Fully Three-Dimensional Bioprinted Skin Equivalent Constructs with Validated Morphology and Barrier Function

Abstract

This article describes a method for the biofabrication of skin tissue equivalents in a multiwell plate format. The technique and results overcome shortcomings of previously published engineering methods, and show good architecture and barrier function from well to well; thus it may be used for compound functional testing and for the development of disease tissue models for screening.

Keywords: barrier function; bioprinting; high-throughput screening; keratinocytes; skin.

FIG. 1.

Schematic of printed construct. (a) Three levels comprise the structure: dermis, laminin/entactin basal layer, and epidermis. (b) Incubation method after printing. BPSEs are incubated for 1.5 h at RT with thrombin/media placed underneath the transwell. Samples are subsequently submerged in a medium and placed in incubator for 1 week. On day 7 medium is lowered and tissues are incubated at air–liquid interface for 1 week. BPSEs, bioprinted skin equivalent.

FIG. 2.

Minimal lateral contraction is observed as tissue matures. Full plate and single-well photos show that tissue maintains full well diameter throughout the time of tissue maturation.

FIG. 3.

Histological comparison of BPSE versus native human skin. (a) H&E staining shows clear layers of differentiated keratinocytes. (b) Dermal cell density of BPSE and human skin was estimated by counting number of nuclei within a 1500 μm² sample area from four representative samples of each tissue type. Density_BPSE = 14,000 ± 4000 cells/mm², Density_Human = 21,000 ± 7000 cells/mm². Scale bars, 100 μm. H&E, Hematoxylin and Eosin.

FIG. 4.

IHC comparison of protein expression between bioprinted tissues and native human skin show comparable morphology. Tight junction proteins ZO-1, Claudin I, and E-Cadherin. Epidermal differentiation proteins Keratin 1, Filaggrin, and Cytokeratin 15. ECM proteins Collagen I and Collagen VII and cell proliferation marker Ki67. Scale bar, 100 μm. ECM, extracellular matrix; IHC, immunohistochemistry.

FIG. 5.

MTT cell viability measurements were taken 2 days after exposure to 5% SDS as PC or water as NC. Mean Optical Density_NC (OD_NC), Ratio of Tissue Viability_PC/NC (TV_PC/NC) and CV were within Acceptance Criteria. n = 6. CV, coefficients of variation; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NC, negative control; PC, positive control; SDS, sodium dodecyl sulfate.

FIG. 6.

Structural Analysis of BPSEs. (a) Confocal imaging of cells in stratum granulosum of BPSE shows double-sided hexagons indicative of f-TKD shape representing TJ formation and allowing the cells to achieve homeostasis of the TJ barrier. Scale bar, 10 μm. (b–d) Optical coherence tomography image of BPSE shows tissue uniformity. (b) 3 × 3 mm² reconstructed section shows uniform distinct tissue layers. (c) Full diameter (∼11 mm) tissue image cross-section and 3D full rendering show extensive epidermal coverage and 3D concave shape. (d) Layer thicknesses are measured for stratum corneum (i), live epidermal layers (ii), and dermis (iii). Scale bar, 200 μm. 3D, three-dimensional; f-TKD, flattened Kelvin's tetrakaidecahedron; TJ, tight junction.

FIG. 7.

Barrier Function Assessment. (a) Electrical conductivity measurement across surface of BPSE. DPM units are arbitrary. Conductivity of SE with epidermis (117 ± 10 DPM, n = 12) is within range of previously reported measurements of native human skin (100 to 168 DPM). BPSE lacking epidermal layer measure conductivity of 831 ± 19 DPM; n = 8. (b) “Inside out” Barrier Function Penetration Assay: Biotin tracer (red) diffused from beneath the tissue does not reach the cornified epidermis. (c) “Outside in” Barrier Function Penetration Assay: Lucifer yellow (green) placed on top of the tissue at days 3 and 14. Scale bar, 100 μm. DPM, Dermal Phase Meter; SE, skin equivalent.