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


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).


CONCLUSIONS
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.
Xu
Qin
Q
Department of Oral and Maxillofacial Surgery, University of Michigan, Ann Arbor, MI 48109-0018, USA.
Izumi
Kenji
K
Tobita
Takayoshi
T
Nakanishi
Yoshitaka
Y
Feinberg
Stephen E
SE
D002470:Cell Survival; D004396:Coloring Agents; D017077:Culture Media, Conditioned; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D017403:In Situ Hybridization; D053582:Interleukin-1alpha / Q000032:analysis / Q000378:metabolism; D015850:Interleukin-6 / Q000032:analysis / Q000378:metabolism; D016209:Interleukin-8 / Q000032:analysis / Q000378:metabolism; D007378:Interleukins / Q000032:analysis / Q000378:metabolism*; D015603:Keratinocytes / Q000378:metabolism*; D009061:Mouth Mucosa / Q000166:cytology / Q000378:metabolism*; D009924:Organ Culture Techniques; D012333:RNA, Messenger / Q000032:analysis; D013778:Tetrazolium Salts; D013844:Thiazoles; D013997:Time Factors; D023822:Tissue Engineering; D054457:Tissue Scaffolds; D042461:Vascular Endothelial Growth Factor A / Q000032:analysis / Q000235:genetics / Q000378:metabolism*
D004396:Coloring Agents; D017077:Culture Media, Conditioned; D053582:Interleukin-1alpha; D015850:Interleukin-6; D016209:Interleukin-8; D007378:Interleukins; D012333:RNA, Messenger; D013778:Tetrazolium Salts; D013844:Thiazoles; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; C022616:thiazolyl blue