Sensitivity evaluation of Sterlab reconstructed human oral epithelium (RHO) using an oral irritation assay
RESULTS AND CONCLUSIONS
INTRODUCTION
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To assess the safety of products for oral and dental hygiene, Sterlab developed oral epithelia from the cell line TR146.
This model allows the in vitro evaluation of irritation potential of products prior to clinical testing. To ensure the
sensitivity of Sterlab epithelia, an oral irritation test is conducted on the reconstructed tissue.
B, C, D, E conditions show cytotoxic effects related to exposure time and concentration
Results of the condition A confirm non-irritating effect of sodium bicarbonate (NaHCO 3)
The different concentrations of H2O2 exhibit a dose-dependent effect on tissue viability.
Table 2: Viability for RHO tissues
MATERIALS AND METHODS
The Sterlab RHO model is a three-dimensional, multilayer culture reconstructed from
the cell line TR146 of human keratinocytes that having equivalent histology to an
in vivo tissue.
Triton X100 1%
Exposition (h)
Figure 1 : Sterlab Reconstructed Human Oral (RHO) epithelium
• RHO tissues aged of 5 days were incubated overnight in 6-well plates containing 1 mL of growth medium at 37 ± 1 ° C in
a humidified atmosphere containing 5 ± 1% CO 2 in air.
0.33
2
NAHCO3 2%
24
H2O2 2%
0.25
4
H2O2 6%
0.25
Designation
Negative control
Positive control
A
B
C
D
E
Untreated tissues
Triton X100
NaHCO3
H2O2
H2O2
H2O2
H2O2
• After treatment, the tissues are extensively rinsed with PBS (without Ca ⁺ ⁺ and without Mg ⁺ ⁺)
• Tissues are then transferred to wells containing 300 µl of a 0.5mg/mL MTT solution and incubated for 3
hours at 37 ° C, 5% CO2.
• After incubation, the tissues are blotted on sterile absorbent paper to remove excess liquid and are
deposited in the wells of a 24-well plates containing 0.8 mL of isopropanol. Then, 0.7 mL of isopropanol is added
to each tissue and incubated for two hours at room temperature.
• After incubation, the extraction solutions are mixed and the absorbance is measured at 550 nm
• Absorbance tissue is determined by subtracting the mean of the blank:
Corrected OD at 550 nm = OD obtained at 550 nm – Blank mean OD at 550 nm
• The percentage of cell viability was calculated for each exposure time:
% viability =
Corrected DO at 550 nm
–----------------------------------------------------------- x 100%
Corrected mean DO550 of negative control
4
75.19% 61.99% 51.41% 35.90% 31.74% 9.22% 49.15% 26.49%
Sterlab 60 µl
75.73% 14.69%
77.87%
62.29% 9.79% 56.25% 9.78% 52.99% 8.19% 32.39%
0.8
0.7
0.6
0.5
0.4
0.3
0.2
Viability by MTT assay:
0.25
92.34%
0.9
Viability
Condition
Sterlab tissues
[C]
Time
1, 4 and 24h
1%
0.33 and 2h
2%
24h
2%
0.25 and 4h
6%
0.25 and 4h
9%
0.25 and 4h
11.4%
0.25 and 4h
4
79.58% 19.42%
1
Table 1: Description of samples tested for the RHO model
0.25
H2O2 11.4%
Sterlab 30 µl
• Prior to testing, the growth medium is changed for each tissue.
• Two tissues were treated topically with 30 or 60 µl of each formulation at each exposure time, and then the viability
was determined for each tissue by MTT assay.
4
H2O2 9%
0.1
0
Sterlab Viability 30 µl
Sterlab Viability 60 µl
5.32%