Construction of functional biliary epithelial branched networks with predefined geometry using digital light stereolithography

Abstract

Cholangiocytes, biliary epithelial cells, are known to spontaneously self-organize into spherical cysts with a central lumen. In this work, we explore a promising biocompatible stereolithographic approach to encapsulate cholangiocytes into geometrically-controlled 3D hydrogel structures to guide them towards the formation of branched tubular networks. We demonstrate that within the appropriate mix of hydrogels, normal rat cholangiocytes can proliferate, migrate and organize into branched tubular structures, form walls consisting of a cell monolayer, transport fluorescent dyes into the luminal space and show markers of epithelial maturation such as primary cilia. The resulting structures have dimensions typically found in the intralobular and intrahepatic bile ducts and are stable for weeks, without any requirement of bulk supporting material, thereby offering total access to the basal side of these biliary epithelial constructs.

Self-organization of Long-lasting Human Endothelial Capillary Networks guided by DLP Bioprinting

“…The same hydrogel structures showed excellent stability over time in the absence of cells, indicating that the lack of stability was directly linked to the presence of living cells. We thus hypothesized that the gel structures were not strong enough to resist degradation by endothelial cells in contrast to what we had observed in a previous study with epithelial cells 26 , and tested other means to increase the stability of our formulations.…”

“…This selforganization is highly disorganized, branching happens randomly, and structures rarely form stable lumens or vascular networks which could subsequently be manipulated and integrated into a bioengineered tissue construct. We thus developed a technique using DLP bioprinting 24 to control the microenvironmental cues necessary to promote the self-organization of endothelial progenitor cells (EPCs) into branched tubes with various size and shape (Fig 1A ) using a similar experimental process described in 26 . Briefly, a photopolymerizable mix of hydrogels, cells and photoinitiator was placed between a glass coverslip and a PDMS roof, and 26 and on published studies using endothelial cells 32 , we first explored various formulations incorporating either methacrylated gelatin (G MA ) or methacrylated type I collagen (C MA ) supplemented with fibrinogen (FG), methacrylated hyaluronic acid (HA MA ), and lithium phenyl-2,4,6 trimethyl-benzoyl phosphinate (LAP)a cytocompatible photoinitiator 33 (Fig 1B Fig S1).…”

“…We thus developed a technique using DLP bioprinting 24 to control the microenvironmental cues necessary to promote the self-organization of endothelial progenitor cells (EPCs) into branched tubes with various size and shape (Fig 1A ) using a similar experimental process described in 26 . Briefly, a photopolymerizable mix of hydrogels, cells and photoinitiator was placed between a glass coverslip and a PDMS roof, and 26 and on published studies using endothelial cells 32 , we first explored various formulations incorporating either methacrylated gelatin (G MA ) or methacrylated type I collagen (C MA ) supplemented with fibrinogen (FG), methacrylated hyaluronic acid (HA MA ), and lithium phenyl-2,4,6 trimethyl-benzoyl phosphinate (LAP)a cytocompatible photoinitiator 33 (Fig 1B Fig S1). Indeed, G MA and C MA are photopolymerizable hydrogels produced from gelatin and collagen respectively which sustain good cell viability following encapsulation and retain natural cell-binding motifs [34][35][36] .…”

“…By mimicking the mechanical properties of the natural cell environment 25 , the 3D photopolymerized hydrogel also provides spatial cues to guide both cell proliferation and polarization. Using this method, we have previously manufactured a branched tubular epithelial network successfully mimicking the intrahepatic biliary tree 26 .…”

Highlights of constructing liver-relevant in vitro models with 3D bioprinting

“…To address this issue, digital Editorial Highlights of constructing liver-relevant in vitro models with 3D bioprinting light stereolithography has been applied to achieve high resolution in 3D bioprinting. For example, biliary epithelial branched networks have been successfully constructed to simulate the structure of bile ducts in the liver (7). Furthermore, digital light stereolithography has been used to construct microscale hexagonal architecture containing hepatic cells, endothelial cells, and adipose-derived stem cells (8).…”