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Because of the functionality of the hCLiP‐derived biliary‐duct‐like structures, they were able to metabolize a cell‐tracking dye to ascertain the extent of the biliary lumen. Therefore, we used the sequential cell‐tracking staining method to determine the tubular lumen in the induced biliary duct structures. Briefly, the induced biliary structures were incubated with 100 µM rhodamine 123 dye for 10 min at 37°C. Subsequently, the biliary‐duct‐like structures were cultured in BIM‐2 medium (BIM‐1 supplemented with 2% Matrigel) for 48 h, followed by incubation with 10 μM cell tracker orange (CTO, C34551; Invitrogen, Tokyo, Japan) dye for 10 min at 37°C. After washing twice with HBSS, images were captured using a microscope (Ti-U and C-HGFI, Nikon, Tokyo, Japan).

The model can be made generalizable to other hardware setups through the use of transfer learning. For our transfer, we utilized a Nikon Ti-E Eclipse inverted fluorescence microscope. Images were acquired using a Nikon CFI Plain Fluor 20× objective and a 21-bit QImaging QIClick Camera. Illumination for brightfield imaging was performed using a 100 W halogen lamp (Ti-Dh Dia Pillar Illuminator). Epifluorescence excitation was performed using a 130 W mercury lamp (Nikon C-HGFI). None of the hardware used in the transfer learning microscope had been previously used in this study. The originally trained models were used as a starting point. The parameters in the convolution layers were fixed, making only the fully connected layers trainable. The final classification layer was replaced with a four-node layer, representative of the four classes (Jurkat, LNCaP, Raji, and THP-1) in the transfer-learning study. These four classes were T-cell lymphoblast Jurkat E6.1 (ATCC TIB-152), prostate derived LNCaP (ATCC CRL-1740), B-cell lymphoma Raji (ATCC CCL-86), and acute monocytic leukemia THP-1 (ATCC TIB-202). Sample preparation and training hyper-parameters were kept the same as previously outlined.

Cells on each grid plate were fixed with 4% (w/v) paraformaldehyde (PFA, Sigma-Aldrich) in 1x phosphate-buffered saline for 20 min at room temperature. Cells were treated for 5 min and thrice washed with the PBS 0.1% (v/v) Triton X-100 (Sigma-Aldrich) in 1 × PBS. To block nonspecific binding, the cells were incubated with 2% (v/v) goat serum (Sigma-Aldrich) in 1 × PBS for 30 min at room temperature and washed thrice with the PBS. The cells were incubated with primary antibodies at 4°C for 3 h. The following primary antibodies (anti-FN antibody IST-9) were used for incubation (1 : 1000; Abcam, England): B-cell lymphoma 2 (BCL2), cytochrome c (Cyt-c), and matrix metalloproteinase 13 (MMP13). After the incubation, the cells were washed thrice with 1 × PBS for 5 min each. Polyclonal secondary antibody (chicken) was added to Anti-Mouse IgG FITC labelled (Abcam, England), and 1 × PBS was added for double staining. These cells were incubated for 1 h at room temperature and counterstained using Hoechst dye and kept for 15 minutes. The signals were observed under a microscope (Nikon C-HGFI, Japan), and the images were taken documented NIS elemental imaging software using the NIS-Elements Documentation software.

The brains were prefixed with 4% paraformaldehyde, dehydrated in the gradient concentration of sucrose solutions (20, 30%), and cut into frozen sections at the thickness of 6 μm. Then primary antibodies including Beclin-1 and LC3B-II, as well neuronal marker anti-NeuN (1:500, Merck Millipore, MAB377), were incubated on the sections. After the primary antibodies were labeled with fluorophore-conjugated secondary antibody, including anti-rabbit secondary antibody (1:200, Abbkine, Dylight 488) and anti-mouse secondary antibody (1:200, Abbkine, Dylight 549), the cell nuclei were counterstained with DAPI. The region of interest was observed with a fluorescence microscope (Nikon, C-HGFI), and the co-staining positive neurons were counted (MediaCybernetics, Image-ProPlus 6.0).

The scaffolds seeded with hMSCs were stained with Hoechst 33342 blue (Invitrogen, USA) and analyzed using fluorescence microscopy (Nikon C-HGFi, Japan) after 20 min of incubation at room temperature. For confocal microscopy (Leica TCS-SP5 II, Leica Microsystem, Mannheim, Germany), the post-fixed (2.5% formalin) scaffold samples were subjected to dual staining using Hoechst dye and acridine orange. The 3D image obtained from the incorporation of multiple series of images collected by confocal laser further assisted in the investigation of cell infiltration up to 0–80 µm into the scaffolds.