Design and fabrication of human skin by three-dimensional bioprinting

Abstract

Three-dimensional (3D) bioprinting, a flexible automated on-demand platform for the free-form fabrication of complex living architectures, is a novel approach for the design and engineering of human organs and tissues. Here, we demonstrate the potential of 3D bioprinting for tissue engineering using human skin as a prototypical example. Keratinocytes and fibroblasts were used as constituent cells to represent the epidermis and dermis, and collagen was used to represent the dermal matrix of the skin. Preliminary studies were conducted to optimize printing parameters for maximum cell viability as well as for the optimization of cell densities in the epidermis and dermis to mimic physiologically relevant attributes of human skin. Printed 3D constructs were cultured in submerged media conditions followed by exposure of the epidermal layer to the air-liquid interface to promote maturation and stratification. Histology and immunofluorescence characterization demonstrated that 3D printed skin tissue was morphologically and biologically representative of in vivo human skin tissue. In comparison with traditional methods for skin engineering, 3D bioprinting offers several advantages in terms of shape- and form retention, flexibility, reproducibility, and high culture throughput. It has a broad range of applications in transdermal and topical formulation discovery, dermal toxicity studies, and in designing autologous grafts for wound healing. The proof-of-concept studies presented here can be further extended for enhancing the complexity of the skin model via the incorporation of secondary and adnexal structures or the inclusion of diseased cells to serve as a model for studying the pathophysiology of skin diseases.

FIG. 1.

Printing scheme employed for the generation of single-cell cultures. Two layers of collagen were printed (i, ii) followed by the printing of a one-cell layer (iii). The dimensions of collagen and cell layers were maintained at 6×6 mm and 4×4 mm, respectively. FB, fibroblasts; KC, kerotinocytes. Color images available online at www.liebertpub.com/tec

FIG. 2.

Construction of three-dimensional (3D) skin tissue. (a) Layer-by-layer printing of collagen matrix, KCs, and FBs to construct the dermal and epidermal compartments in a single structure. (b) Schematic of the 3D printed skin tissue showing the cross-section (left) and top view (right). Color images available online at www.liebertpub.com/tec

FIG. 3.

Optimization of printing parameters for individual cell cultures. Cell suspension density and resolution (spacing between droplets) were varied over a broad range for FBs (a–f) and keratinocytes (KCs) (g–l) printed in monolayers. Cell viability was assessed at each condition after 7 days in culture (m, n). Color images available online at www.liebertpub.com/tec

FIG. 4.

Confocal microscopy imaging of cultured skin. (a) 3D reconstruction of confocal microscopy images of printed skin after 7 days in submerged culture conditions. Live cell nuclei are stained green and present compact and rounded (KC) or large and elongated (FB) morphologies. (b, c) Compressed z-projections of the epidermis and dermis showing KCs and FBs. Color images available online at www.liebertpub.com/tec

FIG. 5.

Thickness of printed skin tissues. Tissue thickness varied significantly over the course of the culture period (3 weeks). A dramatic reduction (2 to 6-fold) in skin thickness was observed at the transition from submerged culture conditions to culture at the air–liquid interface.

FIG. 6.

Shape and form of printed skin tissue. A comparison of skin tissues fabricated via 3D bioprinting and manual deposition indicates that printed skin samples (a, b) retain their form (dimensions) and shape, whereas manually deposited structures (c, d) shrink and form concave shapes (buckle) under submerged culture condition after 7 days. Color images available online at www.liebertpub.com/tec

FIG. 7.

Histology of skin tissue. Printed skin cultures were characterized using hematoxylin and eosin and nuclear staining at day 7 (a, b) and day 14 (c, d) after air–liquid interface (ALI) culture. The epidermal layer is very dense and exhibits compaction with time. The dermis is sparsely populated and shows significant shrinking and compaction with time. Color images available online at www.liebertpub.com/tec

FIG. 8.

Immunofluorescence of skin tissue. Printed skin structures were stained for N-cadherin tight junctions at day 14 of ALI culture. N-cadherin (green) was observed bordering adjacent epidermal cells (nuclei stained in blue), but it was not detected in the dermal compartment. Color images available online at www.liebertpub.com/tec