Recellularized human dermis for testing gene electrotransfer ex vivo

Abstract

Gene electrotransfer (GET) is a proven and valuable tool for in vivo gene delivery to a variety of tissues such as skin, cardiac muscle, skeletal muscle, and tumors, with controllable gene delivery and expression levels. Optimizing gene expression is a challenging hurdle in preclinical studies, particularly for skin indications, due to differences in electrical conductivity of animal compared to human dermis. Therefore, the goal of this study was to develop an ex vivo model for GET using recellularized human dermis to more closely mimic human skin. Decellularized human dermis (DermACELL(®)) was cultured with human dermal fibroblasts and keratinocytes for 4 weeks. After one week of fibroblast culture, fibroblasts infiltrated and dispersed throughout the dermis. Air-liquid interface culture led to epithelial cell proliferation, stratification and terminal differentiation with distinct basal, spinous, granular and cornified strata. Firefly luciferase expression kinetics were evaluated after GET of recellularized constructs for testing gene delivery parameters to skin in vitro. Elevated luciferase expression persisted up to a week following GET compared to controls without electrotransfer. In summary, recellularized dermis structurally and functionally resembled native human skin in tissue histological organization and homeostasis, proving an effective 3D human skin model for preclinical gene delivery studies.

Figure 1.

Diagram of air–liquid interface culture of recellularized dermis.

Figure 2.

Decellularized dermis maintains its basement membrane and supports fibroblast attachment and infiltration from reticular seeding surface only. (A) and (B) Fibroblasts seeded on the papillary surface remained on papillary surface, (DAPI stain for cell nuclei), (B) brightfield image merged with DAPI. (C) DermACELL® without cells is stained for collagen IV (green). (D) Fibroblast infiltration into decellularized dermis after seeding on the reticular surface and one week of cell culture, with fibroblasts stained with vimentin (red). Scale bar = 200 μm. Stars indicate the location of the dermis, while arrows point to the papillary surface and basement membrane.

Figure 3.

Recellularized dermis supports keratinocyte polarization and stratification forming structured epithelium. Serial sections were stained either with hematoxylin and eosin (top row), pan keratin (middle row) for HaCaT keratinocytes, or collagen IV for basement membrane (bottom row). Scale bar = 20 μm.

Figure 4.

Recellularized dermis supports keratinocyte proliferation and differentiation forming functional epithelium. Serial sections were stained for Ki67 for proliferating cells in top and middle rows (merged DAPI and FITC channels or FITC channel alone respectively), or for involucrin for terminal differentiation in bottom row. Scale bar = 20 μm.

Figure 5.

Gene electrotransfer of recellularized dermis enhances luciferase expression. GET + pLuc groups have significantly higher expression than no GET groups for each time point (p < 0.05).

Figure 6.

Metabolic activity of recellularized dermis after gene electrotransfer is reduced compared to no electrotransfer groups for each time point (p < 0.001). There was no significant difference in metabolic activity between GET groups at any time point. There were no significant differences in metabolic activity between no electrotransfer control groups at any time point.