Fei helios nanolab 660

FIB-SEM imaging was performed using FEI Helios NanoLab 660 (Thermo Fisher Scientific) equipped with a cryo-preparation system (PP3010, Quorum). The sample preparation procedures were as follows: hydrogel samples were rinsed with PBS and then fixed with 4% glutaraldehyde at 4 °C overnight and then post-fixed with 1% OsO₄ in ddH₂O for 1 h. The samples were further washed with PBS three times. The hydrated samples were carefully mounted and quickly frozen by dipping them into liquid nitrogen. The frozen samples were transferred onto a cryo-stage (−140 °C) and fractured with a knife in the Quorum chamber. Thereafter, the temperature of the stage was set to −85 °C and then gradually increased to −50 °C (5 °C/min). The temperature was maintained at −50 °C for five more minutes, after which it was reduced to −140 °C. The sublimed samples were sputter-coated with platinum in the SEM (10 mA, 60 s) to increase the conductivity. The images were acquired with a 50-pA beam current at a 5-kV acceleration voltage using the concentric backscatter detector of the SEM.
To calculate the average pore sizes and number densities of hydrogels in the SEM images, we used the Analyze Particle function in ImageJ. For each type of hydrogel, we independently prepared two samples and acquired four SEM images on each sample. The field of view of each image was 31.5 × 22.5 μm².

PANC-1 cells were cultured on well-cleaned and sterilized cover slips (22 mm × 22 mm). After treatment with the macrophage CMs, the cells were rinsed with PBS and then fixed with 3.7% formaldehyde for 15 min and later post-fixed with 1% OsO₄ in PBS for 2 h. After triple PBS flushes, we replaced the medium with ethanol gradually from 10% (v/v in H₂O) to 99.9%. The final samples were dried with a critical point dryer (EM CPD300, Leica Microsystems, Wetzlar, Germany). Before the FIB-SEM operation, the sample surface was sputtered with 10 nm gold to raise the conductivity. FIB-SEM operations were conducted with the FEI Helios NanoLab 660 (Thermo Fisher Scientific). The target area was firstly deposited with a 500 nm platinum protection layer via ion beam-induced deposition, rough-milled with 40 pA and then fine-milled with 7.7 pA gallium ion beams at a 30 kV acceleration voltage. All the SEM images were taken right after the FIB manipulations, acquired with a 25–50 pA beam current at a 1–5 kV acceleration voltage and an “in-lens” detector for the secondary electrons.

An FEI Helios NanoLab 660 (Thermo Fisher Scientific, Waltham, MA, USA) electron microscope was used to obtain SEM images. A Hitachi HT7700 microscope (Hitachi, Tokyo, Japan) at an accelerating voltage of 100 kV was used to record TEM images.