Ultrasonic standing wave systems have previously been used for the generation of 3D constructs for a range of cell types. In the present study, we cultured cells from the human hepatoma Huh7 cell line in a Bulk Acoustic Wave field and studied their viability, their functions, and their response to the anti-cancer drug, 5 Fluorouracil (5FU). We found that cells grown in the acoustofluidic bioreactor (AFB) expressed no reduction in viability up to 6 h of exposure compared to those cultured in a conventional 2D system. In addition, constructs created in the AFB and subsequently cultured outside of it had improved functionality including higher albumin and urea production than 2D or pellet cultures. The viability of Huh7 cells grown in the ultrasound field to 5FU anti-cancer drug was comparable to that of cells cultured in the 2D system, showing rapid diffusion into the aggregate core. We have shown that AFB formed 3D cell constructs have improved functionality over the conventional 2D monolayer and could be a promising model for anti-cancer drug testing.

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SummaryBackgroundTryptase, the most abundant protease of the human mast cell, has been implicated as a key mediator of allergic inflammation that acts through activation of PAR2.ObjectivesTo investigate the contribution of PAR2 in the pro‐inflammatory actions mediated by tryptase in a mice model.MethodsWe have injected recombinant human βII‐tryptase into the peritoneum of PAR2‐deficient and wild‐type C57BL/6 mice. After 6, 12 and 24 hours, mice were killed, peritoneal lavage performed and inflammatory changes investigated.ResultsTryptase stimulated an increase in neutrophil numbers in the peritoneum, but responses did not differ between PAR2‐deficient and wild‐type mice. Heat inactivation of tryptase or pre‐incubation with a selective tryptase inhibitor reduced neutrophilia, but neutrophil accumulation was not elicited with a peptide agonist of PAR2 (SLIGRL‐NH 2). Zymography indicated that tryptase stimulated the release of matrix metalloproteinases (MMP) 2 and 9 in the peritoneum of both mouse strains. Studies involving immunomagnetic isolation of neutrophils suggested that neutrophils represent the major cellular source of tryptase‐induced MMP2 and MMP9. At 24 hours after tryptase injection, there was increased microvascular leakage as indicated by high levels of albumin in peritoneal lavage fluid, and this appeared to be partially abolished by heat‐inactivating tryptase or addition of a protease inhibitor. There was no corresponding increase in levels of histamine or total protein. The extent of tryptase‐induced microvascular leakage or gelatinase release into the peritoneum did not differ between PAR2‐deficient and wild‐type mice.ConclusionsOur findings indicate that tryptase is a potent stimulus for neutrophil accumulation, MMP release and microvascular leakage. Although these actions required an intact catalytic site, the primary mechanism of tryptase in vivo would appear to involve processes independent of PAR2.