Modeling Tissue Polarity in Context

Abstract

Polarity is critical for development and tissue-specific function. However, the acquisition and maintenance of tissue polarity is context dependent. Thus, cell and tissue polarity depend on cell adhesion which is regulated by the cytoskeleton and influenced by the biochemical composition of the extracellular microenvironment and modified by biomechanical cues within the tissue. These biomechanical cues include fluid flow induced shear stresses, cell-density and confinement-mediated compression, and cellular actomyosin tension intrinsic to the tissue or induced in response to morphogens or extracellular matrix stiffness. Here, we discuss how extracellular matrix stiffness and fluid flow influence cell-cell and cell-extracellular matrix adhesion and alter cytoskeletal organization to modulate cell and tissue polarity. We describe model systems that when combined with state of the art molecular screens and high-resolution imaging can be used to investigate how force modulates cell and tissue polarity.

Keywords: cellular mechanics; mechanobiology; microfluidics; polarity; polarized tissue.

Figure 1. Polarity depends on a delicate balance of physical forces

Increasing ECM stiffness causes the loss of apical basolateral polarity. Schematic depicting the effects of increasing mechanical stress on mammary epithelial cells. Chronic exposure to physical forces compromises the ductal structure and is accompanied by the loss of epithelial polarity. These effects are clearly observable with non-malignant MCF10A cell colonies cultured on a reconstituted basement membrane functionalized polyacrylamide gel surfaces of increasing stiffness (150–5,000 Pa). MCF10A cells cultured on surfaces with a biomimetic ECM stiffness similar to that measured in the normal murine mammary gland (150 Pa) form polarized acini organoids which model the terminal ductal lobular units of a differentiated breast. MCF10A organoids synthesize and localize an endogenous laminin 5 basement membrane (red) to the basolateral surface of the acini. These elastically tuned epithelial acini organoid models demonstrate that stiffening of the basement membrane causes a degeneration of polarity, breakdown of luminal structures, stable cell-cell junctions, and loss of the endogenous laminin 5 basement membrane. Nuclei (blue), F-Actin (green), and laminin 5 (red).

Figure 2. Methods to incorporate control of ECM stiffness into culture models of polarity

A. Typical assembly of materials to generate a mechanically tuned 2D surface for cell culture models. Commonly, polyacrylamide gels are mechanically tuned via alterations in polymer and crosslinking density and bonded to γ-aminopropyltriethoxysilane (APTES) functionalized glass coverslips. The resulting gel surface can be functionalized with ECM proteins or bioadhesive ligands via carbodiimide-mediated crosslinking, N-hydroxysuccinimidyl acrylate (NHS-acrylate), N-succinimidyl ester of acrylaminohexanoic acid (N6), Hydrazine, or polydopamine films functioning as an adhesive interface between the polyacrylamide and the desired surface coating ^,. B. Schematic representation of basic cell culture structures which can cast with elastically defined PDMS and are amenable to incorporate fluid flow induced shear stresses. Lumen mimetic tubes or channels may be then be associated with fluid pumps to control the volume and rate of fluid passing through the tube or channel to generate defined fluid flow across cell monolayers or through 3D-cell-lined-lumens.

Figure 3. The utility of microfluidic devices

Presented here is a microfluidic device cast of PDMS of varied stiffness and bonded to a glass surface to create a sealed chamber. The glass surface allows for the device to be monitored with microscopy techniques and the in-line ports allow for additions or removal of substances from the outer surface of the lumen. This device has media inlets and outlets at the terminal ends of the channel so that controlled fluid flow of culture media may be added and modulated to vary fluid flow shear stresses. The inner channel would be lined with a cell monolayer mediated via laminin or collagen coating to foster appropriate polarization. Importantly, these devices may be cast in multiple pieces or multiple devices could be interconnected via flexible hoses .