Development of a 3D human in vitro skin co-culture model for detecting irritants in real-time.
Tissue engineered materials for clinical purposes have led to the development of in vitro models as alternatives to animal testing. The aim of this study was to understand the paracrine interactions arising between keratinocytes and fibroblasts for detecting and discriminating between an irritant-induced inflammatory reaction and cytotoxicity. We used two irritants [sodium dodecyl sulphate (SDS) and potassium diformate (Formi] at sub-toxic concentrations and studied interleukin-1 alpha (IL-1 alpha) release from human keratinocytes and activation of NF-kappaB in human fibroblasts. NF-kappaB activation in fibroblast 2D cultures required soluble factors released by prior incubation of keratinocytes with either SDS or Formi. Neither cell type responded directly to either agent, confirming a paracrine mechanism. Fibroblasts were then cultured in 3D microfiber scaffolds and transfected with an NF-kappaB reporter construct linked to GFP. Findings for 3D cultures were similar to those in 2D in that soluble factors released by prior incubation of keratinocytes with SDS or Formi was required for NF-kappaB activation in fibroblasts. Similarly, direct incubation with either agent did not directly activate NF-kappaB. A technical advantage of using transfected cells in 3D was an ability to detect NF-kappaB activation in live fibroblasts. To confirm paracrine signaling a twofold increase in IL-1 alpha was measured in keratinocyte-conditioned medium after incubation with SDS or Formi, which correlated with fibroblast NF-kappaB activity. In summary, this work has value for developing 3D tissue engineered co-culture models for the in vitro testing of irritant chemicals at sub-toxic concentrations, as an alternative to in vivo models.
Cantòn
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Department of Engineering Materials, University of Sheffield, Kroto Research Institute, Broad Lane, Sheffield, S3 7HQ, UK.
Cole
D M
DM
Kemp
E H
EH
Watson
P F
PF
Chunthapong
J
J
Ryan
A J
AJ
MacNeil
S
S
Haycock
J W
JW
D018929:Cell Culture Techniques; D018920:Coculture Techniques; D005347:Fibroblasts / Q000187:drug effects; D017930:Genes, Reporter; D006801:Humans; D007509:Irritants / Q000633:toxicity*; D015603:Keratinocytes / Q000187:drug effects; D016328:NF-kappa B / Q000378:metabolism; D009924:Organ Culture Techniques; D012867:Skin / Q000187:drug effects*; D013997:Time Factors
D007509:Irritants; D016328:NF-kappa B