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Clinical Trial
. 2015 Jun;21(6):576-84.
doi: 10.1089/ten.TEC.2014.0458. Epub 2014 Dec 17.

Limbal Fibroblasts Maintain Normal Phenotype in 3D RAFT Tissue Equivalents Suggesting Potential for Safe Clinical Use in Treatment of Ocular Surface Failure

Isobel Massie  1 , Sarah B Dale  1 , Julie T Daniels  1
Affiliations
Clinical Trial

Limbal Fibroblasts Maintain Normal Phenotype in 3D RAFT Tissue Equivalents Suggesting Potential for Safe Clinical Use in Treatment of Ocular Surface Failure

Isobel Massie et al. Tissue Eng Part C Methods. 2015 Jun.

Abstract

Limbal epithelial stem cell deficiency can cause blindness, but transplantation of these cells on a carrier such as human amniotic membrane can restore vision. Unfortunately, clinical graft manufacture using amnion can be inconsistent. Therefore, we have developed an alternative substrate, Real Architecture for 3D Tissue (RAFT), which supports human limbal epithelial cells (hLE) expansion. Epithelial organization is improved when human limbal fibroblasts (hLF) are incorporated into RAFT tissue equivalent (TE). However, hLF have the potential to transdifferentiate into a pro-scarring cell type, which would be incompatible with therapeutic transplantation. The aim of this work was to assess the scarring phenotype of hLF in RAFT TEs in hLE+ and hLE- RAFT TEs and in nonairlifted and airlifted RAFT TEs. Diseased fibroblasts (dFib) isolated from the fibrotic conjunctivae of ocular mucous membrane pemphigoid (Oc-MMP) patients were used as a pro-scarring positive control against which hLF were compared using surrogate scarring parameters: matrix metalloproteinase (MMP) activity, de novo collagen synthesis, α-smooth muscle actin (α-SMA) expression, and transforming growth factor-β (TGF-β) secretion. Normal hLF and dFib maintained different phenotypes in RAFT TE. MMP-2 and -9 activity, de novo collagen synthesis, and α-SMA expression were all increased in dFib cf. normal hLF RAFT TEs, although TGF-β1 secretion did not differ between normal hLF and dFib RAFT TEs. Normal hLF do not progress toward a scarring-like phenotype during culture in RAFT TEs and, therefore, may be safe to include in therapeutic RAFT TE, where they can support hLE, although in vivo work is required to confirm this. dFib RAFT TEs (used in this study as a positive control) may be useful toward the development of an ex vivo disease model of Oc-MMP.

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Figures

<b>FIG. 1.</b>
FIG. 1.
MMP-2 and MMP-9 activity. MMP-2 and MMP-9 activity was measured from conditioned medium by gelatin zymography. The three bands on each zymogram correspond to pro MMP-9 (92 kDa), pro MMP-2 (66 kDa), and active MMP-2 (62 kDa) as labeled on the right of each gel. (A) MMP activity from RAFT TEs made with either normal hLF (top) or dFib (bottom) with or without hLE and from nonairlifted and airlifted RAFT TEs (AL). Each lane represents conditioned medium collected from a single RAFT TE construct within one experiment. The zymograms shown in (A) are representative of two further experimental repeats. (B) Comparison between MMP activities from RAFT TEs made with either normal hLF (N) or dFib (D) with hLE or without hLE. MMP activity was compared from nonairlifted (top) or airlifted (bottom) RAFT TEs. Each lane represents conditioned medium pooled from three separate experiments for each RAFT TE condition. In both (A, B), the right hand side lane represents the negative control (conditioned medium was collected from acellular RAFT TEs). dFib, diseased fibroblasts; hLE, human limbal epithelial cells; hLF, human limbal fibroblasts; MMP, matrix metalloproteinase; RAFT, real architecture for 3D tissue; TEs, tissue equivalents. Color images available online at www.liebertpub.com/tec
<b>FIG. 2.</b>
FIG. 2.
Pro MMP-1 secretion. Pro MMP-1 secretion was measured from conditioned medium by ELISA. RAFT TE constructs contained either normal hLF (black bars) or dFib (gray bars). Conditioned medium was collected from RAFT TEs with or without hLE from nonairlifted and airlifted (AL) RAFT TEs. None of these results are statistically significant. n=3±SD. ELISA, enzyme-linked immunosorbent assay; SD, standard deviation.
<b>FIG. 3.</b>
FIG. 3.
Collagen production. Collagen production was measured from conditioned medium by the Sircol colorimetric assay. RAFT TE constructs contained either normal hLF (black bars) or dFib (gray bars). Conditioned medium was collected from RAFT TEs with or without hLE from nonairlifted and airlifted (AL) RAFT TEs. *p<0.05. n=3±SD.
<b>FIG. 4.</b>
FIG. 4.
α-SMA expression. Fibroblast expression of α-SMA (red) in RAFT TE constructs was observed using wholemount immunohistochemistry. RAFT TE constructs were also stained for phalloidin (green) and nuclei were counterstained using DAPI (blue). The positive (forced differentiation under mechanical tension) and negative (omission of anti-α-SMA antibody) controls are shown in (A, B), respectively (top). (C–F) Staining of normal hLF within RAFT TE (middle). (G–J) Staining of dFib within RAFT TE (bottom). (D, F, H, J) hLE+ RAFT TEs (C, E, G, I: hLE− RAFT TEs). (E, F, I, J) Airlifted RAFT TEs (C, D, G, H: nonairlifted RAFT TEs). All images are representative of three fields of view captured per condition per experiment. Scale bars: 50 μm. α-SMA, α-smooth muscle actin. Color images available online at www.liebertpub.com/tec
<b>FIG. 5.</b>
FIG. 5.
TGF-β1 secretion. TGF-β1 secretion was measured from conditioned medium by ELISA. RAFT TE constructs contained either normal hLF (black bars) or dFib (gray bars). Conditioned medium was collected from RAFT TEs with or without hLE from nonairlifted and airlifted (AL) RAFT TEs. *p<0.05. n=3±SD. TGF-β1, transforming growth factor-β1.
<b>FIG. 6.</b>
FIG. 6.
Normal hLF response in RAFT TE to hLE wound. Expression of α-SMA (red) in normal hLF in nonairlifted, hLE+ RAFT TE constructs was observed by wholemount immunohistochemistry. RAFT TE constructs were also stained for phalloidin (green) and nuclei were counterstained using DAPI (blue). RAFT TE constructs were stained either immediately before (A), or 6 days after (B), wounding. Images are representative of five fields of view captured per condition per experiment. Scale bars: 50 μm. Color images available online at www.liebertpub.com/tec
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