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Disc‐shaped hydrogels, not seeded with cells, were immersed in PBS after preparation and kept at 4 °C to the following day for mechanical quantification. After equilibrating them at room temperature for 1 h, gels were mounted onto glass object slides using Cell‐Tak (Thermo Fisher). Gel stiffness was probed by AFM indentation.^[18, ⁶², ^63] Cantilevers (arrow T1, Nanoworld), that had been modified with polystyrene beads of 10 µm diameter (Microparticles GmbH) using epoxy glue (Araldite), were calibrated using the thermal noise method implemented in the AFM software and probed at room temperature in PBS using a speed of 5 µm s⁻¹ and a relative force setpoint of 2.5 nn. Force distance curves were processed using the JPK data processing software. Indentation parts of the force curves (about 1 µm depth) were fitted using the Hertz model for a spherical indenter, assuming a Poisson ratio of 0.5.

Hydrogels bound to the glass coverslips were mounted on a 35 mm dish using vacuum grease and covered with PBS at room temperature. The characterization was performed with a Nanowizard 4 (JPK Instruments) using cantilevers (arrow T1, Nanoworld) equipped with 5 μm diameter polystyrene beads (microParticles GmbH) and calibrated with the thermal noise method. Gels were probed in liquid with an indentation speed of 5 μm/s and a relative force setpoint ranging from 0.6 to 8 nN to achieve comparable indentation depths of approximately 2 μm. The obtained force-distance curves were analyzed using the JPK data processing software. Parts of the curves corresponding to the first 2 μm of indentation depth were fitted using the Hertz/Sneddon model for a spherical indenter and Poisson ratio of 0.5 was assumed (Hertz, 1881 ; Sneddon, 1965 (link)).

After their preparation, hydrogels were immersed in PBS and stored at 4 °C until mechanical characterization on the following day. After equilibrating them at RT for 1 h, gels were mounted using CellTak (Thermofisher, Waltham, MA, USA) onto glass object slides (VWR, Radnor, PA, USA). A Nanowizard I or IV (JPK Instruments, Berlin, Germany) was used to probe the gel stiffness. Cantilevers (arrow T1, Nanoworld, Neuchâtel, Switzerland) that had been modified with polystyrene beads of 10 μm diameter (Microparticles GmbH, Berlin, Germany) using epoxy glue (Araldite, Huntsman Corporation, The Woodlands, TX, USA), were calibrated using the thermal noise method implemented in the AFM software (Version 6.1.159, NanoWizard Control Software, JPK Instruments/Bruker, Berlin, Germany). Hydrogels were probed at RT in PBS using a speed of 5 μm/sec and a relative force setpoint ranging from 2.5 to 4.0 nN in order to obtain comparable indentation depths (0.5–1 µm). Force distance curves were processed using the JPK data processing software (Version 6.1.159, NanoWizard Control Software, JPK Instruments/Bruker, Berlin, Germany). Indentation parts of the force curves (approximately 1.5 μm indentation depth) were fitted using the Hertz/Sneddon model for a spherical indenter, assuming a Poisson ratio of 0.5 [42 ,43 (link)].