In Situ 3D-Printing using a Bio-ink of Protein–photosensitizer Conjugates for Single-cell Manipulation

[Image: see text] Living tissues dynamically modulate their structure and functions through physical and biochemical interactions in the three-dimensional (3D)-microenvironment for their homeostasis or the developmental process of an embryo. However, the manipulation of cellular functions in vitro is still challenging due to the lack of a dynamic material system that can vary the 3D-cellular microenvironment in time and space. Here, we show an in situ 3D-printing technique based on multiphoton lithography using a biocompatible photoresist, bio-ink. The bio-ink composed of protein–photosensitizer conjugates has the ability to cause singlet oxygen and cross-linking reaction to fabricate protein gels with submicrometer-scale precision. Remarkably, the conjugates substantially improve the cytocompatibility and the efficiency of gelation due to the stealth effect of rose bengal (RB) and efficient transfer of singlet oxygen to bovine serum albumin (BSA). 3D-printing in the presence of cells allows for the microfabrication of a protein scaffold and controlled single-cell behavior. This dynamic material system to direct cell fate may offer emerging applications for drug discovery and regenerative medicine.