Human iPS derived progenitors bioengineered into liver organoids using an inverted colloidal crystal poly (ethylene glycol) scaffold.
2018; 182: 299–311
Generation of human organoids from induced pluripotent stem cells (iPSCs) offers exciting possibilities for developmental biology, disease modelling and cell therapy. Significant advances towards those goals have been hampered by dependence on animal derived matrices (e.g. Matrigel), immortalized cell lines and resultant structures that are difficult to control or scale. To address these challenges, we aimed to develop a fully defined liver organoid platform using inverted colloid crystal (ICC) whose 3-dimensional mechanical properties could be engineered to recapitulate the extracellular niche sensed by hepatic progenitors during human development. iPSC derived hepatic progenitors (IH) formed organoids most optimally in ICC scaffolds constructed with 140 mum diameter pores coated with type I collagen in a two-step process mimicking liver bud formation. The resultant organoids were closer to adult tissue, compared to 2D and 3D controls, with respect to morphology, gene expression, protein secretion, drug metabolism and viral infection and could integrate, vascularise and function following implantation into livers of immune-deficient mice. Preliminary interrogation of the underpinning mechanisms highlighted the importance of TGFbeta and hedgehog signalling pathways. The combination of functional relevance with tuneable mechanical properties leads us to propose this bioengineered platform to be ideally suited for a range of future mechanistic and clinical organoid related applications.
View details for PubMedID 30149262
Long-term culture of human liver tissue with advanced hepatic functions.
2017; 2 (11)
A major challenge for studying authentic liver cell function and cell replacement therapies is that primary human hepatocytes rapidly lose their advanced function in conventional, 2-dimensional culture platforms. Here, we describe the fabrication of 3-dimensional hexagonally arrayed lobular human liver tissues inspired by the liver's natural architecture. The engineered liver tissues exhibit key features of advanced differentiation, such as human-specific cytochrome P450-mediated drug metabolism and the ability to support efficient infection with patient-derived inoculums of hepatitis C virus. The tissues permit the assessment of antiviral agents and maintain their advanced functions for over 5 months in culture. This extended functionality enabled the prediction of a fatal human-specific hepatotoxicity caused by fialuridine (FIAU), which had escaped detection by preclinical models and short-term clinical studies. The results obtained with the engineered human liver tissue in this study provide proof-of-concept determination of human-specific drug metabolism, demonstrate the ability to support infection with human hepatitis virus derived from an infected patient and subsequent antiviral drug testing against said infection, and facilitate detection of human-specific drug hepatotoxicity associated with late-onset liver failure. Looking forward, the scalability and biocompatibility of the scaffold are also ideal for future cell replacement therapeutic strategies.
View details for DOI 10.1172/jci.insight.90853
View details for PubMedID 28570275
Viscoelastic lithography for fabricating self-organizing soft micro-honeycomb structures with ultra-high aspect ratios
High-aspect ratio micro- and nano-structures have been used for the production of a variety of applications. In this paper, we describe a simple and cost-effective approach to fabricate an arrayed microarchitecture with an ultra-high aspect ratio using soft materials. The shapes and sizes of the honeycomb structure can be easily modulated by changing the dimensions and position of the base mould pattern and the pressure. The honeycomb structure is used to prepare a drug delivery patch and a microwell array to form cell spheroids without cell loss. The honeycomb structures prepared using natural ECM (collagen-Matrigel) materials are successfully fabricated. The hepatocytes and endothelial cells are seeded and co-cultured in the ECM-based micro-honeycomb to prepare a 3D liver model successfully mimicking an ultrastructure of liver and providing enhanced liver function.
View details for DOI 10.1038/ncomms11269
View details for PubMedID 27157977
A 3D alcoholic liver disease model on a chip
2016; 8 (3): 302-308
Alcohol is one of the main causes of liver diseases, and the development of alcoholic liver disease (ALD) treatment methods has been one of the hottest issues. For this purpose, development of in vitro models mimicking the in vivo physiology is one of the critical requirements, and they help to determine the disease mechanisms and to discover the treatment method. Herein, a three-dimensional (3D) ALD model was developed and its superior features in mimicking the in vivo condition were demonstrated. A spheroid-based microfluidic chip was employed for the development of the 3D in vitro model of ALD progression. We co-cultured rat primary hepatocytes and hepatic stellate cells (HSCs) in a fluidic chip to investigate the role of HSCs in the recovery of liver with ALD. An interstitial level of flow derived by an osmotic pump was applied to the chip to provide in vivo mimicking of fluid activity. Using this in vitro tool, we were able to observe structural changes and decreased hepatic functions with the increase in ethanol concentration. The recovery process of liver injured by alcohol was observed by providing fresh culture medium to the damaged 3D liver tissue for few days. A reversibly- and irreversibly-injured ALD model was established. The proposed model can not only be used for the research of alcoholic disease mechanism, but also has the potential for use in studies of hepatotoxicity and drug screening applications.
View details for DOI 10.1039/c5ib00298b
View details for Web of Science ID 000372256400004
View details for PubMedID 26857817
3D liver models on a microplatform: well-defined culture, engineering of liver tissue and liver-on-a-chip
LAB ON A CHIP
2015; 15 (19): 3822–37
The liver, the largest organ in the human body, is a multi-functional organ with diverse metabolic activities that plays a critical role in maintaining the body and sustaining life. Although the liver has excellent regenerative and recuperative properties, damages caused by chronic liver diseases or viral infection may lead to permanent loss of liver functions. Studies of liver disease mechanism have focused on drug screening and liver tissue engineering techniques, including strategies based on in vitro models. However, conventional liver models are plagued by a number of limitations, which have motivated the development of 'liver-on-a-chip' and microplatform-based bioreactors that can provide well-defined microenvironments. Microtechnology is a promising tool for liver tissue engineering and liver system development, as it can mimic the complex in vivo microenvironment and microlevel ultrastructure, by using a small number of human cells under two-dimensional (2D) and three-dimensional (3D) culture conditions. These systems provided by microtechnology allow improved liver-specific functions and can be expanded to encompass diverse 3D culture methods, which are critical for the maintenance of liver functions and recapitulation of the features of the native liver. In this review, we provide an overview of microtechnologies that have been used for liver studies, describe biomimetic technologies for constructing microscale 2D and 3D liver models as well as liver-on-a-chip systems and microscale bioreactors, and introduce applications of liver microtechnology and future trends in the field.
View details for DOI 10.1039/c5lc00611b
View details for Web of Science ID 000361546200002
View details for PubMedID 26279012
Simple Fabrication Method for a Porous Poly(vinyl alcohol) Matrix by Multisolvent Mixtures for an Air-Exposed Model of the Lung Epithelial System
2014; 30 (41): 12107–13
We introduce a simple and easy method for fabricating a thin and porous matrix that can be used as an extracellular matrix (ECM). A porous poly(vinyl alcohol) (PVA) matrix was created through recrystallization by multiple solvents under distilled water (DW), isopropyl alcohol (IPA), and a combination of DW and IPA. The crysatllization was driven by precipitating and dissolving a solute in a solution of a solvent and a nonsolvent, which induced the formation of microspheres in the IPA. The crystal structure depended on the ratio of the solvent/nonsolvent and the concentration of the PVA aqueous solution; these properties were used to tune the thickness, size, and porosity of the matrices. The resulting PVA matrix was chemically stabilized through a reaction with glutaraldehyde in the IPA solution. We demonstrated that a very thin and porous PVA matrix provided an effective functional model of the lung epithelial system. Lung epithelial cells (A549) displayed a high affinity for this matrix, which was permeable to the culture medium. These properties facilitated culturing under the air environment.
View details for DOI 10.1021/la501453h
View details for Web of Science ID 000343638800001
View details for PubMedID 25260012
Immune-protected xenogeneic bioartificial livers with liver-specific microarchitecture and hydrogel-encapsulated cells
2014; 35 (32): 8983–91
Development of a xenogeneic biological liver support is important in providing a bridge to transplantation or liver regeneration, thus helping to overcome the chronic shortage of liver donors. Among the critical factors in developing biological liver support are the creation of in vivo mimetic micro liver tissue (mLT), especially mLTs containing liver-specific ultrastructure, and an encapsulation method that can package massive numbers of cells while providing immune-protection from the host immune system. We describe here the development of mLTs that include liver microarchitecture and their in situ encapsulation in hydrogel composites. Concave microwells and the tri-culture of three types of primary liver cells were applied for the construction of mLTs showing excellent liver functions and long-term (>1 month) viability in vitro. Large quantities of rat mLTs were encapsulated in collagen-alginate composites, implanted into hepatic failure mice and sustained their survival during regeneration of the remaining liver. The proposed liver support system offers xenogeneic hepatic assistance by mimicking native liver microarchitecture and providing immune-protection without the need for complicated devices or processes, and as such represents a promising system for recovery of organ function.
View details for DOI 10.1016/j.biomaterials.2014.07.009
View details for Web of Science ID 000341476500008
View details for PubMedID 25088727
One-stop microfiber spinning and fabrication of a fibrous cell-encapsulated scaffold on a single microfluidic platform
2014; 6 (2): 024108
This paper provides a method for microscale fiber spinning and the in situ construction of a 3D fibrous scaffold on a single microfluidic platform. This platform was also used to fabricate a variety of fibrous scaffolds with diverse compositions without the use of complicated devices. We explored the potential utility of the fibrous scaffolds for tissue engineering applications by constructing a fibrous scaffold encapsulating primary hepatocytes. The cells in scaffold were cultured over seven days and maintained higher viability comparing with 3D alginate non-fibrous block. The main advantage of this platform is that the fibrous structure used to form a scaffold can be generated without damaging the mechanically weak alginate fibers or encapsulated cells because all procedures are performed in a single platform without the intervention of the operator. In addition, the proposed fibrous scaffold permitted high diffusion capability of molecules, which enabled better viability of encapsulated cells than non-fibrous scaffold even in massive cell culture.
View details for DOI 10.1088/1758-5082/6/2/024108
View details for Web of Science ID 000337705000010
View details for PubMedID 24999513
Application of Concave Microwells to Pancreatic Tumor Spheroids Enabling Anticancer Drug Evaluation in a Clinically Relevant Drug Resistance Model
2013; 8 (9): e73345
Intrinsic drug resistance of pancreatic ductal adenocarcinoma (PDAC) warrants studies using models that are more clinically relevant for identifying novel resistance mechanisms as well as for drug development. Tumor spheroids (TS) mimic in vivo tumor conditions associated with multicellular resistance and represent a promising model for efficient drug screening, however, pancreatic cancer cells often fail to form spheroids using conventional methods such as liquid overlay. This study describes the induction of TS of human pancreatic cancer cells (Panc-1, Aspc-1, Capan-2) in concave polydimethylsiloxane (PDMS) microwell plates and evaluation of their usefulness as an anticancer efficacy test model. All three cell lines showed TS formation with varying degree of necrosis inside TS. Among these, Panc-1 spheroid with spherical morphology, a rather rough surface, and unique adhesion structures were successfully produced with no notable necrosis in concave microwell plates. Panc-1 TS contained growth factors or enzymes such as TGF-β1, CTGF, and MT1-MMP, and extracellular matrix proteins such as collagen type I, fibronectin, and laminin. Panc-1 cells grown as TS showed changes in stem cell populations and in expression levels of miRNAs that may play roles in chemoresistance. Visualization of drug penetration and detection of viability indicators, such as Ki-67 and MitoSOX, were optimized for TS for quantitative analysis. Water-soluble tetrazolium (MTS) and acid phosphatase (APH) assays were also successfully optimized. Overall, we demonstrated that concave PDMS microwell plates are a novel platform for preparation of TS of weakly aggregating cells and that Panc-1 spheroids may represent a novel three-dimensional model for anti-pancreatic cancer drug screening.
View details for DOI 10.1371/journal.pone.0073345
View details for Web of Science ID 000327538600027
View details for PubMedID 24039920
View details for PubMedCentralID PMC3769301
Spheroid-based three-dimensional liver-on-a-chip to investigate hepatocyte-hepatic stellate cell interactions and flow effects
LAB ON A CHIP
2013; 13 (18): 3529–37
We have developed a three-dimensional (3D) liver-on-a-chip to investigate the interaction of hepatocytes and hepatic stellate cells (HSCs) in which primary 3D hepatocyte spheroids and HSCs are co-cultured without direct cell-cell contact. Here, we show that the 3D liver chip offers substantial advantages for the formation and harvesting of spheroids. The most important feature of this liver chip is that it enables continuous flow of medium to the cells through osmotic pumping, and thus requires only minimal handling and no external power source. We also demonstrate that flow assists the formation and long-term maintenance of spheroids. Additionally, we quantitatively and qualitatively investigated the paracrine effects of HSCs, demonstrating that HSCs assist in the maintenance of hepatocyte spheroids and play an important role in the formation of tight cell-cell contacts, thereby improving liver-specific function. Spheroids derived from co-cultures exhibited improved albumin and urea secretion rates compared to mono-cultured spheroids after 9 days. Immunostaining for cytochrome P450 revealed that the enzymatic activity of spheroids co-cultured for 8 days was greater than that of mono-cultured spheroids. These results indicate that this system has the potential for further development as a unique model for studying cellular interactions or as a tool that can be incorporated into other models aimed at creating hepatic structure and prolonging hepatocyte function in culture.
View details for DOI 10.1039/c3lc50197c
View details for Web of Science ID 000323111300007
View details for PubMedID 23657720
Functional 3D Human Primary Hepatocyte Spheroids Made by Co-Culturing Hepatocytes from Partial Hepatectomy Specimens and Human Adipose-Derived Stem Cells
2012; 7 (12): e50723
We have generated human hepatocyte spheroids with uniform size and shape by co-culturing 1∶1 mixtures of primary human hepatocytes (hHeps) from partial hepatectomy specimens and human adipose-derived stem cells (hADSCs) in concave microwells. The hADSCs in spheroids could compensate for the low viability and improve the functional maintenance of hHeps. Co-cultured spheroids aggregated and formed compact spheroidal shapes more rapidly, and with a significantly higher viability than mono-cultured spheroids. The liver-specific functions of co-cultured spheroids were greater, although they contained half the number of hepatocytes as mono-cultured spheroids. Albumin secretion by co-cultured spheroids was 10% higher on day 7, whereas urea secretion was similar, compared with mono-cultured spheroids. A quantitative cytochrome P450 assay showed that the enzymatic activity of co-cultured spheroids cultured for 9 days was 28% higher than that of mono-cultured spheroids. These effects may be due to the transdifferentiation potential and paracrine healing effects of hADSCs on hHeps. These co-cultured spheroids may be useful for creating artificial three-dimensional hepatic tissue constructs and for cell therapy with limited numbers of human hepatocytes.
View details for DOI 10.1371/journal.pone.0050723
View details for Web of Science ID 000312064100029
View details for PubMedID 23236387
View details for PubMedCentralID PMC3517565
Concave microwell based size-controllable hepatosphere as a three-dimensional liver tissue model
2011; 32 (32): 8087–96
We have developed a size-controllable spheroidal hepatosphere and heterosphere model by mono-culturing of primary hepatocytes and by co-culturing primary hepatocytes and hepatic stellate cells (HSCs). We demonstrated that uniform-sized heterospheres, which self-aggregated from primary hepatocytes and HSCs, formed within concave microwell arrays in a rapid and homogeneous manner. The effect of HSCs was quantitatively and qualitatively investigated during spheroid formation, and HSC played an important role in controlling the organization of the spheroidal aggregates and formation of tight cell-cell contacts. An analysis of the metabolic function showed that heterospheres secreted 30% more albumin than hepatospheres on day 8. In contrast, the urea secretion from heterospheres was similar to that of hepatospheres. A quantitative cytochrome P450 assay showed that the enzymatic activity of heterospheres cultured for 9 days was higher as compared with primary hepatospheres. These size-controllable heterospheres could be mass-produced using concave plate and be useful for creating artificial three-dimensional hepatic tissue constructs and regeneration of failed liver.
View details for DOI 10.1016/j.biomaterials.2011.07.028
View details for Web of Science ID 000295241200006
View details for PubMedID 21813175
Diffusion-mediated in situ alginate encapsulation of cell spheroids using microscale concave well and nanoporous membrane
LAB ON A CHIP
2011; 11 (6): 1168–73
Here, we present a novel and simple process of spheroid formation and in situ encapsulation of the formed spheroid without intervention. A hemispherical polydimethylsiloxane (PDMS) micromold was employed for the formation of uniform sized spheroids and two types of nano-porous membrane were used for the control of the crosslinking agent. We characterized the transport properties of the membrane, and the selection of alginate hydrogel as a function of gelation time, alginate concentration, and membrane type. Using the developed process and micromold, HepG2 cell spheroids were successfully formed and encapsulated in alginate without replating. This method allows spheroid encapsulation with minimal damage to the spheroid while maintaining high cell viability. We demonstrate the feasibility of this method in developing a bio-artificial liver (BAL) chip by evaluating viability and function of encapsulated HepG2 spheroids. This method may be applied to the encapsulation of several aggregating cell types, such as β-cells for islet formation and stem cells for embryonic body preservation, or as a model for tumor cell growth and proliferation in a 3D hydrogel environment.
View details for DOI 10.1039/c0lc00540a
View details for Web of Science ID 000287867100028
View details for PubMedID 21298129