Multiphoton-based micropatterning of cell-cell interaction proteins : an in vitro platform for cell niche studies

Abstract

Cells reside in a specialized local microenvironment known as the cell niche. The cell niche is made up of components such as matrix proteins, growth factors, mechanical factors, and neighbouring cells, which together provide essential signals for tissue and cell survival, growth, and function. In particular, the interaction between neighbouring cells, or the cell-cell interaction niche, provide signals for tissue homeostasis, structural integrity, cell proliferation, and cell survival.

One way that cells sense each other is through adherens junctions, which are mechanosensitive junctions mediated by members of the cadherin family. Previous studies utilizing cell niche engineering have largely focused on the effect of extracellular matrix (ECM) mechanical properties on cell behavior such as epithelialization and epithelial-to-mesenchymal transition (EMT); however, mechanosensing at cell-cell interactions may also play a role in these processes.

The Multiphoton Microfabrication and Micropatterning (MMM) platform enables cell niche engineering by fabricating or patterning the individual components, including ECM proteins, growth factors, mechanical stiffness, and elastic modulus, and high-resolution topographical features. Using this platform, it is possible to decouple these different niche factors, which is important when developing a system to investigate cell-cell interaction-mediated mechanosensing.

This study therefore first aimed to develop a cell-cell interaction patterning platform using MMM technology. To this end, E-cadherin was patterned onto bovine serum albumin (BSA) substrates using direct multiphoton crosslinking or indirect immobilization through Protein A/G. Both direct crosslinking and indirect immobilization were found to be controllable spatially and quantitatively via laser power and scan cycle, whilst retaining the protein bioactivity as evaluated using MCF7 and Madin-Darby Canine Kidney (MDCK) cells. Immunofluorescence staining with Protein A/G coatings was also optimized to minimize background staining. E-cadherin was then patterned onto BSA substrates with different moduli and stiffnesses to observe epithelialization and EMT in MDCK cells. E-cadherin-coated matrices and micropillars with lower modulus and lower stiffnesses respectively were found to have poorer epithelialization and junctional integrity, lower spread area, and increased E-cadherin, Vimentin, and N-cadherin expression. Cells on low stiffness conditions also showed elongated morphology and increased aspect ratio. Similarly, laterally-coated E-cadherin with basally-coated Laminin-511 displayed more elongated cells at leading edges under low modulus conditions. Together, these indicate poorer epithelialization on E-cadherin-coated low modulus or stiffness structures and increased partial EMT phenotype on both the above structures as well as laterally-coated E-cadherin low-modulus structures.

Moreover, to enable larger scale fabrication, improved efficiency, and higher throughput of the MMM platform, key steps in the fabrication sequence were automated. Namely, the autofocus, fabrication, and coating parameters on a programmable multiphoton system was investigated, and automated fabrication of a complex 3D villi-like structure, was demonstrated. Technologically, this work provides a method of engineering the cell-cell interaction niche for cell niche studies, and can also be used to immobilize other types of cell niche proteins. This cell niche factor can be combined with other independent niche factors previously developed, thus developing a well-defined complex cell niche for future studies. Biologically, this study demonstrates the importance of cadherin-mediated mechanical properties in cellular activities related to epithelialization and EMT.