Introduction: Currently, limited solutions are available to regenerate the damaged urothelial layer caused by congenital bladder disorders, cancer of the bladder, and trauma. The goal of the present study is to develop a 3D urothelial tissue construct in vitro using the layer-by-layer cell assembly technique. Specifically, urothelial cells differentiated from human adipose-derived stem cells (hADSCs) were subjected to coating with alternating layers of fibronectin and gelatin prior to seeding and culturing. Cell morphology, phenotypic marker expression, and barrier function of the constructs were examined.
Materials and Methods: For urothelial differentiation, hADSCs were cultured in defined keratinocyte serum-free media (KSFM) with supplementation. UROtsa cells were cultured in DMEM supplemented with 10% FBS and 1% Glutamax. Cells were coated via suspension in 9 alternating layers of fibronectin and gelatin at 0.04 mg/mL concentration. Coated cells were placed in either fibronectin-coated or fibronectin-gelatin multilayer-coated Transwell® inserts. To achieve an 8-layered tissue, hADSCs and UROtsa cells were seeded at 8.0x105 and 2.0x106 cells per well, respectively and cultured for at least 24 hours and up to 10 days. At the end of the prescribed time period, cells on insert membranes were fixed and sectioned for H&E staining, and immunostaining for CK-17, CK-20, UPIb, and ZO-1. Permeability across hADSC and UROtsa multilayers was measured using fluorescently labeled dextran (4 kDa, FITC; 70 kDa rhodamine B) and a microplate reader.
Results and Discussion: Fibronectin-gelatin-coated, hADSC-derived urothelial cells on fibronectin-coated membrane showed aggregation after 24 hours indicating incomplete formation of a multilayer. In contrast, fibronectin-gelatin-coated UROtsa cells cultured on a fibronectin-gelatin-coated membrane displayed uniform layering and expression of tight junction protein ZO-1. Uncoated cells or cells seeded on an uncoated membrane exhibited a scattered appearance and less expression of ZO-1. Together, these results suggest that the fibronectin-gelatin coating facilitates cell-cell and membrane adhesion in UROtsa cells, which is critical for formation of 3D tissue constructs.
In addition, permeability studies showed that there was significant difference in transcellular and paracellular transports between cell-seeded and empty culture inserts. However, results were similar between coated and uncoated hADSCs and UROtsa cells indicating that further maturation time may be necessary to create barrier function in the 3D multilayered cell constructs.
Conclusion: The results of the present study provided evidence that the cellular microenvironment modified by ECM proteins would make a significant impact on the formation of 3D multilayered cell structure. Further experimentation is, however, needed to determine the effect of other parameters such as cell maturation and numbers of hADSC-derived urothelial cells on the barrier function of the multilayer construct, which is not only useful for regenerative medicine, but also as test platforms for drug screening and other experimentations in vitro.