Protocol to reconstruct an in vitro 3D full-thickness skin equivalent model with collagen I in 6- and 12-well inserts
Year: 2025
Type: article
Abstract: In vitro 3D full-thickness reconstituted human skin has high physiological relevance due to the presence of differentiation features often lacking in 2D cell cultures. Here, we present a protocol to reconstruct a 3D skin model using human fibroblasts, keratinocytes, and rat-tail collagen I. We describe steps for cell expansion, the casting of cellular and acellular layers, seeding keratinocytes, the air lifting of culture, and incubation. We also demonstrate the use of chitosan to prevent tissue contraction in 12-well inserts. For complete details on the use and execution of this protocol, please refer to Robinson et al. 1 , 2 • Protocol for 3D skin reconstruction using fibroblasts and keratinocytes • Instruction for 2D expansion of human fibroblast and human keratinocyte cells • Differential steps for 6-well and 12-well format of 3D skin culture Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. In vitro 3D full-thickness reconstituted human skin has high physiological relevance due to the presence of differentiation features often lacking in 2D cell cultures. Here, we present a protocol to reconstruct a 3D skin model using human fibroblasts, keratinocytes, and rat-tail collagen I. We describe steps for cell expansion, the casting of cellular and acellular layers, seeding keratinocytes, the air lifting of culture, and incubation. We also demonstrate the use of chitosan to prevent tissue contraction in 12-well inserts.
Source: STAR Protocols
Language: English
Subfield: Cell Biology
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $1,550
Awards: G1415157288, G3486710717, G775246491
OpenAlex ID: W4408134395