Dynamic Culture Substrates That Mimic the Topography of the Epidermal-Dermal Junction

The junction between the epidermal and dermal layers of human skin undulates, the width and depth of the undulations varying with age and disease. We previously showed that when primary human epidermal keratinocytes are seeded on collagen-coated undulating static polydimethylsiloxane (PDMS) elastomer substrates, the stem cells, differentiated cells, and proliferating cells become patterned in response to cues from the underlying substrate. To investigate how patterning occurs over time, we have now created a dynamic model, in which a collagen-coated poly(d,l-lactide-co-glycolide) (PLGA) membrane is placed over a polyimide sheet containing circular holes, differing in diameter and spacing. When a vacuum is applied the membrane is induced to undulate, the heights of the undulations depending on the pressure applied and the size of the holes. We observed clustering of cells with high levels of beta 1 integrin expression, a stem cell marker, in the base of the undulations within 48 h of applying the vacuum. Differentiating involucrin-positive cells did not cluster; however, there was clustering of cells with high E-cadherin expression and nuclear YAP. Rho kinase inhibition resulted in loss of clustering, suggesting a role for Rho family members in the process. Impact Statement In human skin the junction between the epidermis and dermis undulates. Epidermal stem cells pattern according to their position relative to those undulations. Here we describe a rig in which epidermal cells are cultured on a collagen-coated poly(d,l-lactide-co-glycolide) (PLGA) membrane. When a vacuum is applied the membrane is induced to undulate. Stem cells cluster in response to the vacuum, whereas differentiating cells do not. Rho kinase inhibition results in loss of clustering, suggesting a role for Rho family members in the process. This dynamic platform is a new tool for investigating changes in the skin with age and disease.