Construction of three-dimensional liver tissue models by cell accumulation technique and maintaining their metabolic functions for long-term culture without medium change

Abstract

Three-dimensional (3D) hepatocyte cultures have attracted much attention to obtain high biological functions of hepatocyte for pharmaceutical drug assessment. However, maintaining the high functions for over one month is still a key challenge although many approaches have been reported. In this study, we demonstrate for the first time simple and rapid construction of 3D-hepatocyte constructs by our cell accumulation technique and their high biological functions for one month, without any medium change. The human hepatocyte carcinoma (HepG2) cells were coated with ∼ 7 nm-sized extracellular matrix (ECM) films consisting of fibronectin (FN) and gelatin (G), and then incubated in cell culture insert to construct 3D-tissue constructs for 24 h. The thickness of obtained 3D-HepG2 constructs was easily controlled by altering seeding cell number and the maximum is over 100 μm. When a large volume of culture media was employed, the 3D-constructs showed higher mRNA expression of albumin and some cytochrome P450 (CYP) enzymes as compared to general two-dimensional (2D) culture. Surprisingly, their high cell viabilities (over 80%) and high mRNA expressions were successfully maintained without medium change for at least 27 days. These results demonstrate novel easy and rapid technique to construct 3D-human liver tissue models which can maintain their high functions and viability for 1 month without medium change.

In situ patterned micro 3D liver constructs for parallel toxicology testing in a fluidic device

“…In fact, it has been shown that simply transitioning from 2D to 3D resulted in increased HEPG2 albumin expression and secretion [28]. This is not limited to HEPG2s either.…”

Advanced Biomaterials and Processing Methods for Liver Regeneration: State‐of‐the‐Art and Future Trends

“…Although typically used in surface engineering applications, or to tune membrane's permeability, LbL methodologies are being used in liver TE. Despite the number studies using LbL are scarce, LbL deposition of fibronectin and gelatin on the surface of HepG2 cells renders a multilayered adhesive film that is fundamental for further cell accumulation . Surprisingly, the cell constructs obtained by this method were able to sustain cell viability up to 27 d of culture, with high albumin and cytochrome P450 enzyme expressions ( Figure ).…”

“…Adapted with permission. [218] Copyright 2014, John Wiley and Sons. PDMS Decellularized liver matrix + GelMA HepG2 cells [234][235][236] PEG hiPSCs [232,237] Liver organoids Matrigel Primary hepatocytes [238] Alginate Decellularized liver matrix + GelMA HepG2 cells [229,230] PEG PDMS + PEG hiPSCs [232,237,239] www.advancedsciencenews.com www.advhealthmat.de…”

Dual‐chambered membrane bioreactor for coculture of stratified cell populations

“…If different scaffolds are grown in separate bioreactors, they each develop a cell population with distinct characteristics. These scaffolds can be bound together in vitro or in vivo using a variety of layering techniques, usually crosslinking (Gleghorn, Lee, Cabodi, Stroock, & Bonassar, ; Gurkan et al, ; Harley et al, ; B. S. Soo Kim et al, ; M. Kim & Kim, ; Levingstone et al, ; Pi et al, ; G. Yang, Lin, Rothrauff, Yu, & Tuan, ), sintering (Camarero‐Espinosa, Rothen‐Rutishauser, Weder, & Foster, ; Costa et al, ; Mosher, Spalazzi, & Lu, ; Spalazzi, Doty, Moffat, Levine, & Lu, ), or by inducing extracellular matrix (ECM) deposition (Asano, Shimoda, Okano, Matsusaki, & Akashi, ; Matsusaki et al, ; Matsuzawa, Matsusaki, & Akashi, ; Ng, Qi, Yeong, & Naing, ; Nishiguchi, Yoshida, Matsusaki, & Akashi, ; Papenburg et al, ), but it is difficult to predict the interface that will form (Figure b‐i). In the DCB, two parallel flow inlets connect to a common chamber, followed by two flow outlets (Figure b‐ii).…”