Three-dimensional (3D) control over the placement of bioactive cues is fundamental to understand cell guidance and develop engineered tissues. Two-photon patterning (2PP) provides such placement at micro-to millimeter scale, but non-specific interactions between proteins and functionalized extracellular matrices (ECMs) restrict its use. Here we report a 2PP system based on non-fouling hydrophilic photocages and Sortase A-based enzymatic coupling offering unprecedented orthogonality and signal-to-noise ratio in both inert hydrogels and complex mammalian matrices. Improved photocaged peptide synthesis, and protein functionalization protocols with broad applicability are introduced. Importantly, the method enables 2PP in a single step and in the presence of fragile biomolecules and cells. As a corollary, we demonstrate the guidance of axons through 3D-patterned nerve growth factor (NGF) within brain-mimetic ECMs. Our approach allows for the interrogation of the role of complex signaling molecules in 3D matrices, thus helping to better understand biological guidance in tissue development and regeneration.
Nanoindentation refers to a class of experimental techniques where a micrometric force probe is used to quantify the local mechanical properties of soft biomaterials and cells. This approach has gained a central role in the fields of mechanobiology, biomaterials design and tissue engineering, to obtain a proper mechanical characterization of soft materials with a resolution comparable to the size of single cells (μm). The most popular strategy to acquire such experimental data is to employ an atomic force microscope (AFM); while this instrument offers an unprecedented resolution in force (down to pN) and space (sub-nm), its usability is often limited by its complexity that prevents routine measurements of integral indicators of mechanical properties, such as Young's Modulus (E). A new generation of nanoindenters, such as those based on optical fiber sensing technology, has recently gained popularity for its ease of integration while allowing to apply sub-nN forces with µm spatial resolution, therefore being suitable to probe local mechanical properties of hydrogels and cells.In this protocol, a step-by-step guide detailing the experimental procedure to acquire nanoindentation data on hydrogels and cells using a commercially available ferruletop optical fiber sensing nanoindenter is presented. Whereas some steps are specific to the instrument used herein, the proposed protocol can be taken as a guide for other nanoindentation devices, granted some steps are adapted according to the manufacturer's guidelines. Further, a new open-source Python software equipped with a user-friendly graphical user interface for the analysis of nanoindentation data is presented, which allows for screening of incorrectly acquired curves, data filtering, computation of the contact point through different numerical procedures, the conventional computation of E, as well as a more advanced analysis particularly suited for single-cell nanoindentation data.
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