Visualizing cell arrangements
In vivo , ECM molecules including fibronectins, collagens, and laminins not only affect signaling properties and cell function but also modulate overall tissue architecture with direct consequences on cell arrangements and tissue organization.[57] To better understand the effects of specific ECM proteins on the cell organization, we used immunohistochemistry staining for HepG2 and HSC cells in whole mount spheroids to visualize the impact of ECM modified MPs on arrangements (Figure 7) . Although HepG2 cell distribution was homogenous, we observed distinct localization of HSCs toward the periphery of spheroids, generating a distinct outer layer of similar thickness in all groups. This also correlates with E-cadherin staining results (Figure 6B ). The contacting area of the hepatocytes contains collagen fibrils, [58]resulting in a membrane thickening which we also observed in our images (the purple ring at the edge of all groups). We observed that groups treated with laminin-511 and 521 resulted in localization of some HSCs toward the center of spheroids, perhaps because PFC-laminin 511/521 MPs stimulate cells to change position relative to their neighbors toward contact strengthening. This finding lends support to prior findings and confirms that interactions between cells and the ECM at integrin-based adhesion sites allow cells to sense their physical surroundings and adjust mechanisms of migration and anchorage.[59]Thus, besides organization, our co culture system provides a similar physicochemical structure that more closely mimics in vivo tissue counterparts.