Figure 3. Development and characterization of a synthetic endometrial stromal cell culture.

(A) Schematic of the workflow for culturing ESCs in 3D using a synthetic hydrogel. (B) Maximum intensity projection images of F-actin (green) reveal ESC remodeling (cell elongation and dispersion) at day 7 of culture in stromal (serum containing) media impacted primarily by hydrogel stiffness (20X magnification) as quantified in (C). (D) Time-lapse images of embedded ESCs in synthetic matrix (PEG) compared to the natural-derivedhydrogels Collagen I (~4 mg/mL) and Matrigel (8 mg/mL) allow for long-term stable cultures. (E) Brightfield images of stromal decidualization morphology in synthetic ECMs as a function of matrix stiffness (3wt%, 5wt% and 7wt% MIX) after 14 days of EP treatment and in standard ESC media or EEO medium (see Methods). (F) Biochemical measurement of prolactin (PRL) in the spent media (EEO media) from 15-day cultures in (E) compared to standard 2D polystyrene cultures (N=4). (G) Represenatitive fluorescent staining of DNA synthesis (EdU incorporation for 24 hrs hrs prior to fixation at day 15 as a function of simulated cycle phase and inflammation cue, IL1B (H) Quantification of mean EdU⁺ staining from cultures shown in (G) shows IL1B suppresses DNA synthesis of ESCs (n=3) (I) Quantification of apoptosis (cleaved caspase-3⁺) in ESC cells stimulated with homrone and IL1B treatment groups at day 15 of culture relative to total nuclei count (N=3) (J) Inflammatory challenge with IL1B significantly suppressed PRL secretion in ESC cultures (E+ = E + IL1B, PE+= PE+IL1B, W = hormone withdrawal) (N=6). (K) Vimentin stain (red) of ESC morphology cultured in synthetic and natural hydrogels. IL1B (1ng/mL) treated ESC in synthetic hydrogel provided as a control for inflamed morphology after 6 days. Significance is indicated as *p<0.05, **p<0.01, ***p<0.001. Scale bars are 50 mm, unless otherwise noted.