Department of Engineering Materials, University of Sheffield, Kroto Research Institute, Broad Lane, Sheffield, S3 7HQ, UK.
Biotechnol Bioeng. 2010 Aug 1;106(5):794-803. doi: 10.1002/bit.22742.
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.
用于临床目的的组织工程材料已经导致了体外模型的发展，作为动物试验的替代方法。本研究的目的是了解角质形成细胞和成纤维细胞之间产生的旁分泌相互作用，以检测和区分刺激性炎症反应和细胞毒性。我们使用两种刺激性物质[十二烷基硫酸钠（SDS）和二甲醛钾（Formi]在亚毒性浓度下，并研究了人角质形成细胞释放的白细胞介素-1α（IL-1α）和人成纤维细胞中 NF-κB 的激活。成纤维细胞 2D 培养物中 NF-κB 的激活需要角质形成细胞预先与 SDS 或 Formi 孵育释放的可溶性因子。两种细胞类型都没有直接对任何一种物质作出反应，证实了旁分泌机制。然后，将成纤维细胞在 3D 微纤维支架中培养，并转染与 GFP 相连的 NF-κB 报告基因构建体。3D 培养物的发现与 2D 培养物相似，即需要预先孵育 SDS 或 Formi 的角质形成细胞释放的可溶性因子才能激活成纤维细胞中的 NF-κB。同样，直接与任一试剂孵育不会直接激活 NF-κB。在 3D 中使用转染细胞的一个技术优势是能够检测活成纤维细胞中 NF-κB 的激活。为了证实旁分泌信号，在用 SDS 或 Formi 孵育后，在角质形成细胞条件培养基中测量到 IL-1α 的两倍增加，这与成纤维细胞 NF-κB 活性相关。总之，这项工作对于开发用于亚毒性浓度下刺激性化学物质的体外测试的 3D 组织工程共培养模型具有重要价值，可替代体内模型。
