Research ArticleTISSUE ENGINEERING

Platform technology for scalable assembly of instantaneously functional mosaic tissues

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Science Advances  28 Aug 2015:
Vol. 1, no. 7, e1500423
DOI: 10.1126/sciadv.1500423
  • Fig. 1 Fabrication and physical characterization of the Tissue-Velcro platform.

    (A) The hooks and loops of the conventional Velcro system inspired the Tissue-Velcro design, based on a biocompatible and biodegradable polymer, POMaC. Red arrows indicate the built-in hooks. Scale bar, 1 mm. (B) Illustration of the fabrication process of the scaffold including a microinjection step followed by the stamping step. (C) Cell seeding process. A Matrigel-based cell suspension is allowed to gel on the scaffold, and when removed from the tissue culture, substrate holes are formed. After self-assembly, the compacted tissues can be handled and patterned. (D) SEM images revealed detailed scaffold architecture with the T-shaped hooks and an accordion mesh. Scale bar, 1 mm. Inset, high-magnification SEM of T-shaped hooks. Scale bar, 500 μm. (E) SEM image captures two scaffolds interlocking. The hooks from the bottom scaffold (dull gray) protrude above the struts of the top scaffold (white). Immediate detachment is prevented by these hooks of the bottom scaffolds catching on the struts of the top scaffold. Scale bar, 500 μm (left image); 300 μm (right image).

  • Fig. 2 Mechanical properties of Tissue-Velcro.

    (A) Representative force curve from the mechanical pull-off test of the scaffold (n = 4). Inset scale bar, 5 mm. (B) Representative uniaxial tensile stress-strain plots of the scaffold in the x direction (xD) and y direction (yD) (n = 4). (C) Summary of the measured apparent modulus of the scaffold in the x direction (xD), y direction (yD), and the anisotropic ratio (xD/yD) (mean ± SD, n = 4). (D and E) Representative 3D renderings of profilometry data of the preassembled scaffold components. (D) Bottom mesh and post (n = 3); (E) top hook (n = 3). (F) Illustration of the cross-sectional view of an assembled scaffold labeled with measured heights (n = 3).

  • Fig. 3 Cardiac Tissue-Velcro characterization.

    (A) Cardiac cell assembly around a mesh over 7 days. Scale bar, 100 μm. (B) Area decrease (%) during 1-Hz paced contraction derived from scaffold deformation increased from day 4 to 6 (day 4: 0.9 ± 0.3%, day 6: 1.4 ± 0.07%, mean ± SD, n = 3). Representative plots of electrically paced (1-Hz) cardiac tissue contracting and compressing the scaffold on days 4 and 6 of culture (n = 3). (C) Immunostaining of cardiac Tissue-Velcro on day 7 with sarcomeric α-actinin (red) and F-actin (green) (n = 4). Scale bar, 30 μm. (D) SEM of a Tissue-Velcro showing tissue bundles (day 7); scale bar, 100 μm. Inset, high-magnification SEM of a segment of Tissue-Velcro; scale bar, 100 μm. (E) EC coating around 7-day-old cardiac tissue grew to confluence in 24 hours (CD31, red). Scale bar, 100 μm. (F) CFDA cell tracker (green)–labeled endothelial cells; scale bar, 50 μm. (G) Representative images of nuclear staining (DAPI, blue) overlaid with nuclear orientation vectors along the long nuclear axis (n = 3). Scale bar, 50 μm. (H) Normalized distribution of orientation angles for cell nuclei and scaffold struts, respectively (representative trace of n = 3).

  • Fig. 4 Tissue function and viability upon assembly and disassembly.

    (A) Coculture conditions were instantaneously established in the z direction by assembling two layers of Tissue-Velcro (day 7): one consisting of cardiac FBs (red) and the second consisting of CMs (green). Scale bar, 800 μm. Tissue interlocking was visualized with high-magnification fluorescent images focusing on layer 1 (L1) and layer 2 (L2). Scale bar, 200 μm. (B) Assembly into a three-layer CM tissue construct. Scale bar, 800 μm. High-magnification fluorescent images focused on L1 and L3 confirm Tissue-Velcro interlocking. Scale bar, 200 μm. Arrowheads point to T-shaped microhooks protruding from the middle layer (L2) into the top layer (L1). (C) Electrical excitability parameters of the cardiac Tissue-Velcro (day 7) before assembly (mean ± SD, n = 8), after assembly (two-layer, mean ± SD, n = 4), after disassembly (mean ± SD, n = 8), and 1 day after disassembly (mean ± SD, n = 8). (D and E) Viability staining of CM Tissue-Velcro (day 4) (D) before (n = 3) and (E) after the tissue assembly/disassembly process (n = 4). Scale bar, 200 μm. CFDA, green; propidium iodide (PI), red. Scaffold struts exhibit autofluorescence in the red channel. (F) Quantification of tissue viability from LDH activity in tissue culture media collected before (mean ± SD, n = 8) and after the tissue assembly/disassembly process (mean ± SD, n = 4).

  • Fig. 5 Patterned mosaic tissue assembly.

    (A to C) SEM of two cardiac tissues (day 4) assembled together and then cultured for an additional 3 days (n = 4). (B and C) White arrows indicate locations where cells spread through a pathway created by the hook and loop configuration linking the two tissues together. Scale bars, 1 mm (A); 300 μm (B and C). (D and E) Tissues (day 7) composed of cardiac FBs were labeled either green or red and arranged into (D) a 2D pattern (scale bar, 800 μm) and (E) an offset 2D pattern to extend the length of the construct (scale bar, 800 μm). (F) Two cardiac tissues (day 7) were labeled either green or red and assembled together approximately at 45° angle. Scale bar, 800 μm.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/1/7/e1500423/DC1

    Fig. S1. Base material physical properties under cell culture conditions.

    Fig. S2. Hook and loop interlocking mechanism is a dominant factor governing the mechanical stability of the assembled two-layer structures.

    Fig. S3. Cardiac tissue contractility.

    Fig. S4. Immunostaining of cardiac Tissue-Velcro on day 7 for sarcomeric α-actinin (red) and F-actin (green) at various locations of the tissues.

    Fig. S5. Drug response.

    Fig. S6. Coculture of cardiac and endothelial cells.

    Fig. S7. Scanning electron micrograph of the assembled two-layer cardiac tissue cultivated for 3 days.

    Fig. S8. Scanning electron micrograph of an additional Tissue-Velcro design with spring-like structures that could potentially be used to enhance scaffold anisotropic mechanical properties and tissue anisotropic contraction.

    Movie S1. The 3D confocal reconstruction of two interlocked scaffolds shows the hooks from the scaffold on the lower layer catching on the struts of the scaffold on the upper layer.

    Movie S2. Recording of a mechanical pull-off test to measure the force required to detach interlocked scaffolds.

    Movie S3. Two interlocked cardiac tissue layers were manipulated with tweezers, demonstrating that assembled multilayer tissue constructs can be handled and manipulated.

    Movie S4. Time lapse of seeded CMs remodeling and compacting over a 3-day period on a single layer scaffold mesh (no hooks).

    Movie S5. Contraction of cardiac tissue mesh after tissue remodeling, day 4.

    Movie S6. Electrical field stimulation applied to a single scaffold mesh (no hooks) seeded with CMs, after 7 days in culture.

    Movie S7. Autofluorescent scaffold contraction recorded and processed to measure fractional shortening.

    Movie S8. Vertical scan of a three-layer Tissue-Velcro.

    Movie S9. Electrically paced cardiac tissues contracting before assembly, after assembly, after disassembly, and 1 day after disassembly.

    Movie S10. Spontaneous contraction of a two-layer cardiac tissue cultured for an additional 3 days after assembly.

    Movie S11. Response of Tissue-Velcro (day 5) to epinephrine (300 nM) stimulation.

    Reference (54)

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. Base material physical properties under cell culture conditions.
    • Fig. S2. Hook and loop interlocking mechanism is a dominant factor governing the mechanical stability of the assembled two-layer structures.
    • Fig. S3. Cardiac tissue contractility.
    • Fig. S4. Immunostaining of cardiac Tissue-Velcro on day 7 for sarcomeric α-actinin (red) and F-actin (green) at various locations of the tissues.
    • Fig. S5. Drug response.
    • Fig. S6. Coculture of cardiac and endothelial cells.
    • Fig. S7. Scanning electron micrograph of the assembled two-layer cardiac tissue cultivated for 3 days.
    • Fig. S8. Scanning electron micrograph of an additional Tissue-Velcro design with spring-like structures that could potentially be used to enhance scaffold anisotropic mechanical properties and tissue anisotropic contraction.
    • Reference (54)

    Download PDF

    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.mov format). The 3D confocal reconstruction of two interlocked scaffolds shows the hooks from the scaffold on the lower layer catching on the struts of the scaffold on the upper layer.
    • Movie S2 (.mov format). Recording of a mechanical pull-off test to measure the force required to detach interlocked scaffolds.
    • Movie S3 (.mov format). Two interlocked cardiac tissue layers were manipulated with tweezers, demonstrating that assembled multilayer tissue constructs can be handled and manipulated.
    • Movie S4 (.mov format). Time lapse of seeded CMs remodeling and compacting over a 3-day period on a single layer scaffold mesh (no hooks).
    • Movie S5 (.mov format). Contraction of cardiac tissue mesh after tissue remodeling, day 4.
    • Movie S6 (.mov format). Electrical field stimulation applied to a single scaffold mesh (no hooks) seeded with CMs, after 7 days in culture.
    • Movie S7 (.mov format). Autofluorescent scaffold contraction recorded and processed to measure fractional shortening.
    • Movie S8 (.mov format). Vertical scan of a three-layer Tissue-Velcro.
    • Movie S9 (.mov format). Electrically paced cardiac tissues contracting before assembly, after assembly, after disassembly, and 1 day after disassembly.
    • Movie S10 (.mov format). Spontaneous contraction of a two-layer cardiac tissue cultured for an additional 3 days after assembly.
    • Movie S11 (.mov format). Response of Tissue-Velcro (day 5) to epinephrine (300 nM) stimulation.

    Files in this Data Supplement:

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