Protocol to reconstruct an in vitro 3D full-thickness skin equivalent model with collagen I in 6- and 12-well inserts

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.

Keywords: Cell Biology; Cell Differentiation; Cell culture; Tissue Engineering.

Graphical abstract

Figure 1

Bright-field image of 3T3-J2 fibroblasts At this near confluent density, the cells are ready for detachment and gamma irradiation. Scale bar, 150 μm.

Figure 2

Bright-field image of human fibroblasts and keratinocytes Both images show optimal density for passaging. Typically, (A) human fibroblasts appear as long and slender, while (B) proliferative human keratinocytes appear as small, round and adhering with each other. The irradiated 3T3-J2 feeder cells are labeled with red asterisks, while the differentiated keratinocytes are labeled with green asterisks. Scale bar, 150 μm.

Figure 3

Severe contraction accompanied with abnormal skin morphology occurs with 12-well insert (A) Top view images of three skin tissues reconstructed with 12-well inserts cultured in the absence of chitosan on the 14^th day of air lifting. 13-S-017 is the de-identified donor ID. (B) Cross-section H&E staining of the least contracted tissues. Scale bar, 100 μm.

Figure 4

3D skin models reconstructed with donor-matched human fibroblasts and keratinocytes in 6-well inserts (A) Top view images of the 6-well tissues cultured with cells derived from 4 different human donors on the 14^th day of air lifting. 12-S-012, 14-S-001, 17-S-017 and 17-S-028 are the de-identified donor IDs. (B) H&E staining of the 6-well cross section tissues. Scale bar, 50 μm.

Figure 5

3D skin models reconstructed in 12-well inserts with chitosan-collagen I layer (A) Top view images of three skin tissues reconstructed with 12-well inserts in the presence of chitosan on the 14^th day of air lifting. 13-S-017 is the de-identified donor ID. (B) Cross-section H&E staining of the 3 replicate tissues. Scale bar, 50 μm.

Figure 6

Representative immunofluorescence images of K14 (green) and Vimentin (red) expressed in the 6-well tissues 12-S-012, 14-S-001, 17-S-017 and 17-S-028 are the deidentified donor IDs. Scale bar, 100 μm.

Figure 7

Representative immunofluorescence images of K10 (green) and Ki67 (red) expressed in the 6-well tissues 12-S-012, 14-S-001, 17-S-017 and 17-S-028 are the deidentified donor IDs. Scale bar, 100 μm.

Figure 8

Representative immunofluorescence images of Filaggrin (red) expressed in the 6-well tissues 12-S-012, 14-S-001, 17-S-017 and 17-S-028 are the deidentified donor IDs. Scale bar, 100 μm.

Figure 9

Representative immunofluorescence images of Loricrin (green) expressed in the 6-well tissues 12-S-012, 14-S-001, 17-S-017 and 17-S-028 are the deidentified donor IDs. Scale bar, 100 μm.

Figure 10

Representative immunofluorescence images of Claudin-1 (green) expressed in the 6-well tissues 12-S-012, 14-S-001, 17-S-017 and 17-S-028 are the deidentified donor IDs. Scale bar, 100 μm.