2.1. Cell Culture
Corneal keratocytes were isolated from rabbit eyes obtained from Pel Freez (Rogers, AR, USA) as previously described. Cells were cultured in tissue culture flasks with medium consisting of Dulbecco’s modified Eagle’s minimum essential medium with pyruvate (DMEM; Invtrogen, Carlsbad, CA), supplemented with 1% RPMI vitamin mix (Sigma-Aldrich, St. Louis, MO), 100µM nonessential amino acids (Invitrogen, Carlsbad, CA), 100µg/ml ascorbic acid, and 1% penicillin/streptomycin amphotericin B (Fungizone; BioWhittaker, Inc., Walkersville, MD) to maintain the keratocyte phenotype.
2.2. Preparation of Cell-Seeded Compressed Collagen Matrices
To prepare compressed collagen matrices, 10mg/ml of Type I Rat Tail collagen (BD Biosciences, San Jose, CA) was diluted to a final concentration of 4mg/ml. 0.6ml of 10X DMEM was added to 5ml of the collagen solution and after drop-wise neutralization with 1N sodium hydroxide, a suspension of 8×106
keratocytes in 0.6ml serum-free media was added to the collagen mixture. The solution containing cells and the collagen was poured into a 3×2×1cm well and allowed to set for 30 minutes at 37°C. Following polymerization, the matrix was compacted by external compression as previously described [34
]. Briefly, a layer of nylon mesh (~50µm mesh size) was placed on a double layer of filter paper. The matrices were placed on the nylon mesh, covered with another layer of nylon mesh and a pane of glass, and loaded with a 130g stainless steel block for 5 minutes at room temperature. This led to the formation of flat collagen sheet approximately 400 microns thick, with a cell density of ~33,000 cells/mm3
which is somewhat higher than that of the human cornea (20,522 ± 2,981 cells/mm3
2.3. Preparation of Sandwiched Compressed Collagen Matrices
To prepare compressed sandwich collagen matrices, 0.3ml of 10X DMEM was added to 2.5ml of collagen solution and after drop-wise neutralization with 1N sodium hydroxide, 0.3ml serum-free media was added to the collagen mixture. The collagen mixture with no cells was poured into a 3×2×1cm well and allowed to set for 30 minutes at 37°C. Following polymerization, a suspension of 2×106 or 5×105 keratocytes in 1 ml serum-free media was poured over the polymerized collagen. After 30 minutes to allow cell attachment to the matrix, media (including unattached cells) was removed from the top of the collagen. Another layer of collagen was poured over the layer of cells and incubated for 30 minutes at 37°C. Following polymerization, the matrix was compacted by external compression as described above.
2.4. In Vitro Injuries
Following compression, 8mm diameter buttons were punched out of the compressed matrix and incubated in serum-free media for 24 hours to allow cell spreading. We then simulated an injury by either: (1) immersing a 3 mm diameter cylindrical stainless steel probe (25 g) with a polished flat tip in liquid nitrogen for 20 seconds, then placing the tip against the surface of the construct for 5 seconds using only gravitational force (Freeze injury), or (2) pushing on the surface near the center of the matrices for approximately 1 second using either a 3 mm diameter flat tip probe or a glass probe with a 5 mm diameter spherical tip (Mechanical Injury). Following injury, the buttons were then cultured in serum-free media supplemented with either 10% FBS, TGFβ1 (10ng/ml, Sigma Aldrich, St. Louis, MO), PDGF BB (50ng/ml, Millipore, Temecula, CA), or no growth factor (control) for up to 4 days. Growth factor concentrations were determined from previous studies, and represent the lowest concentration to give a maximal effect [37
2.5. Live/Dead Staining
To determine the effects of injury simulation, the Live/Dead® Viability/Cytotoxicity Assay Kit (Invitrogen; CA) was used. This assay is based on the simultaneous determination of live and dead cells with two probes that measure recognized parameters of cell viability—intracellular esterase activity and plasma membrane integrity. One day after in vitro injury to the compressed matrix buttons, LIVE/DEAD® labeling was performed according to the manufacturer’s instructions. Labeled cells were then imaged using confocal microscopy.
2.6. F-Actin and DNA Labeling
One to seven days after injury, constructs were fixed using 3% paraformaldehyde in phosphate buffer for 10 min and permeabilized with 0.5% Triton X-100 in phosphate buffer for 3min. Cells were labeled with Alexa Fluor 488 Phalloidin (1:50; Molecular Probes, Eugene, OR) for 1 hour and then washed in phosphate buffer saline (PBS; 3 times for 5 minutes). To stain the cell nuclei, TOTO-3 iodide (1:200; Molecular Probes, Eugene, OR) was then added to each sample for 15 minutes, and samples were washed a final time with PBS.
2.7. Laser Confocal Microscopy
After labeling the cells, fluorescent (for f-actin and nuclei) and reflected light (for collagen) 3-D optical section images were acquired simultaneously using laser confocal microscopy (Leica SP2, Heidelberg, Germany). Stacks of optical sections (z-series) were acquired by changing the position of the focal plane in 4µm steps for a 20X objective (non-immersion, 0.7 NA, 590µm free working distance) or 1µm steps for a 63X objective (water immersion, 1.2 NA, 220µm free working distance). Overalapping image stacks were collected across the entire wound area, as shown in .
Schematic showing where the montage of 3-D image stacks was collected for each sample.
2.8. Assessment of Cell Migration
Maximum intensity projections of the F-actin and TOTO-3 image stacks were created, and color overlays were generated using Metamorph (Molecular Devices, Sunnyvale, CA). Photoshop (Adobe, Mountain View, CA) was then used to align the overlapped images to create a montage of the injured area. Each montage included the wound area along with border region outside the injury. The distance that cells had traveled into the wound area was calculated by drawing a straight line between the edge of the injury and cells at the leading edge of the migratory front.