title: Reconstruction of hepatic stellate cell-incorporated liver capillary structures in small hepatocyte tri-culture using microporous membranes.
abstract: In liver sinusoids, hepatic stellate cells (HSCs) locate the outer surface of microvessels to form a functional unit with endothelia and hepatocytes. To reconstruct functional liver tissue in vitro, formation of the HSC-incorporated sinusoidal structure is essential. We previously demonstrated capillary formation of endothelial cells (ECs) in tri-culture, where a polyethylene terephthalate (PET) microporous membrane was intercalated between the ECs and hepatic organoids composed of small hepatocytes (SHs), i.e. hepatic progenitor cells, and HSCs. However, the high thickness and low porosity of the membranes limited heterotypic cell-cell interactions, which are essential to form HSC-EC hybrid structures. Here, we focused on the effective use of the thin and highly porous poly( d, l-lactide-co-glycolide) (PLGA) microporous membranes in SH-HSC-EC tri-culture to reconstruct the HSC-incorporated liver capillary structures in vitro. First, the formation of EC capillary-like structures was induced on Matrigel-coated PLGA microporous membranes. Next, the membranes were stacked on hepatic organoids composed of small SHs and HSCs. When the pore size and porosity of the membranes were optimized, HSCs selectively migrated to the EC capillary-like structures. This process was mediated in part by platelet-derived growth factor (PDGF) signalling. In addition, the HSCs were located along the outer surface of the EC capillary-like structures with their long cytoplasmic processes. In the HSC-incorporated capillary tissues, SHs acquired high levels of differentiated functions, compared to those without ECs. This model will provide a basis for the construction of functional, thick, vascularized liver tissues in vitro.
authors.affiliation: Centre for System Integration Engineering, School of Integrated Design Engineering, Keio University, 3-1-14-1 Kohoku, Yokohama, 223-0061, Japan.
authors.forename: Junichi
authors.initials: J
authors.lastname: Kasuya
authors.forename: Ryo
authors.initials: R
authors.lastname: Sudo
authors.forename: Genta
authors.initials: G
authors.lastname: Masuda
authors.forename: Toshihiro
authors.initials: T
authors.lastname: Mitaka
authors.forename: Mariko
authors.initials: M
authors.lastname: Ikeda
authors.forename: Kazuo
authors.initials: K
authors.lastname: Tanishita
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D020099:Coated Materials, Biocompatible / Q000737:chemistry*; D018920:Coculture Techniques / Q000379:methods; D042783:Endothelial Cells / Q000166:cytology / Q000378:metabolism*; D055166:Hepatic Stellate Cells / Q000166:cytology / Q000378:metabolism*; D022781:Hepatocytes / Q000166:cytology / Q000378:metabolism*; D019344:Lactic Acid / Q000737:chemistry*; D008297:Male; D008567:Membranes, Artificial; D011100:Polyglycolic Acid / Q000737:chemistry*; D000077182:Polylactic Acid-Polyglycolic Acid Copolymer; D016062:Porosity; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: D020099:Coated Materials, Biocompatible; D008567:Membranes, Artificial; D000077182:Polylactic Acid-Polyglycolic Acid Copolymer; D011100:Polyglycolic Acid; D019344:Lactic Acid
keywords: endothelial cell; hepatic stellate cell; liver sinusoid; microporous membrane; poly(d, l-lactide-co-glycolide); small hepatocyte; three-dimensional; tri-culture