The development of a protocol for isolating and expanding the cell population that lines bile ducts has enabled the in vitro generation of bioengineered ducts. These can replace native bile ducts when transplanted into mice.
Bile, which facilitates the absorption of lipids in the small intestine, is highly toxic to cells of the liver, where it is produced. This fluid must therefore be drained from the organ through the intra- and extrahepatic bile ducts, which lie respectively within and outside the liver. Damage to bile ducts can prevent proper drainage, causing often-fatal liver diseases1. One possible treatment is implantation of artificial, bioengineered bile ducts. Writing in Nature Medicine, Sampaziotis et al.2 report a protocol for generating such ducts, which they used to replace common bile ducts in mice.
The inner lining of bile ducts consists of cells called cholangiocytes. The first step in generating bile ducts in vitro is therefore to procure extrahepatic cholangiocytes. Protocols3,4 have been successfully developed for generating intrahepatic cholangiocytes from human pluripotent stem cells, which have the potential to give rise to any cell type in the body. But intrahepatic and extrahepatic cholangiocytes have different origins and functions5, and these protocols cannot be used to generate the latter cells. An alternative method is to isolate extrahepatic cholangiocytes from the gall bladder (in which bile is stored) using enzymatic digestion processes6. However, the isolated cells have limited potential to proliferate when grown in two-dimensional layers, making it hard to produce enough cells to form a bile duct.
Sampaziotis et al. showed that extrahepatic cholangiocytes could be isolated mechanically by brushing or scraping cells from the inside of human extrahepatic bile ducts (Fig. 1). This method is minimally invasive, in contrast to isolation from the gall bladder, which requires surgery. In addition, it allows easy access to human cells — often a considerable limitation for therapies.
The 2D nature of cell culture limits cholangiocyte proliferation, but cells in vitro can also be directed to form 3D mini-organs called organoids, which develop into structures similar to their in vivo counterparts7. Sampaziotis and colleagues adapted an organoid culture system for intrahepatic cholangiocytes3 by screening for growth-factor proteins that support the growth of extrahepatic cholangiocyte organoids (ECOs). They alighted on three — EGF, R-spondin and DKK1.
Cells from ECOs grown with these factors could function as mature cholangiocytes following long-term culture (20 cycles of cell growth in culture dishes). Furthermore, this approach generated many more cells than could 2D cultures, opening up the possibility of clinical uses.
Sampaziotis et al. next evaluated the potential of ECO cells to form sheets of biliary tissue. The authors seeded cholangiocytes from ECOs onto biodegradable scaffolds made from polyglycolic acid, which provide support for tissue regeneration. ECO-derived cholangiocytes grew well, covering the scaffold, whereas cholangiocytes cultured in 2D conditions did not.
Can ECO cells be used to repair injured tissue in vivo? The researchers damaged the gall bladders of mice and transplanted scaffolds harbouring ECO-derived cholangiocytes into the organs. The gall bladders underwent full remodelling, with ECO-derived cells adopting the shape and structure of the native tissue, and the animals survived without complications. By contrast, control mice that received cell-free scaffolds died, owing to leakage of bile from the gall bladder.
Sampaziotis and colleagues then moved a step farther, preparing bioengineered tissue in the shape of a bile duct. Previously, a transplanted, biodegradable polymer tube alone has been used to replace the common bile duct in pigs8. In that system, native cholangiocytes migrated to the graft site to form the new bile duct. But the pigs used had healthy cholangiocytes, and it was unclear whether this approach would work in people with cholangiocyte-related diseases. A better solution would therefore be to populate the tubes with functional cholangiocytes before transplantation. The authors did just this, using tubular collagen scaffolds hosting ECO-derived cholangiocytes. These bioengineered ducts could successfully replace common bile ducts in mice in which the native ducts had been removed (Fig. 1).
The long-term survival of ECO-populated scaffolds and bile ducts in vivo depends on them forming true biliary structures and being infiltrated by the host's blood supply. After transplantation, the authors' bioengineered ducts did develop proper bile-duct structures, and showed mature cholangiocyte function. Moreover, host cells other than cholangiocytes migrated to the scaffolds, and eventually formed supporting tissue to promote the survival of ECO cells.
This is a remarkable step in the development of extrahepatic bile ducts for regenerative medicine. Current surgical techniques that remove extrahepatic bile ducts and connect the liver directly to the small intestine are effective for treating bile-duct disorders, but often result in infections that spread from the intestine back along the intrahepatic bile duct7. These could be largely avoided by using ECO-derived, bioengineered ducts that would act as a bridge between the liver and small intestine. The next generation of ECO-derived bile ducts should provide a controllable release of bile — something not achieved in the current study.
One potential pitfall is the length of time that would be needed to generate bio-artificial bile ducts for patients, because some cholangiocyte-related diseases lead to aggressive liver disorders. In addition, the ducts of people with these diseases often contain large amounts of connective and inflammatory tissues, preventing the collection of normal cholangiocytes1. Fast production or on-the-shelf supplies of bioengineered bile ducts will thus be necessary in the future.
The work has obvious implications for studies of tissue regeneration. ECO cells formed biliary cell layers and adopted the tissue architecture of bile ducts in vivo, whereas they underwent simple proliferation on scaffolds in vitro. Researchers could therefore use the current study's protocols to search for signalling molecules that enable the self-assembly of ECO cells into bile ducts in vitro.
Advances in stem-cell research have led to the successful generation of many liver cell types in vitro. Hepatocytes (the liver's main cell type) can be developed from pluripotent stem cells, or by direct conversion from other mature cells9. Researchers have even constructed vascularized liver buds in vitro, although these are smaller than their in vivo counterparts10. With the production of extrahepatic bile ducts, all the necessary building blocks for constructing a liver that can be connected to the intestine are now in hand. The next challenge is to assemble these pieces into a functional liver in the laboratory.