Girish K Srivastava 1, 2,
José Carlos Pastor 1, 2
1.
Instituto Universitario de
Oftalmobiologia Aplicada (IOBA), Universidad de Valladolid, Valladolid, Spain.
2.
Centro en Red de Medicina
Regenerativa y Terapia Celular de Castilla y León, Spain.
Abstract
Cytotoxicity test is a primary indictor of
biocompatibility and safety of medical devices. The ISO 10993 has recommended
several in vitro cytototoxicity test methods and parameters. One of them is
possibility to apply phenol as a positive control. Phenol is cytotoxic to cell
and tissue cultures over determined concentrations. The paper describes errors
generated in experimental results if phenol is used for long period in
cytototoxic text methods. The ARPE-19 and L929 cell cultures are prepared in 96
wells plate following ISO 10993 recommendations and exposed to positive,
negative and test samples. Different concentrations of phenol (0.025, 0.1, 0.2.
0.4, 1.6 mg/ml as well as 1x (1.6 mg/ml), 3x, 66.6% of 1x and 3x) are prepared
with culture medium and used in different experimental plans. The cell cultures
were exposed, following direct contact cytotoxicity method, directly to phenol
concentration for 30 and 60 minutes and then grown for 24 and 72 hours, or
were, following extract contact cytotoxicity method, incubated 72 hours with
phenol concentrations. The cell culture viability was measured using the MTT
and XTT assay. The data are analyzed statistically and errors generated due to
phenol samples were detected. The results showed the cell culture lost their
viability (<1%) at 1.6 mg / ml concentration of phenol on 30 minutes
exposure. On short period exposure (up to 60 minutes in this study), there were
no effects on viability of cell cultures of neighboring wells of a culture
plate. However, where cell cultures were incubated for a long period, 72 hours,
with phenol, the cell culture of neighboring wells also lost their viability.
This lost decreases as far as wells are from the wells that contained phenol.
The results showed the L929 cell cultures were more sensitive to phenol than
ARPE-19 cell cultures. The study concludes that including phenol presence as a
positive control for long period in an experimental plan can generate errors in
data; which concludes into false positive for a test sample.
Keywords: Phenol, cytotoxicity, ISO
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Publication
Ms. Ref. No.: A0101003
Reviewer 1 Reviewer 2
Received: 23022017
Accepted: 03042018
Article in Press: 04042018
Article in Press: 04042018