Thermofluidic heat exchangers for actuation of transcription in artificial tissues

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Science Advances  30 Sep 2020:
Vol. 6, no. 40, eabb9062
DOI: 10.1126/sciadv.abb9062
  • Fig. 1 Thermofluidic heating in 3D bioprinted hydrogels.

    (A) Schematic of thermofluidic workflow. A biocompatible fluid flows around a power supplied heating element to preheat the fluid before entry in perfusable channel networks within hydrogel tissue constructs laden with heat-sensitive cells. During perfusive heating, hydrogel temperature is continuously monitored using an infrared camera. (B) Perfusable channel networks of varying spatial geometries can be bioprinted within biocompatible 3D hydrogels. Top: 3D rendering of network architectures. Middle: Hydrogel channels infused with tonic water fluoresce when imaged under ultraviolet backlight. Bottom: Infrared thermography of heat-perfused hydrogels demonstrates that during perfusion, heat traces the path of fluid flow and dissipates into the bulk hydrogel. Scale bars, 5 mm.

  • Fig. 2 Thermal profile characterization in perfused hydrogels.

    (A) Photograph of a single-channel bioprinted hydrogel used for initial thermal characterization. Scale bar, 5 mm. (B) Representative infrared images from controlled perfusion of heated fluid through the channel over time (left). Scale bars, 5 mm. (C) Representative finite-element modeling images depicting steady-state predictions on the surface of perfused hydrogels at varying flow rates and constant heater power (left; full dataset in fig. S1B). Computational modeling predicts that flow rate can achieve maximal hydrogel temperatures in the mild hyperthermia temperature range (right, gray shading denotes mild hyperthermia range). (D) Hydrogels were experimentally perfused at flow rates of 0.5 and 1.0 ml min−1 and imaged using infrared thermography. Scale bars, 5 mm. (E) Hydrogel temperature plotted orthogonal (x) to the flow direction at inlet and outlet positions show agreement between thermal gradients in computational and experimental measurements (computational, dashed lines; experimental, solid lines). (F) Hydrogel temperature plotted parallel (y) to flow direction demonstrates a larger temperature drop from inlet to outlet (y) during flow at 0.5 ml min−1 (∆T0.5) compared to flow at 1.0 ml min−1 (∆T1.0) in computational and experimental models (computational, dashed lines; experimental, solid lines; n = 5, data are mean temperature ± standard error, **P < 0.01 by Student’s t test). Photo credit: Daniel Corbett, University of Washington.

  • Fig. 3 Fluidic heating induces gene expression in 3D artificial tissues.

    (A) HEK293T cells were engineered to express fLuc under the HSPA6 promoter. (B) Schematic of thermofluidic activation of encapsulated cells. (C) Single-channel tissue used for 3D heat activation (left). Scale bar, 3 mm. Transmittance image of cellularized hydrogel after printing (middle). Scale bar, 500 μm. HEK293T cells in bioprinted tissues stained with calcein-AM (“live,” green) and ethidium homodimer (“dead,” red; right). Scale bars, 200 μm. (D) Representative infrared images of thermofluidic perfusion in single-channel hydrogels. Scale bars, 2 mm. (E) Hydrogel temperatures are tuned by changing heater power at constant flow rate (n = 3, mean temperature ± standard error). (F) Representative bioluminescence images of hydrogels (top; scale bars, 2 mm) and intensity traces at three positions (A to C) across the width (x) of the hydrogel after 30 min of perfused heating. (G) Fold change in bioluminescence after 30 min of heating relative to 25°C controls. (H) Representative bioluminescence images of hydrogels (top; scale bars, 2 mm) and intensity traces after 60 min of perfused heating (bottom; scale bars, 2 mm). (I) Fold change in bioluminescence after 60 min of heating demonstrates a temperature-dependent dosage response in gene expression [(G and I); n = 3, mean fold luminescence ± standard error; *P < 0.05 and **P < 0.01 by one-way ANOVA followed by Dunnett’s multiple comparison test]. (J) Temperature-expression response curve (black) shows mean bioluminescent radiance across temperature; shaded regions (gray) indicate ± SD. n = 3. Photo credit: Daniel Corbett, University of Washington.

  • Fig. 4 Heated perfusion of complex network architectures localizes gene expression in space and time.

    (A) Heat exchanger inspired designs for various flow directions, fluid temperatures, and channel architectures (schematics; left and center). Representative thermal (middle) and bioluminescent (right) images demonstrate spatial tunability of thermal and gene expression patterning. Scale bars, 5 mm. (B) Photographic image of four-armed clock-inspired hydrogel used for dynamic activation (top; channel filled with red dye). Each inlet is assigned to a local region (A to D). Schematic shows the spatial and dynamic heating pattern for the 4-day study (bottom). (C) Representative infrared (top) and bioluminescence expression (bottom) images for dynamic hydrogel activation at each day during the time course. (D) Quantification of local bioluminescent signals from regions of interest corresponding to each day of heating. Across all 4 days, regions corresponding to perfused arms had higher bioluminescent signals than nonperfused arms (n = 5, data are mean luminescence ± standard error; *P < 0.05 and **P < 0.01 by one-way ANOVA followed by Tukey’s post hoc test).

  • Fig. 5 HEAT gene patterning is maintained after tissue implant in vivo.

    (A) Artificial tissues with embedded heat-inducible fLuc HEK293T cells received 44°C thermofluidic heating (channel heat, n = 5), 44°C global heating (bulk heat, n = 3), or remained at 37°C (no heat, n = 3) for 1 hour before immediate implantation into athymic mice. (B) Bioluminescence from implanted hydrogels (dashed lines) showed region specific signal only in channel heated hydrogels. (C) Average line profiles (top) across the width (x) of the hydrogel for inlet, middle, and outlet positions show that only channel heated gels induced a spatially coordinated response that was statistically significant (bottom) between the center (position B) and edges of the hydrogel (position A and C; channel heat, n = 5; bulk heat, n = 3; no heat, n = 3; data are mean luminescence ± standard error; **P < 0.01, by one-way ANOVA.

  • 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.

Supplementary Materials

  • Supplementary Materials

    Thermofluidic heat exchangers for actuation of transcription in artificial tissues

    Daniel C. Corbett, Wesley B. Fabyan, Bagrat Grigoryan, Colleen E. O’Connor, Fredrik Johansson, Ivan Batalov, Mary C. Regier, Cole A. DeForest, Jordan S. Miller, Kelly R. Stevens

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