Organized cellular alignment is critical for variety of biological phenomenon as well as necessary for several tissue engineering applications. Although a variety of methods have been used to control cellular alignment in 2D, recapitulating the organized 3D cellular alignment found within native tissues remains a challenge. In this study, we present a new method to align cells in localized user-defined orientations using femtosecond (fs) laser enabled hydrogel densification. Fs laser direct writing was used to induce densification within partially crosslinked gelatin methacrylate (GelMA) hydrogel. Densified line patterns were used to preferential align variety of cells such as mouse 10T1/2s fibroblasts and IDG-SW3 osteocytes, and human HUVECs and hiPSC-derived MSCs. Cellular alignment as a function of cell-culture time, line spacing, and modification-depth were characterized. As compared to the current technology, this method can be applied to any photocrosslinkable hydrogel, as it does not require specialized chemical or physical modifications or any external guidance cues. Additionally, densification can be introduced during active cell culture providing temporal flexibility in experimental design. This method can be potentially used for the creation of organized engineered tissues.
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