Figure 3. Migration of Cardiac Fibroblasts in Response to Varying Levels of Ischemia.

A) Schematic of three stacks used to induce an environment of low ischemia (left), medium ischemia (middle), and high ischemia (right) for two layers containing cardiomyocytes (100,000 cells/zone) at the bottom of the stack. Each stack contains a different number of layers of immortalized 3T3 fibroblast (50,000 cells/zone) placed on top of the stack (the cells in these upper layers deplete nutrients diffusing toward the cardiomyocytes). Each stack contained a single layer of primary cardiac fibroblasts (10,000 cells/zone) between the 3T3 fibroblasts and cardiomyocytes. Cardiac fibroblasts were labeled with Cell Tracker Orange prior to seeding. Each stack was cultured for three days. B) Viability (determined with calcein AM) of the top layer of cardiomyocytes (e.g., cardiomyocytes in the second from bottom layer of the stack shown in A). C) Relative amount of migration of the cardiac fibroblasts into the top layer of cardiomyocytes, determined by the fluorescence intensity of Cell Tracker Orange in the second from bottom layer that contains either cardiomyocytes (in matrigel) or only matrigel. Error bars are the standard deviation of 10 replicates. Means with different letters are significantly different based on Holm-Sidak method, (P = <0.001). Detailed statistics of each value available in the SI.