title: Multi-layered culture of human skin fibroblasts and keratinocytes through three-dimensional freeform fabrication.
abstract: 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.
authors.affiliation: Department of Radiology, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Wonhye
authors.initials: W
authors.lastname: Lee
authors.forename: Jason Cushing
authors.initials: JC
authors.lastname: Debasitis
authors.forename: Vivian Kim
authors.initials: VK
authors.lastname: Lee
authors.forename: Jong-Hwan
authors.initials: JH
authors.lastname: Lee
authors.forename: Krisztina
authors.initials: K
authors.lastname: Fischer
authors.forename: Karl
authors.initials: K
authors.lastname: Edminster
authors.forename: Je-Kyun
authors.initials: JK
authors.lastname: Park
authors.forename: Seung-Schik
authors.initials: SS
authors.lastname: Yoo
mesh_terms: D018929:Cell Culture Techniques / Q000379:methods*; D002470:Cell Survival / Q000187:drug effects; D003094:Collagen / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004129:Dimethylpolysiloxanes / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology* / Q000187:drug effects; D006801:Humans; D020100:Hydrogels; D015603:Keratinocytes / Q000166:cytology* / Q000187:drug effects; D008954:Models, Biological; D012867:Skin / Q000166:cytology*
chemical_list: D003432:Cross-Linking Reagents; D004129:Dimethylpolysiloxanes; D020100:Hydrogels; D003094:Collagen