*3.4. JAR Cells Exhibit Durotaxis*

Spatial changes in ECM stiffness were shown to induce migration toward increased stiffness in numerous cell types both in vitro and in vivo [49]. This process, which is a key regulator of cell migration and invasion, is called durotaxis [50–52]. Although durotaxis was observed in many cell types [53–56], few studies described durotaxis in the context of human trophoblast cells. Therefore, we performed tracing for JAR cells grown on simulated ECM with a large stiffness gradient for 9.5 h (Figure 4A). By analyzing these trajectories, we found that most cells tended to migrate toward the stiffer simulated ECM region (Figure 4B,D). PIV analysis that was performed on two adjacent frames also demonstrated that the migratory direction of most cells coincided with the positive direction of the stiffness gradient (Figure 4C). These results demonstrate that JAR cells exhibit durotaxis.

**Figure 4.** JAR cells exhibit durotaxis. (**A**) Tracking of JAR cells cultured on stiff region (scale bar: 50 µm, time bar: 9.5 h). E indicates the apparent Young's modulus. Arrows indicated the displacement of each cell, (**B**) Representative JAR cell migration plots on stiff region over 9.5 h. The total cell number *n* = 56, number of independent experiments *N* = 3. (**C**) Vector map of JAR cell migration on stiff region (scale bar: 150 µm). Arrows indicated the vector of velocity. (**D**) Rose diagram of cell migration direction, which displays the angular between migration and stiffness gradient and the frequency of each class. The total cell number *n* = 56, number of independent experiments *N* = 3.
