3D Hepatic Organoid-Based Advancements in LIVER Tissue Engineering

Abstract

Liver-associated diseases and tissue engineering approaches based on in vitro culture of functional Primary human hepatocytes (PHH) had been restricted by the rapid de-differentiation in 2D culture conditions which restricted their usability. It was proven that cells growing in 3D format can better mimic the in vivo microenvironment, and thus help in maintaining metabolic activity, phenotypic properties, and longevity of the in vitro cultures. Again, the culture method and type of cell population are also recognized as important parameters for functional maintenance of primary hepatocytes. Hepatic organoids formed by self-assembly of hepatic cells are microtissues, and were able to show long-term in vitro maintenance of hepato-specific characteristics. Thus, hepatic organoids were recognized as an effective tool for screening potential cures and modeling liver diseases effectively. The current review summarizes the importance of 3D hepatic organoid culture over other conventional 2D and 3D culture models and its applicability in Liver tissue engineering.

Keywords: 3D culture; hepatic function; hepatic organoids; hepatocytes; liver tissue engineering.

Figure 1

Structures of the liver lobule and liver sinusoids. Hepatocytes are aligned radially to form the liver plate along with the sinusoids. The portal veins and hepatic artery branches terminate in the sinusoids, draining blood into the sinusoids and through the acinus to the central vein. The sinusoids are lined by fenestrated liver sinusoidal endothelial cells with Kupffer cells interspersed onto the endothelium. Between the liver plate and the sinusoids is the space of Disse, containing extracellular matrix components and hepatic stellate cells [5].

Figure 2

Functions of activated HSCs in diseased livers [19].

Figure 3

Cell polarity and bile canaliculi network formation in hepatocyte sandwich culture and the comparison of the endocytosis markers with liver slides, representing the in vivo conditions. (A) Protocol for fabrication of sandwich culture and removal of the soft collagen top layer for immunofluorescence staining. (B) Immunofluorescence images for re-establishment of cell polarity by staining markers for (C) tight junctions (ZO-1, F-Actin) and CD13, a transcytotic marker; A comparison of (D) tight junction markers (ZO-1, F-Actin) (E) cell polarity marker; (F) apical bile acid transporter (MRP2); and endosomal markers i.e., (G) EEA, Early endosome antigen 1, and (H) APPL1, adaptor protein containing a pleckstrin-homology domain, phosphotyrosine binding domain, and leucine zipper motif 1 between monolayer culture and sandwich culture was visualized and compared with liver slices. Scale bar = 20 µm [41].