Multi-layered culture of human skin fibroblasts and keratinocytes through three-dimensional freeform fabrication

Biomaterials. 2009 Mar;30(8):1587-95. doi: 10.1016/j.biomaterials.2008.12.009. Epub 2008 Dec 23.


We present a method to create multi-layered engineered tissue composites consisting of human skin fibroblasts and keratinocytes which mimic skin layers. Three-dimensional (3D) freeform fabrication (FF) technique, based on direct cell dispensing, was implemented using a robotic platform that prints collagen hydrogel precursor, fibroblasts and keratinocytes. A printed layer of cell-containing collagen was crosslinked by coating the layer with nebulized aqueous sodium bicarbonate. The process was repeated in layer-by-layer fashion on a planar tissue culture dish, resulting in two distinct cell layers of inner fibroblasts and outer keratinocytes. In order to demonstrate the ability to print and culture multi-layered cell-hydrogel composites on a non-planar surface for potential applications including skin wound repair, the technique was tested on a poly(dimethylsiloxane) (PDMS) mold with 3D surface contours as a target substrate. Highly viable proliferation of each cell layer was observed on both planar and non-planar surfaces. Our results suggest that organotypic skin tissue culture is feasible using on-demand cell printing technique with future potential application in creating skin grafts tailored for wound shape or artificial tissue assay for disease modeling and drug testing.

MeSH terms

  • Cell Culture Techniques / methods*
  • Cell Survival / drug effects
  • Collagen / metabolism
  • Cross-Linking Reagents / pharmacology
  • Dimethylpolysiloxanes / metabolism
  • Fibroblasts / cytology*
  • Fibroblasts / drug effects
  • Humans
  • Hydrogels
  • Keratinocytes / cytology*
  • Keratinocytes / drug effects
  • Models, Biological
  • Skin / cytology*


  • Cross-Linking Reagents
  • Dimethylpolysiloxanes
  • Hydrogels
  • Collagen