Experimental models for investigating intra-stromal migration of corneal keratocytes, fibroblasts and myofibroblasts

Abstract

Following laser vision correction, corneal keratocytes must repopulate areas of cell loss by migrating through the intact corneal stroma, and this can impact corneal shape and transparency. In this study, we evaluate 3D culture models for simulating this process in vitro. Buttons (8 mm diameter) were first punched out of keratocyte populated compressed collagen matrices, exposed to a 3mm diameter freeze injury, and cultured in serum-free media (basal media) or media supplemented with 10% FBS, TGFβ1 or PDGF BB. Following freeze injury, a region of cell death was observed in the center of the constructs. Although cells readily migrated on top of the matrices to cover the wound area, a limited amount of cell migration was observed within the constructs. We next developed a novel "sandwich" model, which better mimics the native lamellar architecture of the cornea. Using this model, significant migration was observed under all conditions studied. In both models, cells in TGFβ and 10% FBS developed stress fibers; whereas cells in PDGF were more dendritic. PDGF stimulated the most inter-lamellar migration in the sandwich construct. Overall, these models provide insights into the complex interplay between growth factors, cell mechanical phenotypes and the structural properties of the ECM.

Keywords: 3D Culture; Cell Mechanics; Corneal Keratocytes; Extracellular Matrix; Growth Factors.

Figure 1

Schematic showing where the montage of 3-D image stacks was collected for each sample.

Figure 2

Maximum intensity projection images (~50 microns thick) of Live/Dead staining after 1 day of culture in 10% FBS. Live cells are labeled green and dead cells are labeled red. (A) 1 day after mechanical injury (MI), induced by pushing on the surface of the matrix using a probe with a 5mm diameter spherical tip; (B) 1 day control sample, which was left untouched.

Figure 3

Maximum intensity projection images (~50 microns thick) of Live/Dead staining in standard compressed matrices after 1 day of culture in 10% FBS. Live cells are labeled green and dead cells are labeled red. (A) 1 day after freeze injury (FI), induced by pushing on the surface of the matrix using a cold 3 mm diameter probe; (B) 1 day control sample, in which a room temperature probe was used.

Figure 4

High magnification images of f-actin, 4 days after freeze injury in standard compressed matrices. (A) Migrating cells in basal media have a more stellate morphology and rarely exhibit stress fibers; (B) Following culture in 10% FBS, migrating cells become fibroblastic, as indicated by the development of a bipolar morphology and stress fiber formation; (C) Following culture in TGFβ1, cells developed a broader morphology and prominent intracellular stress fibers were observed; (D) Migrating cells in PDGF appeared more elongated with branching, dendritic cell processes.

Figure 5

Maximum intensity projection images (~50 microns thick) of f-actin (green) and TOTO-3 (red), 4 days after freeze injury (FI) in standard compressed matrices and culture in (A) basal media; (B) 10% FBS; (C) TGFβ1 or (D) PDGF. Migrating cells are observed in the wounded area under 10% FBS and TGFβ1 conditions. However, most of these cells are on or near the surface of the construct. Few migrating cells are observed under serum-free or PDGF culture conditions.

Figure 6

Maximum intensity projection images (~50 microns thick) of Live/Dead staining after 1 day of culture following freeze injury using sandwich construct. Live cells are labeled green and dead cells are labeled red. (A) 1 day after freeze injury, induced by pushing on the surface of the matrix using a cold 3 mm diameter probe; (B) 1 day control sample, in which a room temperature probe was used.

Figure 7

Maximum intensity projection images of f-actin (green) and toto (red), 4 days after freeze injury (FI) on sandwiched matrix constructs, and culture in basal media (A); 10% FBS (B); TGFβ (C) or PDGF BB (D); Migrating cells are observed in the wounded area under PDGF (D) and 10% FBS (B) conditions. Few migrating cells are observed under serum-free (A) or TGFβ1 (C) culture conditions. E. Quantitative analysis of the distance cells traveled into the injury. Traveling the farthest were cells in PDGF (** P < 0.001 as compared to the other three conditions) and 10% FBS (* P < 0.01 as compared to basal media and TGFβ1).

Figure 8

High magnification images of f-actin, 4 days after freeze injury using sandwiched matrix construct. (A) Migrating cells in basal media have a more stellate morphology and rarely exhibit stress fibers; (B) Following culture in 10% FBS, migrating cells become fibroblastic, as indicated by the development of a bipolar morphology and stress fiber formation; (C) Following culture in TGFβ1, cells developed a broader morphology and prominent intracellular stress fibers were observed; (D) Migrating cells in PDGF appeared more elongated with branching, dendritic cell processes.

Figure S1

Maximum intensity projection images (~50 microns thick) of f-actin (green) and TOTO-3 (red), 7 days after freeze injury on standard compressed matrices and culture in (A) basal media; (B) 10% FBS; (C) TGFβ1 or (D) PDGF. Migrating cells have completely covered the wounded area under 10% FBS conditions. However, most of these cells are on or near the surface of the construct. Few migrating cells are observed under either serum-free or PDGF culture conditions.