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, 2010 (4), pdb.prot5416

The Mouse Cornea as a Transplantation Site for Live Imaging of Engineered Tissue Constructs

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The Mouse Cornea as a Transplantation Site for Live Imaging of Engineered Tissue Constructs

Ross A Poché et al. Cold Spring Harb Protoc.

Abstract

The field of tissue engineering aims to recapitulate healthy human organs and 3-D tissue structures in vitro and then transplant these constructs in vivo where they can be effectively integrated within the recipient patient and become perfused by the host circulation. To improve the design of materials for artificial tissue scaffolds, it would be ideal to have a high-throughput imaging system that allows one to directly monitor transplanted tissue constructs in live animals over an extended period of time. By combining such an assay with transgenic, cell-specific fluorescent reporters, one could monitor such parameters as tissue construct perfusion, donor cell survival, and donor-host cell interaction/integration. Here, we describe a protocol for a modified version of the classical corneal micropocket angiogenesis assay, employing it as a live imaging "window" to monitor angiogenic poly(ethylene glycol) (PEG)-based hydrogel tissue constructs.

Figures

Figure 1
Figure 1
Polymerization of hydrogel discs. See text for detailed procedure.
Figure 2
Figure 2
Modified corneal micropocket assay. (A–E) Shown here is an illustration detailing the steps involved in surgical implantation of fluorescently labeled hydrogel discs into the mouse cornea. By incorporating a fluorescent dye such as fluorescein isothiocyanate (FITC), the hydrogel is easily observed within the corneas of live mice (E, arrowhead). See text for details. Adapted by permission from MacMillan Publishers Ltd: [Nature Protocols] (Rogers et al. 2007), copyright 2007.
Figure 3
Figure 3
High-speed confocal imaging of circulating fluorescent microspheres among an implanted corneal hydrogel. Images were acquired at 50 frames/sec using a Zeiss LSM 5 LIVE high-speed confocal microscope with a Zeiss Plan-Neofluar 5X/0.15 NA objective lens, resulting in a movie showing the circulation of microspheres through the newly induced vessels surrounding the hydrogel. (A) A single frame taken from the image series. Bright green microspheres are located within the induced vessels and surrounding the hydrogel in the cornea of a live animal. (B–D) Images from the Imaris software in which individual spheres (yellow and blue arrows) are tracked in successive frames in an area of interest (white box in A). The tracks shown represent the trajectories of 23 microspheres imaged over 15.62 sec (753 images) and are shown as colored lines. The time stamp shows that the elapsed time between each frame (B to D) is 100 msec. The velocities of fluorescent spheres that were tracked ranged from 0.65 to 1.15 mm/sec (Average = 980.58±147.89 µm/sec).
Figure 4
Figure 4
Corneal flat mounts from implanted Flk1-myr::mCherry transgenic mice. (A) Schematic depiction of a corneal flat mount showing how the tissue is separated into quadrants, with one quadrant containing the implanted hydrogel and induced host vessels. (B) Blown-up view of the boxed region in panel A. (C,D) Tiled, confocal images of an actual corneal flat mount show a green fluorescently labeled hydrogel (containing VEGF) sitting adjacent to the limbic region (C) and Flk1-myr::mCherry+ (magenta) neovessels extending toward the hydrogel (D).
Movie 1
Movie 1
High-speed confocal imaging of circulating fluorescent microspheres among an implanted corneal hydrogel. A hydrogel disc releasing PDGF-BB (320 ng) and FGF-2 (80 ng) was implanted into the cornea and fluorescent microspheres within the circulation were imaged after 11 d.

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