Taking advantage of the spatial resolution afforded by this hydrogel system using orthogonal wavelengths of light, unique cell populations were encapsulated and recovered from individual hydrogels. Human bone marrow-derived stromal cells (hS5) stably expressing either mCherry, GFP, or BFP were encapsulated in Rubiq, Rubpy, and oNB-crosslinked hydrogels, respectively (Fig. ). Upon irradiation with red, green, or blue light, selective hydrogel degradation was observed by confocal microscopy (Fig. ). Following degradation, the gel-liberated cell populations were collected and analyzed via flow cytometry. Harvested cell collections matched the expected color composition: 617 nm light released only mCherry + cells, 530 nm yielded matched populations of mCherry + and GFP + , and 405 nm light degraded all three hydrogels and gave equivalent counts of mCherry + , GFP + , and BFP + cells (Fig. ). Results further demonstrate wavelength orthogonality and multiplexability of cell release from tricolor visible lightresponsive gels. Encapsulated cell viability at 24 hours post encapsulation. Cell counting revealed no statistical difference in cell viability across all crosslinkers at 24 hours. c. Rubiq, Rubpy, and oNB hydrogels were exposed to red, green, or blue light respectively (617 nm, 530 nm, 405 nm; 10 mW cm -2 ) and the released fibroblasts allowed to settle to the bottom of the well over 24 hr prior to imaging. Surviving cells exhibited classical fibroblast morphology upon calcein green/EtHD staining. Scale bars = 50 µm. d. High levels of viable cells were recovered from each hydrogel formulation, though cells released from Rubiq hydrogels showed lower levels of viability compared to Rubpy and oNB. ** p<0.0036 e. hS5-mCherry + , hS5-GFP + , and hS5-BFP + cells were encapsulated in Rubiq, Rubpy, and oNB hydrogels respectively. 24 hours after encapsulation, hydrogels were exposed to 617 nm, 530 nm, or 405 nm light. f. Images of hydrogels bearing mCherry + , GFP + , or BFP + hS5 cells following light exposure. Scale bars = 500 µm. g. Flow cytometry histograms show only expected cell populations were released into the media, demonstrating the orthogonality of the hydrogel system.