*2.10. Durotaxis Assays*

Cells were seeded on PA hydrogels with stiffness gradients. The cells were mounted and maintained on the microscope as described above. After live cell imaging and cell tracking, the direction of cell movement was calculated based on the results of the PIV analysis (fractional velocities in the x and y directions).

#### *2.11. Spheroid Spreading Assay*

Multicellular spheroids were generated as described above. Diameters of JAR spheroids were similar to those of human embryos at the periods of implantation (5–6 days after fertilization), with an average of 150 ± 15 µm [34,35]. By observation under a stereomicroscope (Olympus, Japan), each spheroid at the bottom of the well was carefully aspirated with a disposable pipette tip and transferred into a small dish (LABSELECT 12111). Spheroids with proper size were gently collected and evenly dispersed into six-well plates containing hydrogel-coated coverslips. Spheroids were then incubated for 24 h for attachment and spreading before imaging with 10× or 20× objectives. To quantify the degree of dispersion, images were firstly converted into 8-bit image and then thresholded using ImageJ (Image > Adjust > Threshold) to outline the periphery of the aggregate, and the spreading area (total area–spheroid area) of the 24 h image was divided by the area of the spheroid, which was considered the spreading ratio.

#### *2.12. Statistical Analysis*

All statistical analyses were performed using R software (version 4.1.1). Results were expressed as the mean ± standard deviation (SD). Every experiment was repeated three times (*n* = 3). After confirming that the data were normally distributed and homogeneous in variance using the Shapiro–Wilk significance test as well as the Bartlett test, Student's *t*-test was used for analysis. For all comparisons, *p* < 0.05 was considered statistically significant.

#### **3. Results**

#### *3.1. Simulated ECM Stiffness Regulates Trophoblast Cell Morphology and Spreading Area*

Numerous studies demonstrated that cell spreading and focal adhesion maturation are positively correlated with ECM rigidity in various cells [36–42]. However, this correlation was not investigated in cytotrophoblasts. Therefore, we fabricated PA hydrogels (Figure S1), examined the surface morphology of the gels by SEM (Figure S2), and measured the apparent elastic modulus by AFM (Figure S3), which represent varying ECM stiffnesses independently of topographical and compositional cues [28]. We cultured JAR cells on simulated ECM with stiffness gradients ranging from 10 kPa to 100 kPa (Figure 1A,B). We then divided the hydrogel into three regions according to their stiffness: a stiff region (46.7 ± 25 kPa), an intermediate region (14.6 ± 8.0 kPa), and a soft region (6.9 ± 0.5 kPa).

After 24 h incubation, the cells were observed by phase-contrast microscope (Figure 1C). Cells growing on simulated ECM with different stiffness showed significant changes in cell morphology and spreading area. On the stiffer simulated ECM, JAR cells were polygonal with a larger cell spreading area, while the spreading area of the cells tended to decrease as stiffness decreased, and the cell morphology gradually became round (Figure 1D). These results demonstrate that JAR cell morphology and spreading area are regulated by simulated ECM stiffness.
