Research ArticleENGINEERING

Ultrathin micromolded 3D scaffolds for high-density photoreceptor layer reconstruction

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Science Advances  21 Apr 2021:
Vol. 7, no. 17, eabf0344
DOI: 10.1126/sciadv.abf0344
  • Fig. 1 PGS ice cube tray scaffold manufacturing process.

    (A) Schematic illustration of the ice cube tray PR scaffolds designed to have a reservoir layer for cell capture and retention and a through-hole layer for exchange of fluid, waste products, and nutrients both in vitro and during scaffold degradation in vivo. (B to G) Schematic illustration of the procedure to fabricate the ice cube tray PR scaffolds using a poly(glycerol sebacate) (PGS) prepolymer. (B) Through-hole and (C) reservoir etching processes of a Si master mold. (D) Molding and demolding processes of a hard-polydimethylsiloxane (h-PDMS) stamp from the Si master mold. (E) Mounting and demounting processes of the h-PDMS stamp for fabricating a PGS ice cube tray PR scaffold. (F) Delamination process of the scaffold using a razor blade. (G) Final PGS ice cube tray PR scaffold.

  • Fig. 2 Fabrication of Si master mold and h-PDMS stamp.

    Scanning electron microscopy (SEM) images of the ice cube tray–shaped (A) Si master mold, and (B) h-PDMS stamp showing (i) a tilted view, (ii) a top view, and (iii) a cross-sectional view, respectively. The inset images show a magnified view of the microstructures of the fabricated Si master mold and h-PDMS stamp.

  • Fig. 3 Fabrication of the PGS ice cube tray PR scaffold.

    (A to C) Low-magnification photographic images depicting the fabrication process of the PGS ice cube tray PR scaffold. (A) h-PDMS stamp ready to be demounted from the scaffold on a Si wafer after complete PGS curing. (B) A PGS scaffold on the Si wafer after stamp removal. After removing scaffold edges, the scaffold was delaminated from the Si wafer using a single-edge razor blade. (C) Fabricated PGS ice cube tray scaffold held with fine forceps. (D to G) SEM images of the fabricated ice cube tray retinal scaffold showing (D) a top view, (E) a bottom view, and (F) a cross-sectional view. (G) Large-area SEM image of the fabricated scaffold and a magnified view of a scaffold reservoir wall (inset).

  • Fig. 4 Finite element analysis showing equivalent von Mises stress distribution in the PGS scaffolds.

    (A) Wineglass and (B) ice cube tray design under 5 N of tensile force in the x and y directions: (i) isometric view, (ii) top view, (iii) bottom view, and (iv) orthogonal view. The color bar shows the von Mises stress (in newton per square meter) for an applied tensile force.

  • Fig. 5 Generation of PGS ice cube tray PR scaffold constructs.

    (A to C) Low-magnification photographic images depicting scaffold mounting into the transwell insert. (A) Transwell insert with PGS scaffold below. The outer edge of the scaffold was glued to the transwell insert with soft PDMS. The area of the transwell insert removed to mount scaffolds was 19.6 mm2 (internal diameter, 5 mm). (B) Transwell insert holder with a PGS ice cube tray scaffold mounted into a transwell insert. (C) Six-transwell scaffold cell culture system. (D to F) Laminin-coated ice cube tray scaffolds are readily filled with hPSC-derived CRX+/tdTomato-expressing PRs. (D) 3D rendering of a scaffold (176 μm by 185 μm by 22 μm) confirms successful capture of multiple PRs (labeled in red) in individual capture wells. Cell nuclei are labeled with 4′,6-diamidino-2-phenylindole (DAPI) (blue). (E) Cells were seeded onto scaffolds at varying densities to determine the minimum number required to achieve the maximum carrying capacity of CRX+/tdTomato-PRs per well. Median (bold dashes) and quartiles (fine dashes) are shown within individual violin plots. (F) Scaffolds seeded with CRX+/tdTomato-PRs (RFP+, red) contain both ARR3-expressing cone PRs (green) and NR2E3-expressing rod PRs (pink). A 3D lateral view of the scaffold demonstrates relatively even distribution of ARR3+ cones and NR2E3+ rods. 3D rendering is 644 μm by 644 μm by 20 μm. Photo Credit: In-Kyu Lee, Department of Electrical and Computer Engineering, University of Wisconsin–Madison.

  • Fig. 6 Micropatterned ice cube tray scaffolds support prearranged orientation of seeded PRs.

    (A and B) Maximum intensity projections of scaffold whole mounts seeded with CRX+/tdTomato-PRs (red) revealed that PRs plated on scaffolds express PRPH2 (6A, green) and VGLUT1 (6B, green). DAPI-labeled cell nuclei and PGS autofluorescence are shown in blue. (C) PRPH2+ outer segments were often oriented perpendicular to the base of the scaffold (magnified in underlying image). (D) Expression of presynaptic marker VGLUT1 (green) primarily localizes to the top portion of the scaffold. 3D renderings (C and D) are 644 μm by 644 μm by 20 μm.

  • Table 1 Structural and mechanical specifications for wineglass and ice cube tray scaffolds.

    Wineglass scaffold
    Ice cube tray
    scaffold design
    Overall thickness (μm)2530
    Space between
    through-holes (μm)
    Through-holes (μm)5 diameter, 10 depth
    (1 hole per capture
    5 diameter, 5 depth
    (9 holes per capture
    Capture well reservoir
    volume (mm3)
    0.177 x 10−52.103 x 10−5
    Scaffold biomaterial
    volume (based on a
    scaffold) (mm3)
    0.340.169 (50% less
    Young’s modulus1.18 MPa1.3 MPa
  • Table 2 Cell payload advantages of ice cube tray versus wineglass scaffold designs.

    Wineglass scaffold
    design (26)
    Ice cube tray
    scaffold design
    Average number of PRs
    per capture well
    1.3 ± 0.517.8 ± 2.4
    PRs within a single
    scaffold (5 mm in
    diameter or
    19.63 mm2)
    1.005 × 1053.412 × 105
    Scaffold PR density
    (cells/mm2) (6.0 × 104
    to 20.0 × 104 cells/mm2
    within the macula in
    healthy retina)
    0.512 × 1041.74 × 104

Supplementary Materials

  • Supplementary Materials

    Ultrathin micromolded 3D scaffolds for high-density photoreceptor layer reconstruction

    In-Kyu Lee, Allison L. Ludwig, M. Joseph Phillips, Juhwan Lee, Ruosen Xie, Benjamin S. Sajdak, Lindsey D. Jager, Shaoqin Gong, David M. Gamm, Zhenqiang Ma

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