Constitutive release of cytokines by human oral keratinocytes in an organotypic culture

Abstract

Purpose: The Food and Drug Administration requires an accurate determination of the dose and potency of tissue-engineered or combination products as is required for drugs. This needs to be done as a rapid, quantitative, and noninvasive measurement of biologic function/activity in a way so as not to perturb the tissue-engineered product being developed. The aim of this study was to correlate constitutive release of cytokine(s) from unstimulated cells, at different stages of development, within a 3-dimensional (3D) organotypic ex vivo produced oral mucosa equivalent (EVPOME) to be used for intraoral grafting, with oral keratinocyte cell viability of the EVPOME.

Materials and methods: Tissue culture medium was assayed with an enzyme-linked immunosorbent assay from monolayer culture of oral keratinocytes and a 3D EVPOME to determine the constitutive release of interleukin (IL) 1alpha, IL-6, IL-8, and vascular endothelial growth factor (VEGF). VEGF messenger ribonucleic acid expression by oral keratinocytes within the 3D EVPOME was detected by in situ hybridization at days 4, 7, and 11. The number of viable oral keratinocytes within the EVPOME was extrapolated from VEGF release by use of a modified MTT assay.

Results: Both VEGF release level and the number of viable cells in the monolayer cultures and 3D EVPOME as measured by MTT assay significantly increased in a time-dependent manner (P < .001, r = 0.743).

Conclusion: These results suggest that the increasing detectable levels of VEGF associated with the increasing number of viable cells in the EVPOME may provide a useful noninvasive/nondestructive means of assessing both cellular viability (dose) and biologic function/activity (potency) of a combination cell-based device such as the EVPOME.

FIGURE 1

Representative MTT assay for a primary cell culture for oral keratinocytes (a) Evidence of the presence of MTT formazan crystals developed in a monolayer of cultured viable oral keratinocytes (arrows). Note absence of formazan crystals in non-viable cells (arrowheads). (b) A typical calibration curve for primary cells showing a linear relationship between optical densities (cell viability) and the known plated cell numbers.

FIGURE 2

Histology of: (a) Day 4, (b) Day 7, and (c) Day 11 EVPOMEs. Note difference in stratification of the epithelial layer that is correlated with the number of cells and thickness of basal and parabasal cell layers. (Hematoxylin and eosin staining; Scale bar = 50μm)

FIGURE 3

Detection of cell viability on the EVPOME using a MTT assay. (a) Formazon crystals (arrows) from the MTT assay are visible and localized in the basal and suprabasal layer cells of a Day 11 EVPOME. (Frozen section, Scale bar=50um; Original magnification; × 200) (b) An increase in numbers of viable cells within the EVPOME is detected as the culturing period (day 4, 7 and 11) and increase in stratification of the epithelial layer occurs. A repeated-measure one-way ANOVA and the Tukey post-hoc test were used to compare differences among these three groups. Significant differences between each two groups were observed (*, p< 0.001, n =20).

FIGURE 4

Time-dependent release of VEGF by oral keratinocytes on the EVPOME. A repeated-measure one-way ANOVA and the Tukey post-hoc test were used to compare differences among these three groups. Significant differences between each two groups were observed (*, p< 0.001, n =20).

FIGURE 5

Significant correlation was observed between cell viability, days in culture and VEGF release into the culture medium. Correlation coefficient was 0.743 (p< 0.001).

FIGURE 6

In situ hybridization of Day 11 EVPOME. (a) anti-sense probe: Note VEGF mRNA expression is localized in basal and parabasal cell layers. The uppermost keratinized layer lacked any expression of VEGF mRNA. (b) sense probe: As a negative control, hybridization with the VEGF sense probe does not show any signal. (Scale bar = 50um; Original magnification; x400)