Effects of interstitial flow in cell morphology, differentiation, and organization in 3-dimensional tissue cultures

Physiological interstitial flow, the movement of fluid through porous tissues, plays an important role in governing interstitial viability, organization and architecture by providing convective macromolecular transport and a specific mechanical environment to cells in the interstitium. Despite its importance, the biological regulation of interstitial fluid balance is poorly understood, largely because of the lack of experimental models. In this work, to examine the effects of interstitial flow on cell organization in soft tissue cultures over relatively long periods of time (days to weeks), we developed an in vitro flow chamber where we can (i) specifically control the flow environment, (ii) directly observe cell organization, and (iii) measure the mechanical properties (e.g. hydraulic resistance) 'online'. We observed velocity-dependent alignment of fibroblasts perpendicular to interstitial flow in collagen cultures within 48 hours. In addition, we found that flow differentially stimulates blood and lymphatic endothelial morphogenesis in 6 days with the lymphatic endothelial cells forming mostly unicellular structures with large vacuoles and long extensions, while blood endothelial cells formed multicellular branched lumen-containing networks. Further investigations on the fibroblast remodeling revealed that interstitial flow induced collagen fiber alignment perpendicular to flow within 12 hours and that both cell and matrix alignments were alpha1beta1 integrin-mediated, suggesting a possible alignment pathway which (i) interstitial flow initially induced matrix anisotropy that (ii) provided contact guidance for the fibroblasts to orient along and (iii) further aligned their surrounding matrix fibers. Since aligned structures and angiogenesis were often seen in differentiated wound and fibrotic tissues, we hypothesized that interstitial flow could itself contribute to the wound repair process. We subsequently demonstrated that interstitial flow itself could induce alpha11 integrin-mediated fibroblast-to-myofibroblast differentiation characterized by alpha-SMA via the autocrine upregulation of TGF-beta1. These novel findings, along with the model we developed, helped to form an experimental basis for understanding the mechanobiological regulation of interstitial fluid balance in soft tissues. Our findings here also suggest that biomechanical factors such as interstitial flow are equally as important as the generally emphasized biochemical stimuli in regulating and governing cell morphology, differentiation and organization.