Presentation Information
[1ACCE-13]Effects of three-dimensional hollow fiber culture on the hepatic differentiation of induced pluripotent stem cells
○Hiroshi Mizumoto1, Otsubo Kotaro2, Masamichi Kamihira1 (1. Department of Chemical Engineering , Faculty of Engineering, Kyushu University (Japan), 2. Graduate School of Engineering, Kyushu University (Japan))
Keywords:
stem cell differentiation,three-dimensional culture
[Purpose]
The increasing demand for reliable human liver models in biopharmaceutical development and regenerative medicine has intensified interest in induced pluripotent stem cell (iPSC)-derived hepatocytes as alternatives to primary hepatocytes. However, conventional two-dimensional (2D) differentiation protocols often produce hepatocyte-like cells with insufficient functional maturity. This study aimed to evaluate whether a three-dimensional (3D) hollow fiber (HF) culture system could enhance hepatic maturation of iPSC-derived cells and to determine the optimal stage for transitioning from 2D to 3D HF culture within a stepwise differentiation protocol.
[Methods]
Human iPSCs (201B7 line) were differentiated through three sequential stages: endodermal induction, hepatoblast differentiation, and hepatocyte maturation. Three experimental groups were established: (1) continuous monolayer culture (Mono), (2) HF culture initiated at day 3 following endodermal induction (HF1), and (3) HF culture initiated at day 8 following hepatoblast differentiation (HF2). HF culture was performed using hollow fibers with an inner diameter of 112 µm. For evaluation, changes in cell number and expression of hepatic marker genes were analyzed, and functional assays of urea synthesis and albumin secretion were conducted for comparison.
[Results]
Cell numbers increased during the hepatoblast differentiation phase in the monolayer group, while both HF groups exhibited reductions in cell number due to mechanical and enzymatic stress associated with cell re-seeding. Gene expression analysis confirmed successful differentiation in all groups, with albumin expression increasing more prominently in HF1 than in HF2. Despite reduced cell yields, HF-cultured cells exhibited significantly enhanced functional performance compared with the monolayer control. Both HF groups demonstrated markedly elevated urea synthesis and albumin secretion, indicating improved hepatic functionality. However, although HF2 achieved high levels of urea synthesis, the results showed considerable variability and lower albumin secretion compared wtih HF1.
[Discussion]
These findings indicate that three-dimensional hollow fiber culture significantly enhances the functional maturation of iPSC-derived hepatocytes compared with conventional monolayer culture. This improvement is likely attributable to enhanced cell-cell interactions and the optimized fiber diameter (112 µm), which facilitates efficient nutrient and oxygen exchange. Transitioning to HF culture immediately after endodermal induction (HF1) was more effective than introducing HF culture at the hepatoblast stage (HF2). This is likely because increased cellular fragility during later stages of differentiation makes dissociation more detrimental, contributing to the functional variability observed in HF2.
[Conclusion]
In summary, early-stage implementation of hollow fiber culture provides a robust and reproducible strategy for generating functionally mature hepatic tissues, with significant translational potential in regenerative medicine.
The increasing demand for reliable human liver models in biopharmaceutical development and regenerative medicine has intensified interest in induced pluripotent stem cell (iPSC)-derived hepatocytes as alternatives to primary hepatocytes. However, conventional two-dimensional (2D) differentiation protocols often produce hepatocyte-like cells with insufficient functional maturity. This study aimed to evaluate whether a three-dimensional (3D) hollow fiber (HF) culture system could enhance hepatic maturation of iPSC-derived cells and to determine the optimal stage for transitioning from 2D to 3D HF culture within a stepwise differentiation protocol.
[Methods]
Human iPSCs (201B7 line) were differentiated through three sequential stages: endodermal induction, hepatoblast differentiation, and hepatocyte maturation. Three experimental groups were established: (1) continuous monolayer culture (Mono), (2) HF culture initiated at day 3 following endodermal induction (HF1), and (3) HF culture initiated at day 8 following hepatoblast differentiation (HF2). HF culture was performed using hollow fibers with an inner diameter of 112 µm. For evaluation, changes in cell number and expression of hepatic marker genes were analyzed, and functional assays of urea synthesis and albumin secretion were conducted for comparison.
[Results]
Cell numbers increased during the hepatoblast differentiation phase in the monolayer group, while both HF groups exhibited reductions in cell number due to mechanical and enzymatic stress associated with cell re-seeding. Gene expression analysis confirmed successful differentiation in all groups, with albumin expression increasing more prominently in HF1 than in HF2. Despite reduced cell yields, HF-cultured cells exhibited significantly enhanced functional performance compared with the monolayer control. Both HF groups demonstrated markedly elevated urea synthesis and albumin secretion, indicating improved hepatic functionality. However, although HF2 achieved high levels of urea synthesis, the results showed considerable variability and lower albumin secretion compared wtih HF1.
[Discussion]
These findings indicate that three-dimensional hollow fiber culture significantly enhances the functional maturation of iPSC-derived hepatocytes compared with conventional monolayer culture. This improvement is likely attributable to enhanced cell-cell interactions and the optimized fiber diameter (112 µm), which facilitates efficient nutrient and oxygen exchange. Transitioning to HF culture immediately after endodermal induction (HF1) was more effective than introducing HF culture at the hepatoblast stage (HF2). This is likely because increased cellular fragility during later stages of differentiation makes dissociation more detrimental, contributing to the functional variability observed in HF2.
[Conclusion]
In summary, early-stage implementation of hollow fiber culture provides a robust and reproducible strategy for generating functionally mature hepatic tissues, with significant translational potential in regenerative medicine.
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