116 Colon adenocarcinoma Studies of absorption via intestinal epithelium, toxicity tests

16HBE140 Bronchial epithelium Studies of absorption and excretion through the bronchial

epithelium

Breast cancer Studies of transport and absorption of drugs, screening anticancer compounds

respiratory epithelium

Studies of absorption via bronchial epithelium, metabolic and transport model to study drug delivery to the

U2OS Osteosarcoma cells Studies of transport and absorption of drugs

HaCaT Adult human skin Penetration of drug through the skin

reproduce their own number, thus, they are no longer sufficient for cell viability tests.

selected. A good summarizing table was created by Amelian et al. [20] (Table 1).

viability tests.

134 Cytotoxicity

counterparts.

SkBr3, MCF-7, MDA-MB231,

ZR-75-1

2.2. Advantages of cytotoxicity tests

Cell line Origin Application

Calu-3 Serous cells of submucosal

gland

Table 1. Most common cell lines used in cytotoxicity studies [20].

The results of the cytotoxicity tests require additional consideration. They—even if the right cell lines were used—are not an automatic green light for in vivo application. If a given chemical proves to be non-toxic, it only means that we do not necessarily have to start our next experiment in an animal with the smallest dosage, but from a medium or a high concentration, to determine the maximum single dose, maximum daily dose and the LD50 value. Also, if we do not use the appropriate cell line—like testing an ointment preservative on enterocytes—then the scientific value of our study will be questioned. The cytotoxicity tests usually end up in a specific IC50 value. These values usually cannot be compared, because these tests are highly dependent on the parameters of the test system. Such parameters can be: cell line, passage number of the cells, number of cells/well, volume of medium/well, growth time of a plate, concentration/volume of reagent, manufacturer of the reagent, length of incubation, reaction time, solubilization solution (if needed) even the performance of the spectrophotometer. We suggest that instead of using static IC50 values, trends, comparisons should be seen. Stating that compound A has an IC50 value of 0.5 mg/ml and B has 0.25 mg/ml should be changed that A is about twice as tolerable, than B. This fact does not decrease the significance of the cytotoxicity tests, but prevents to deny of a scientifically accurate study, because the written IC50 value cannot be reproduced. Another issue is that a specific compound can interact with the reagents or the mechanism of the method, thus a false positive or negative result can be detected. Such interaction can only be found if we use more than one method or in the scientific literature. If we only use one type of cytotoxicity test, the scientific value will be low and the chance of detecting false results will be high. This is not necessarily caused by a specific interaction, but because of the given test system, the whole method will over- or underestimate cytotoxicity. Thus, it is advisable to use different types of tests, so link MTT with LDH or RT-CES, but not with XTT, because they are both tetrazolium based assays and have the same limitations.
