Results: 4

1.
Figure 3

Figure 3. From: InVERT molding for scalable control of tissue microarchitecture.

Multi-compartmental placement dictates hepatic tissue function. (a) Liver sinusoidal endothelial cells (green) were patterned in ‘juxtaposed’, or compartmentally distinct ‘paracrine’ conformations relative to hepatic aggregates using InVERT molding (scale bar 200 μm). Paracrine configurations for primary hepatic aggregates and liver endothelial cells yield significantly greater albumin secretion than juxtaposed configurations [P = 0.0087, n = 5 and 6 and 160 ± 56 and 69 ± 22 μg/106 hepatocytes (S.D.) for each respective group, Mann Whitney test], and similar trends were observed for iPS-Hep aggregates though this data was not significant [P = 0.3500, n = 3 for both groups, 77 ± 45 and 33 ± 11 ng/106 hepatocytes (S.D.) for paracrine and juxtaposed groups, respectively; Mann Whitney test]. (b) Stromal cells (green) were patterned either in paracrine, juxtaposed’ or ‘interpenetrating’ conformation relative to iPS-hepatocyte-like cells using InVERT molding (scale bars 200 μm). Interpenetrating configurations yield higher albumin secretion relative to other conformations (P = 0.0250, n = 5, 4, 6, 6 and 0.2 ± 0.1, 0.3 ± 0.2, 0.2 ± 0.2, 1.9 ± 1.3 μg/106 iHeps (S.D.) for each respective group; One-way ANOVA with Tukey post-hoc test).

KR Stevens, et al. Nat Commun. ;4:1847-1847.
2.
Figure 1

Figure 1. From: InVERT molding for scalable control of tissue microarchitecture.

Fabrication method and versatility. (a) Process flow diagram for InVERT molding. Seeded cell populations are green (Intaglio phase) and red (Relief phase). (b) Substrate-based molding produces hydrogels from 1.5 cm- (inset) to 14 cm-diameter. Here, size and spacing (800 μm) of cell clusters enables macroscopic visualization after hematoxylin staining. (c) InVERT molding produces multi-compartmental patterning and is compatible with various substrates and materials. Labeled cells (green endothelial cells, calcein-AM; red fibroblasts, calcein red-orange AM) are patterned in fibrin gel using a custom-fabricated branching pattern substrate (top) or in agarose using a substrate molded using a corner cube bike reflector (middle). Substrate-molded cell layers were stacked manually for multi-layer patterning (bottom; hepatocyte aggregates, calcein red-orange AM, and endothelial cell cords, green calcein-AM; scale bars 500 μm unless otherwise denoted). (d) InVERT molding is compatible with many cell types. Mouse C2C12 skeletal myoblasts, mouse J2-3T3 fibroblasts, human ovarian carcinoma cells (OVCAR-8), stromal mouse embryonic fibroblasts (10T1/2), human cervical cancer cells (HeLa), normal human dermal fibroblasts (NHDF), and human iPS-hepatocyte-like cells with liver endothelial cells (TMNK1; LEC) were labeled with calcein dyes and patterned in fibrin gel using InVERT molding (scale bars 300 μm).

KR Stevens, et al. Nat Commun. ;4:1847-1847.
3.
Figure 2

Figure 2. From: InVERT molding for scalable control of tissue microarchitecture.

Multi-level and multicellular microorganization can be controlled across and within distinct compartments. (a) Hepatic aggregates (red) were embedded within an endothelial lattice (green) using InveRT molding. (b) Each 1.5 cm diameter hydrogel contained approximately 1000 aggregates (top left, representative phase images, scale bar 1 mm; inset 200 μm). Hepatic aggregates (red, calcein red-orange AM) were patterned within the endothelial lattice (green; calcein-AM, top right; scale bar 200 μm). 3D patterning is evident from opaque oblique and cross sectional-rendering of stacked multiphoton images (middle left; grid scale bar 127 μm). A single cross-section slice reveals overlap between populations in the Z plane (blue lines) but no overlap in the XY plane (middle right). Altering cell seeding density of J2 fibroblast aggregates and liver endothelial cells can eliminate overlap (bottom left; scale bars are 110 μm). Edge-to-edge distance (dotted white line) between cellular compartments followed distinct Poisson distributions for different spacings (bottom right) and was 116 ± 25 μm S.D. and 366 ± 52 μm (S.D.), respectively. (c) An average of 10 – 500 hepatocytes were seeded per microwell. Resultant aggregates were either encapsulated in hydrogels or removed for immunostaining analysis. Aggregates encapsulated in hydrogels (left, scale bar 200 μm) exhibit Poisson distribution (center; average cross-sectional area of 50 ± 15×102 μm2, 145 ± 28×102 μm2, and 590 ± 110×102 μm2 (S.D.) for 10, 100, and 500 cell aggregates, respectively). Cytokeratin immunostaining of isolated aggregates reveals distinct morphology across aggregate sizes (right, scale bar 50 μm). (d) Multiphoton imaging of hydrogels with patterned aggregates containing both hepatocytes and J2 fibroblasts demonstrates that fibroblasts were dispersed throughout each hepatic aggregate (fibroblasts, mCherry; hepatocytes, calcein-AM, scale bar 50 μm). Representative aggregates show fibroblasts located at both the edges and center of the aggregates (bottom).

KR Stevens, et al. Nat Commun. ;4:1847-1847.
4.
Figure 4

Figure 4. From: InVERT molding for scalable control of tissue microarchitecture.

Optimization of tissue architecture modulates hepatic function in vitro and in vivo. (a) The number of hepatocytes per aggregate (10 – 500) resulted in distinct hepatic tissue function in vitro (*p < 0.05, SEM, n = 4, 3, 3 and total albumin of 9 ± 2, 104 ± 12, 21 ± 9 (S.D.) for each respective group, Kruskal-Wallace test). (b) Addition of J2 fibroblasts to hepatic aggregates in hydrogels sustained (left) and improved cumulative (right) hepatic functions in a dose-dependent manner in vitro (100 hepatocytes per aggregate + fibroblasts at 1: 0, 1: 1, 1: 2 hepatocytes: fibroblast, *p < 0.05, SEM, n = 5, 5, 4 and total albumin of 15 ± 4, 300 ± 132, 602 ± 179 μg/106 hepatocytes (S.D.) for each respective group, One-way ANOVA with Tukey post-hoc test). (c) Representative bioluminescence images of nude mice that received patterned tissue containing no cells (“Blank”), 500 rat primary hepatocytes per aggregate (“500”), 100 hepatocytes per aggregate (“100”), or 100 hepatocytes + J2 fibroblasts per aggregate (“100+J2”) implanted in the intra-peritoneal space. (d) Optimal in vitro tissue configurations result in sustained hepatic functions to over four weeks following implantation (*p < 0.05, SEM, n = 3, 4, 5, 6 and total bioluminescence of 1.2e6 ± 0.3e6, 1.8e6 ± 0.5e6, 3.2e6 ± 0.8e6, 5.4e6 ± 2.3e6 (S.D.) for each respective group, Kruskal-Wallace test with Dunn's Multiple Comparison post-hoc test). (e) Representative histological images of patterned tissues containing 100 crypreserved human hepatocytes per aggregate with or without J2 fibroblasts (“100” or “100+J2”) that were extracted seven days after intra-peritoneal implantation in nude mice. Patterned arrays of aggregates (left, hematoxylin and eosin) that contained arginase-1 positive hepatocytes (right, ARG-1, red) were identified in all animals with cellular implants (scale bars 100 μm). (f) The addition of fibroblasts sustained human hepatic function for at least four weeks (*p < 0.05, n = 4, 6, 6 and total bioluminescence of 0.0e8 ± 0.0e8, 3.4e8 ± 2.1e8, 7.0e8 ± 3.3e8 (S.D.) for each respective group, Mann-Whitney test, ‘blank’ excluded from statistical analysis).

KR Stevens, et al. Nat Commun. ;4:1847-1847.

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