Development of three-dimensional human skin models by cell coating technology for alternative systems to animal testing

摘要

Introduction: The appropriate construction technique for a three-dimensional (3D) tissue is required to enhance the potential of cells to form engineered tissues. Recently, various techniques have been developed to construct 3D multilayered tissues, for example cell sheet engineering and multilayer scaffolds. Although these methods can construct cells into 3D multilayers, they have limitations due to the complicated manipulation of the fragile cell sheet or residual scaffolds in the cells. Moreover, precise control over the thickness and components of an extracellular matrix is difficult. We recently reported a simple and unique bottom-up approach, termed "cell-accumulation technique', to develop 3D cellular multilayers with the desired layer number and location by the fabrication of nanometer-sized layer-by-layer (LbL) fibronectin (FN)-gelatin (G) (FN-G) films as a nano-ECM onto the single cell surfaces. Less than 10 nm thickness of ECM films composed of FN-G allowed all cells to adhere to each other through interactions between the FN-G nanofilms and the cell membrane proteins to create various types of tissues such as blood vessel walls and livers. Using this technique and a sandwich culture, highly dense and homogeneous endothelial tubular networks were formed in fibroblast tissues. In this study, 3D-layered skin model consisting of human keratinocytes (KC), normal human dermal fibroblast (NHDF), and human umbilical vein endothelial cells (HUVEC) were fabricated by the cell-accumulation technique and their barrier function was evaluated via TEER measurements, tight-junction formation and drug permeability test. Materials and Methods: NHDF were suspended in 0.04 mg/ml of FN and G/PBS solution, and alternately incubated for 1 min with a washing step. The centrifugation was performed at 200 x g for 1 min at each step. After 9 steps of coating, about 10 nm of the FN-G nanofilms were coated onto single cell surfaces. The cells were suspended in D-MEM with 5% FBS, and were seeded onto 24 well trans-well inserts. After 1 day, NHDF tissues from 10 to 20 layers thickness were constructed. The epidermal layers were prepared on the surface of the obtained NHDF dermis. To enhance adhesion of KC, the outermost surface of the NHDF dermis was coated with type Ⅳ collagen (Col Ⅳ) (0.2 mg/ml) for 30 min of incubation. KC was then seeded onto the surface of the dermis. After 1 days of incubation, the constructs were lifted to the air-liquid interface and ramification medium was added (air-lift culture). The morphology, differentiation, and thickness change were evaluated from the hematoxylin and eosin (HE) and immunohistochemical staining images of histological sections. Results and Discussion: The thickness of dermis consisting of NHDF was easily controlled from approximately 50 to 150 urn by altering the seeded cell number. KC seeded on the surface of the dermis showed homogeneous differentiation by lifting to air-liquid interface for 7 days. Histological analysis revealed four distinct layers such as basal layer, spinous layer, granular layer, and cornified cell layer in the epidermis. The co-sandwich culture of HUVEC within 10-layered dermis showed in vitro co-network formation of blood capillaries inside the dermis. After the sandwich culture of HUVEC with NHDF, KC was seeded onto the top and lifted to the air-liquid interface for KC differentiation. After 7 day incubation, obvious lumen structures of HUVEC networks in the dermal and epidermal layers of KC on the vascularized dermis was observed. Histological images stained with HE and immunostained with antibody for ZO-1, claudin and occuldin (tight-junction proteins) clearly revealed the formation of tight-junction in the epidermal layers consisting of KC. TEER profiles of constructed skin model continuously increased with increasing culture times and reached from 2000 to 4000 Ω cm2 value, suggesting formation of the cornified cell layer with barrier function. Conclusions: The 3D-layered skin models with blood capillary networks have valuable potential for applications in drug effect and toxicology and tissue engineering. Especially, it is expected to be applied to alternative systems to animal testing.