Directing Cholangiocyte Morphogenesis in Natural Biomaterial Scaffolds

Adv. Sci. 2022, 9, 2102698 DOI: 10.1002/advs.202102698 Several key pieces of information are inadvertently missing in the original manuscript. Please find below the updated Figure 4 caption in which the gel conditions are given and a new method detail, that describes the assembly and seeding protocol for the microfluidic device used in the manuscript. The authors apologize for any inconvenience this may have caused. Figure 4. Intrahepatic Biliary Ducts on a Chip. A) Immunofluorescence images, characterizing marker expression and morphology of normal rat cholangiocytes grown on planar 2D surfaces and in 3D type 1 collagen gels (3 mg mL−1). B) Experimental schematic describing the co-culture of J2 fibroblasts and NRCs separated by a collagen gel. In addition, representative confocal images of NRCs after 9 days of culture are shown (with NRCs cultured at low density [20,000 cells per well] and high density [50,000 cells per well]), highlighting the ability to form branched lumenized structures with stromal support (n = 3 gels). C) (i) Device schematic and representative brightfield images, showing top and cross-sectional views of a dual channel microfluidic platform; (ii) encased within the biomaterial scaffolds are two parallel 300 µm open lumenal structures spaced 1 mm apart; (iii) the arrangement provides capacity to seed IHCs and flow media in the patent channels; (iv) and encapsulate IHCs in the bulk compartment of the device. D) Representative time course images of LifeAct RFP-IHCs grown in microfluidic device with 20 ng mL−1 EGF, showing collapse of open structures after six days, but anastomosis between bulk networks with channel structures. E) Representative maximum intensity projection images of LifeAct RFP-IHCs cultured for 6 days with 20 ng mL−1 EGF in 5 mg mL−1 fibrin scaffolds, showing maintenance of open cell-laden channels compared to Matrigel/collagen type 1 scaffolds (Nuclei stained with DAPI). F) Representative brightfield image of microfluidic cultured NRCs, grown over the course of 7 days (n = 3 devices). G) Confocal z-projection alongside an inset H) of microfluidic cultured NRCs showing polarized epithelium along the channel walls (n = 3 devices). Biliary Network Formation. Collagen gels were formed as previously described. In brief, 1.0 × 106 cells mL −1 were encapsulated in gels composed of Matrigel mixed at equal parts with 3.0 mg mL −1 rat tail collagen type I (Corning), that was titrated to pH 7.0-7.5 with 1M NaOH. 100 µl of the collagen mixture was added to wells of a 96 well plate and allowed to polymerize at 37°C for 30 minutes. For fibrin gels, 1.0 × 106 cells mL−1 were resuspended in fibrinogen (5 mg mL−1; Sigma) and crosslinked with thrombin (3 U mL−1; Sigma). An additional 100 µl of epithelial media was added to the gels after solidification. Microfluidic Device Assembly. Two-channel microfluidic devices were fabricated as previously described[1]. In short, tubular channels were formed in the PDMS devices, after functionalizing the surfaces with 0.01% poly-l-lysine and 1% glutaraldehyde to promote ECM adhesion. Two 300 µm diameter acupuncture needles (Hwato), blocked with 0.1% (w/v) bovine serum albumin (Sigma), were inserted into the needle guides of the devices. ECM solutions (collagen, collagen/Matrigel or fibrin) containing cells, were cast into the device, and allowed to polymerize for 30 minutes at 37°C. Needles were removed following gel polymerization and seeded with cells to line the channels. In experiments with NRCs, channels were coated with 50 µg mL−1 laminin for at least 1 hour at 37°C following needle removal.