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A Bile Duct-on-a-Chip with Organ-Level Functions

Yu Du, Gauri Khandekar, Jessica Llewellyn, William Polacheck, Christopher S. Chen, Rebecca G. Wells

Preprint posted on March 30, 2019 https://www.biorxiv.org/content/10.1101/594291v1

Article now published in Hepatology at http://dx.doi.org/10.1002/hep.30918

Un poco biliar conducto: Researchers invent a bile duct-on-a-chip

Selected by Zhang-He Goh

Background of preprint

Alterations in the tight junctions of bile duct epithelial cells, or cholangiocytes, have been associated with chronic cholestatic liver diseases both in humans and in mouse models. However, most in vitro biliary research currently involves the use of cultured cells in either 2D monolayers or 3D organoids, both of which are not representative of bile duct structural organisation and cannot perform biliary physiological functions like compartmentalising bile. To bridge this gap between biliary physiology and existing biliary models, Du et al. apply organ-on-chip technology to develop a micro-engineered bile duct. As described by the authors, the significance of this is threefold:

  • The bile duct-on-a-chip performs key functions of the bile duct,
  • The bile duct-on-a-chip is a useful tool that can be used to study the barrier function of the cholangiocyte monolayer quantitatively, and
  • The barrier function of the cholangiocyte monolayer at the apical or basolateral side can be studied independently.

Finally, the authors provide a proof-of-concept by using their invention to demonstrate the protective role of the cholangiocyte apical glycocalyx.

Key findings of preprint

The findings of this preprint can be categorised into four main sections (Fig. 1). First, the authors fabricated and characterised the bile duct-on-a-chip. Second, Du et al. proved that independent access to the apical and basal surfaces of the cholangiocyte monolayer could be achieved in their invention. Third, the authors used the bile duct-on-a-chip to show that the glycocalyx protects cholangiocytes from bile acid-induced damage. Fourth, the authors demonstrated that their device could be used with cells from other sources.

Figure 1. Four key findings in the preprint by Du et al.

(A) Fabrication and characterisation of bile duct-on-a-chip

Du et al. first developed the bile duct-on-a-chip by lining a channel with mouse cholangiocytes, which formed a confluent and compact epithelial monolayer as shown using F-actin staining. Characterising these cholangiocytes yielded four major observations:

  • These cholangiocytes maintained the expression of the cholangiocyte marker K19.
  • The expression of ZO-1 and E-cadherin 1 at cell-cell junctions demonstrated the formation of tight junctions.
  • ASBT staining confirmed the polarisation of cells on the bile duct-on-a-chip.
  • Cholangiocytes had to be superconfluent and compact in order to give rise to full barrier function with a significant decrease in permeability. To demonstrate that the tight junction formation indeed led to the barrier function of the bile duct, the authors perfused the lumen with a range of sizes of FITC-dextran (4-70 kDa) and showed that there was no clear leakage of this fluorescent dextran into the collagen matrix after 10 minutes.

 (B) Apical and basal surfaces of the cholangiocyte monolayer can be accessed independently

To show that the apical and basal surfaces of the cholangiocyte monolayer react differently to different toxins, Du et al. applied both the toxic isoflavonoid biliatresone [1] and the bile acid glycochenodeoxycholic acid (GCDC) independently to each side of the bile duct-on-a-chip. The authors found that this indeed increased the permeability of monolayers in the bile duct-on-a-chip. Furthermore, this damage was worse with basal than apical administration, an observation ascribed to the lower tolerance of bile from the basolateral side [2-4]. Based on this finding, the authors posited that leakage of toxic bile through the epithelial monolayer can give rise to a feedback loop that results in increasingly severe toxicity.

(C) Proof-of-concept: The apical glycocalyx protects cholangiocytes from bile acid-induced damage

Du et al. then used their invention to show that demonstrate the cholangiocyte-lined channel is resistant to bile acid toxicity. This resistance could be attributed to the apical glycocalyx, which is known to protect against bile acid toxicity [5,6]—this protective effect against GCDC was lost after removal with neuraminidase, and the permeability of the cholangiocyte-lined channels towards 4 and 10 kDa (but not 70 kDa) FITC-dextran increased.

(D) Demonstration of compatibility of bile duct-on-a-chip with cells from other sources

When Du et al. seeded and characterised their device using primary murine extrahepatic cholangiocytes via a similar procedure, they found that the cholangiocytes in the device (a) expressed K19, (b) exhibited cell junctions that were even tighter than those previously investigated, and (c) exhibited polarisation.

Significance of this preprint: Current developments and future directions

Before the invention of the first organ-on-a-chip a decade ago [7], the idea may have sounded—to borrow the words from Miguel Rivera from Pixar’s Cocoun poco loco (a little crazy). But the technology has since come a long way. Today, organs-on-a-chip are highly valuable miniaturisation technologies that can fill many roles. The same can be said about the bile duct-on-a-chip invented by preprint authors. It adds to a veritable trove of bioengineered bile ducts [8,9]. As the authors point out in their preprint, the device has four distinctive benefits:

  • Two sets of ports that enable sampling of luminal contents,
  • Independent and selective exposure of the apical and basolateral sides to drugs or toxins,
  • Variation of the chemical composition and mechanics of the surrounding matrix, and
  • Variation of the fluid flow rate.

The potential applications of the bile duct-on-a-chip are many. It could be used to further explore the hepatobiliary tree, whose complex environment makes the pathophysiology of associated diseases particularly baffling. In pharmacokinetic testing phases of drug discovery, too, the bile duct-on-a-chip could prove useful: the hepatobiliary system plays a crucial role in drug disposition, and a validated model of a bile duct-on-a-chip could open a host of exciting in vitro pharmaceutical biotesting applications. The bile duct-on-a-chip could even be used to understand the hepatobiliary system more holistically, which would aid in efforts to characterise how drugs and toxins might affect the entire hepatobiliary system.

Questions for authors

  • What are some potential reasons for the better barrier function demonstrated by the primary cell-lined device compared to the cholangiocyte cell line? What might be the physiological significance of this difference?
  • What are some challenges in biliary pathophysiology that you hope to solve using this device?

References

[1] Waisbourd‐Zinman O, Koh H, Tsai S, Lavrut PM, Dang C, Zhao X, Pack M, Cave J, Hawes M, Koo KA, The toxin biliatresone causes mouse extrahepatic cholangiocyte damage and fibrosis through decreased glutathione and SOX17, Hepatology 64(3) (2016) 880-893.

[2] Hopkins AM, Walsh SV, Verkade P, Boquet P, Nusrat A, Constitutive activation of Rho proteins by CNF-1 influences tight junction structure and epithelial barrier function, Journal of cell science 116(4) (2003) 725-742.

[3] Xia X, Francis H, Glaser S, Alpini G, LeSage G, Bile acid interactions with cholangiocytes, World journal of gastroenterology: WJG 12(22) (2006) 3553.

[4] Benedetti A, Alvaro D, Bassotti C, Gigliozzi A, Ferretti G, La Rosa T, Di Sario A, Baiocchi L, Jezequel AM, Cytotoxicity of bile salts against biliary epithelium: a study in isolated bile ductule fragments and isolated perfused rat liver, Hepatology 26(1) (1997) 9-21.

[5] Hohenester S, Maillette de Buy Wenniger L, Paulusma CC, van Vliet SJ, Jefferson DM, Oude Elferink RP, Beuers U, A biliary HCO3− umbrella constitutes a protective mechanism against bile acid‐induced injury in human cholangiocytes, Hepatology 55(1) (2012) 173-183.

[6] de Buy Wenniger LJM, Hohenester S, Maroni L, van Vliet SJ, Elferink RPO, Beuers U, The Cholangiocyte Glycocalyx Stabilizes the ‘Biliary HCO3-Umbrella’: An Integrated Line of Defense against Toxic Bile Acids, Digestive Diseases 33(3) (2015) 397-407.

[7] Huh D, Matthews BD, Mammoto A, Montoya-Zavala M, Hsin HY, Ingber DE, Reconstituting Organ-Level Lung Functions on a Chip, Science 328(5986) (2010) 1662-1668.

[8] Sampaziotis F, Justin AW, Tysoe OC, Sawiak S, Godfrey EM, Upponi SS, Gieseck III RL, de Brito MC, Berntsen NL, Gómez-Vázquez MJ, Reconstruction of the mouse extrahepatic biliary tree using primary human extrahepatic cholangiocyte organoids, Nature medicine 23(8) (2017) 954.

[9] Chen C, Jochems PG, Salz L, Schneeberger K, Penning LC, Van De Graaf SF, Beuers U, Clevers H, Geijsen N, Masereeuw R, Bioengineered bile ducts recapitulate key cholangiocyte functions, Biofabrication 10(3) (2018) 034103.

Tags: bile duct, microfluidics, organ models, organ on a chip

Posted on: 19th April 2019 , updated on: 29th September 2019

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  • Author's response

    Rebecca Wells and Yu Du shared

    1. What are some potential reasons for the better barrier function demonstrated by the primary cell-lined device compared to the cholangiocyte cell line? What might be the physiological significance of this difference?

    One possible reason is the inherent difference between cell lines and primary cells. Cell lines are convenient to use yet lack biological relevance. In contrast, primary cells have limited capacity of expansion, but maintain many important markers and functions. As shown by E-cadherin and ZO-1 immunostaining, primary cells maintained more intact cell-cell junctions, which play a crucial role in barrier function. Additionally, primary cells may be able to form a more compact monolayer, which we showed leads to improved barrier function – we believe these findings are physiologically important primarily because they demonstrate the importance of a compact (beyond confluent) monolayer.

    2. What are some challenges in biliary pathophysiology that you hope to solve using this device?

    Cholangiopathies are always associated with alterations in bile duct barrier function. By replicating the structure and barrier functions of the bile duct, this device will be useful in studying different cholangiopathies. We are applying this model to study biliary atresia, in particular the increased susceptibility of the neonatal duct to injury compared to the adult duct. In the future, we will incorporate a vascular system into this device in order to model the immune response in response to cholangiocyte damage; this may be particularly useful in studying primary sclerosing cholangitis.

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