Figure 2. Primary sphere formation by keratocytes from the peripheral and central regions of the rabbit cornea. (A, B) Stromal cells from the peripheral or central cornea form spheres. Stromal tissue was disaggregated into single cells, which were plated at a density of 10 viable cells/μl in a basal medium containing methylcellulose gel matrix to prevent reaggregation. More than 99% of the cells were single cells on day 0. Growth of a representative sphere is shown until day 7. Scale bar=200 μm. (C) The number of primary spheres derived from stromal tissue was compared between the periphery and center of the cornea. The number of sphere colonies obtained from samples of the peripheral stroma (n=10) was significantly higher than that for samples of the central stroma (n=10) after seven days of culture (The asterisk indicates that p=0.00021 and unpaired t-test was performed). (D) The size of primary sphere colonies derived from samples of peripheral (n=10) and central (n=10) corneal stroma was compared. The mean size of spheres from both regions gradually increased during culture to exceed 250 μm on day 7 (periphery: 258±63 μm versus center: 203±71 μm after seven days, mean±SD). n.s.=not significant.