Fig. 6. Thermofluidic Wnt regulation in engineered HEK293T and HepaRG cells.

(A) Schematics of lentiviral constructs (left) and thermofluidic HEK293T tissue experiments (right). (B) Transmittance image of cellularized construct after printing (left; zones indicated by dashed lines). Infrared image of construct during heating (right). Scale bars, 1 mm. (C) mCherry⁺ HEK293T cells in printed tissues (left). Scale bars, 1 mm. Images of thermofluidically heated Wnt2 constructs after immunostaining for V5 tag (coexpressed with Wnt2; right; images taken near the tissue’s channel and periphery as indicated by insets). Scale bars, 200 μm. (D) Wnt family genes were up-regulated in zone 3 of thermofluidically perfused gels compared to controls (n = 4, mean fold change ± standard error; *P < 0.05 and **P < 0.01 by two-way ANOVA followed by Tukey’s multiple comparison test). (E) Differentiated HepaRG cells were engineered with a heat-inducible RSPO1 construct (schematic, top) and printed in single-channel hydrogels (photograph, left). Scale bars, 1 mm. After heating (infrared), HepaRGs remained viable in printed constructs (calcein). Scale bar, 200 μm. (F) Thermofluidically heated RSPO-1 HepaRG hydrogels were dissected into zones 1 to 3 based on distance from the heat channel for RT-qPCR analysis at 1, 24, and 48 hours after heating. Expression fold change was normalized to no heat control samples. qPCR analysis of RSPO-1 across dissected zones (n = 5 to 10, data are mean fold change ± standard error; *P < 0.05 by one-way ANOVA followed by Tukey’s multiple comparison test). (G) RT-qPCR analysis of pooled RNA across all zones at each time point for pericentral associated genes, glutamine synthetase, CYP1A2, CYP1A1, CYP2E1, and CYP3A4, and periportal/midzonal genes, Arg1 and E-cadherin (n = 15 to 30, data are mean fold change ± standard error, **P < 0.01 and *P < 0.05 by one-way ANOVA followed by Tukey’s multiple comparison test). Photo credit: Daniel Corbett, University of Washington. n.s., not significant.