A Novel Skin Whole Organ Culture Technique Maintains In Vivo Cellular Characteristics and Population Profiles

PURPOSE: In reconstructive surgery, large areas of tissue loss represent a major surgical obstacle. Where split-thickness skin grafting and flap transfers fail, cell-based treatments represent a promising therapeutic option. Currently, cell therapies are limited to transplants from non-autologous donors, or expanded isolated skin components (e.g., keratinocytes only). However, literature suggests that keratinocytes and fibroblasts act synergistically to restore functional tissue.¹ Typical culture conditions poorly mimic in vivo conditions, and skin cells change dramatically after plating.² Thus, there is a demand for techniques to expand multiple autologous cell types without fundamentally altering cell behavior. Here, we describe methods for the ex vivo culture of skin cells that allow for efficient expansion while maintaining in vivo cell characteristics.

 

METHODS: Adult mouse skin was harvested and sterilized using gradated iodine solutions. Tissue was chopped with sterile scissors followed by digestion with 0.5 mg/mL Liberase^TM DL (Roche). Cells were grown in DMEM/F12 with 10% fetal bovine serum and 1% penicillin-streptomycin, on polystyrene coated with 0.1% gelatin (EmbryoMax) or in 3D collagen hydrogels of varying stiffness. Morphology was assessed via imaging and analysis using Photoshop CS6 (Adobe). Relative cell populations were quantified using fluorescence-activated cell sorting (FACS). Isolation of Engrailed-positive fibroblasts (EPFs), the dermal fibroblast population responsible for wound healing (collagen deposition), was achieved by FACS of cells from En1^Cre;R26^mTmG mice.

 

RESULTS: Skin cells grown via whole organ culture on gelatin-coated polystyrene had no significant change in resident cell population density over multiple passages (2-4% fibroblasts; of non-fibroblasts, 50-60% blood cells; remainder keratinocytes; P>0.05). Upon isolation from whole organ culture, fibroblasts of a single population (EPFs) demonstrated expansion by over 20-fold in two passages. With traditional culture methods, fibroblasts demonstrate increased cell size over repeated passages; in contrast, these phenotypic shifts in EPFs were rescued by culturing in 3D hydrogels or on gelatin-coated polystyrene. Specifically, EPFs grown on gelatin-coated polystyrene demonstrated no significant change in cell size from passage (P)1 to P3 (average fold change=0.879, n=3 biological replicates, all P>0.05).

 

CONCLUSIONS: By removing many of the artificial selection pressures that cells experience in culture, we accomplished efficient ex vivo expansion of in vivo-like skin cells. Specifically, by employing whole organ culture rather than culturing cells in isolation, nonselective media, and 3D hydrogels to mimic in vivo mechanical tensions, cells retained their in vivo morphology and population densities. Autologous cell-based therapies hold increasing promise for complex reconstructive surgery, and our results signify a therapeutically relevant advancement that may enable improved cosmesis and functionality of transplanted skin organs. With similar expansion of human skin, a 4mm punch biopsy alone could yield the equivalent of over 250 mm² of skin for transplantation. In the future, we will verify our technique using epigenetic studies and machine learning-based assessment of cell morphology, and will use our technique to create 3D organoids capable of transplantation.

 

REFERENCE LIST:

1. Werner S, Krieg T, Smola H. Keratinocyte-fibroblast interactions in wound healing. J Invest Dermatol. 2007;127(5):998-1008.
2. Walmsley GG, Rinkevich Y, Hu MS. Live fibroblast harvest reveals surface marker shift in vitro. Tissue Eng Part C Methods. 2015;21(3):314-21.