Preserved liver-specific functions of hepatocytes in 3D co-culture with endothelial cell sheets

Kim, Kyungsook; Ohashi, Kazuhiko; Utoh, Rie; Kanô, Kyoko; Okano, Teruo

doi:10.1016/j.biomaterials.2011.10.084

Cited by 134 publications

(115 citation statements)

References 33 publications

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“…Studies have demonstrated that the non-parenchymal cells are important for maintaining the function of hepatocytes. [21][22][23] Consequently, our subsequent work is to co-culture non-parenchymal cells with hepatocytes to improve the hepatocytes' growth and functions.…”

Section: Discussionmentioning

confidence: 99%

Hepatocyte Culture in Autologous Decellularized Spleen Matrix

Gao¹,

Xiang³

et al. 2015

Organogenesis

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ABSTRACT. Background and Aims: Using decellularized scaffold to reengineer liver tissue is a promising alternative therapy for end-stage liver diseases. Though the decellularized human liver matrix is the ideal scaffold for reconstruction of the liver theoretically, the shortage of liver donors is still an obstacle for potential clinical application. Therefore, an appropriate alternative scaffold is needed. In the present study, we used a tissue engineering approach to prepare a rat decellularized spleen matrix (DSM) and evaluate the effectiveness of this DSM for primary rat hepatocytes culture. Methods: Rat decellularized spleen matrix (DSM) was prepared by perfusion of a series of detergents through spleen vasculature. DSM was characterized by residual DNA and specific extracellular matrix distribution. Thereafter, primary rat hepatocytes were cultured in the DSM in a 3-dimensional dynamic culture system, and liver cell survival and biological functions were evaluated by comparison with 3-dimensional sandwich culture and also with cultured in decellularized liver matrix (DLM). Results: Our research found that DSM did not exhibit any cellular components, but preserved the main extracellular matrix and the intact vasculature evaluated by DNA detection, histology, immunohistochemical staining, vessel corrosion cast and upright metallurgical microscope. *Correspondence to: Yi Lv; Email: luyi169@126.com Received July 7, 2014; Revised September 10, 2014; Accepted January 18, 2015.16 Organogenesis, 11:16-29, 2015 Ó 2015 Taylor & Francis Group, LLC ISSN: 1547-6278 print / 1555-8592 online DOI: 10.1080/15476278.2015 Moreover, the method of DSM preparation procedure was relatively simple with high success rate (100%). After seeding primary hepatocytes in DSM, the cultured hepatocytes survived inside DSM with albumin synthesis and urea secretion within 10 d. Additionally, hepatocytes in dynamic culture medium had better biological functions at day 10 than that in sandwich culture. Albumin synthesis was 85.67 § 6.34 mg/10 7 cell/24h in dynamic culture in DSM compared to 62.43 § 4.59 mg/10 7 cell/ 24h in sandwich culture (P < 0.01) and to 87.54 § 5.25 mg/10 7 cell/24h in DLM culture (P > 0.05); urea release was 32.14 § 8.62 mg/10 7 cell/24h in dynamic culture in DSM compared to 20.47 § 4.98 mg/10 7 cell/24h in sandwich culture (P < 0.05) and to 37.38 § 7.29 mg/10 7 cell/24h cultured in DLM (P > 0.05). Conclusion: The present study demonstrates that DSM can be prepared successfully using a tissue engineering approach. The DSM is an appropriate scaffold for primary hepatocytes culture.

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Section: Discussionmentioning

confidence: 99%

Hepatocyte Culture in Autologous Decellularized Spleen Matrix

Gao¹,

Xiang³

et al. 2015

Organogenesis

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“…Current approaches to use HLCs for an in vitro liver model, however, are mostly limited to 2D culture or simple 3D spheroid cultures (3,7,(10)(11)(12)(13)(14). The lack of a biomimetic microenvironment provided from the 3D interactions of parenchymal and nonparenchymal cell types along the hepatic differentiation stages may potentially be one of the limiting factors to functional maturation of the hepatic progenitor cells (HPCs), as well as the functional preservation of HLCs in vitro (15,16).Over the last decades, microtechnology tools have emerged to forge the advances in tissue engineering (3,(17)(18)(19)(20). Although a majority of these microfabrication techniques are limited to the generation of simple 2D geometries with selected materials, digital light processing (DLP)-based 3D printing provides superior speed and scalability for the fabrication of complex 3D microstructure (21,22).…”

mentioning

confidence: 99%

mentioning

confidence: 99%

Deterministically patterned biomimetic human iPSC-derived hepatic model via rapid 3D bioprinting

Zhu

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

726

661

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The functional maturation and preservation of hepatic cells derived from human induced pluripotent stem cells (hiPSCs) are essential to personalized in vitro drug screening and disease study. Major liver functions are tightly linked to the 3D assembly of hepatocytes, with the supporting cell types from both endodermal and mesodermal origins in a hexagonal lobule unit. Although there are many reports on functional 2D cell differentiation, few studies have demonstrated the in vitro maturation of hiPSC-derived hepatic progenitor cells (hiPSC-HPCs) in a 3D environment that depicts the physiologically relevant cell combination and microarchitecture. The application of rapid, digital 3D bioprinting to tissue engineering has allowed 3D patterning of multiple cell types in a predefined biomimetic manner. Here we present a 3D hydrogel-based triculture model that embeds hiPSC-HPCs with human umbilical vein endothelial cells and adiposederived stem cells in a microscale hexagonal architecture. In comparison with 2D monolayer culture and a 3D HPC-only model, our 3D triculture model shows both phenotypic and functional enhancements in the hiPSC-HPCs over weeks of in vitro culture. Specifically, we find improved morphological organization, higher liver-specific gene expression levels, increased metabolic product secretion, and enhanced cytochrome P450 induction. The application of bioprinting technology in tissue engineering enables the development of a 3D biomimetic liver model that recapitulates the native liver module architecture and could be used for various applications such as early drug screening and disease modeling.3D bioprinting | in vitro hepatic model | iPSC | tissue engineering | biomaterials T he liver plays a critical role in the synthesis of important proteins and the metabolism of xenobiotic; the failure of these functions is closely related to disease development and drug-induced toxicity (1). For these reasons, in vitro liver models have been extensively developed to serve as platforms for pathophysiological studies and as alternatives to animal models in drug screening and hepatotoxicity prediction (2-4). Human primary hepatocytes, considered one of the most mature liver cell sources, lose many liver-specific functions rapidly when cultured in vitro due to the great discrepancies between the native and culture environments (5, 6). In addition, the practical difficulties in obtaining liver biopsy samples from every patient further hinder their use in personalized liver models. Consequently, hepatocytes derived from human induced pluripotent stem cells (hiPSCs), with the potential to be patient specific and easily accessible, have been widely acknowledged as the most promising cell source for developing personalized human hepatic models (4, 7).Many groups have reported monolayer differentiation of hiPSCs into hepatocyte-like cells (HLCs) and their ability to metabolize drugs (7-9). Nevertheless, hiPSC-derived HLCs are still considered immature in terms of many liver-specific gene expressions, functions, and...

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“…Furthermore, primary rat hepatocytes maintained liver-specific functions for up to 28 days on the temperature-responsive sheets when cultured with endothelial cells [114] . These sheets attach to the bottoms of culture dishes at 37℃ and detach at 25℃, and provide a system for easy culturing and handling of cells.…”

Section: Tomizawa M Et Al Differentiation Of Hepatocytes From Hips mentioning

confidence: 99%

Induction of Hepatocyte Differentiation in Human Pluripotent Stem Cells

Tomizawa

Shinozaki

Motoyoshi

et al. 2015

Journal of Gastroenterology and Hepatology Research

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be advantageous in that it could resolve the ethical problems associated with stem cell research and could eliminate the need for administration of immunosuppresants in hepatocyte transplantation. To establish a method for the production of hepatocytes from hiPS cells, it is necessary to understand both the mechanism of hepatocyte differentiation as well as the current techniques for culturing of primary hepatocytes. Meanwhile, hepatocytes can also be differentiated from endodermal cells upon stimulation with growth factors that induce expression of specific transcriptional regulators. Current protocols for the generation of hepatocytes from hiPS cells include the sequential application of growth factors to mimic the environment of fetal liver. Initially, cells are treated with activin and bone morphogenetic proteins, followed by the application of hepatocyte growth factor and, at the final differentiation stage, Oncostatin M. Expression of transcription factors is necessary for hepatocyte differentiation. However, because the efficiency at which vectors expressing these factors can be transfected into hiPS cells is disappointingly low, adenoviral vectors have been used, which have a transduction efficiency of 100%. This method is used to sequentially introduce sex-determining region Y (Sry) HMG box (Sox) 17, hematopoietically expressed homeobox, and hepatocyte nuclear factor-4α into hiPS cells. These factors, in concert with the stimulation with growth factors, promote differentiation of these cells to hepatocytes. Despite the development of these procedures, however, the resulting cells remain immature and only perform certain hepatocyte functions. In this review, we describe each of the above points, and then discuss novel approaches and future directions for the In vitro production of hepatocytes. ABSTRACTCulturing of hepatocytes is used in both experimental and clinical procedures. Human induced pluripotent stem (hiPS) cells can be established from the somatic cells of individual patients. The In vitro production of hepatocytes from these hiPS cells would

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Preserved liver-specific functions of hepatocytes in 3D co-culture with endothelial cell sheets

Cited by 134 publications

References 33 publications

Hepatocyte Culture in Autologous Decellularized Spleen Matrix

Hepatocyte Culture in Autologous Decellularized Spleen Matrix

Deterministically patterned biomimetic human iPSC-derived hepatic model via rapid 3D bioprinting

Induction of Hepatocyte Differentiation in Human Pluripotent Stem Cells

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