Engineering liver tissue spheroids with inverted colloidal crystal scaffolds

Department of Biomedical Engineering, University of Michigan, 3074 H.H. Dow Building, 2300 Hayward Street, Ann Arbor, MI 48109, USA
Biomaterials (Impact Factor: 8.56). 09/2009; 30(27):4687-4694. DOI: 10.1016/j.biomaterials.2009.05.024


Multicellular spheroids provide a new three-dimensional (3D) level of control over morphology and function of ex vivo cultured tissues. They also represent a valuable experimental technique for drug discovery and cell biology. Nevertheless, the dependence of many cellular processes on the cluster diameter remains unclear. To provide a tool for the systematic evaluation of such dependences, we introduce here inverted colloidal crystal (ICC) scaffolds. Uniformly sized pores in ICC cell matrixes afford a high yield production of controlled size spheroids in standard 96 well-plates. Transparent hydrogel matrix and ship-in-bottle effect also allows for convenient monitoring of cell processes by traditional optical techniques. Different developmental stages of 46.5–151.6 μm spheroids from HepG2 hepatocytes with vivid morphological similarities to liver tissue (bile canaliculi) were observed. The liver-specific functions of HepG2 cells were systematically investigated and compared for spheroids of different diameters as well as 2D cultures. Clear trends of albumin production and CYP450 activity were observed; diffusion processes and effect of cellular aggregation on metabolic activity were identified to be the primary contributors to the size dependence of the liver functions in HepG2 spheroids in ICC scaffolds. Since the aggregation of cells into clusters is a universal biological process, these findings and scaffolds can be applied to many other relevant cell types.

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    • "Moreover, the 3D architecture of spheroids also presents a barrier for the diffusion of nutrients, oxygen, drugs and metabolic waste, especially when spheroids reach the critical size limit [31]. While scaffolds such as polyurethane foams [32] [33] and inverted colloidal crystal scaffolds [34] have been developed to limit spheroid size, the use of this approach is limited by potential drug adsorption onto the scaffold surface. Microfabricated substrates such as microwells [20] [26] or ligand-link thin films [29] minimizes the problem of drug adsorption but spheroids cultured in these platforms tend to detach from the surface during routine medium change. "
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    ABSTRACT: Liver-specific functions in primary hepatocytes can be maintained over extended duration in vitro using spheroid culture. However, the undesired loss of cells over time is still a major unaddressed problem, which consequently generates large variations in downstream assays such as drug screening. In static culture, the turbulence generated by medium change can cause spheroids to detach from the culture substrate. Under perfusion, the momentum generated by Stokes force similarly results in spheroid detachment. To overcome this problem, we developed a Constrained Spheroids (CS) culture system that immobilizes spheroids between a glass coverslip and an ultra-thin porous Parylene C membrane, both surface-modified with poly(ethylene glycol) and galactose ligands for optimum spheroid formation and maintenance. In this configuration, cell loss was minimized even when perfusion was introduced. When compared to the standard collagen sandwich model, hepatocytes cultured as CS under perfusion exhibited significantly enhanced hepatocyte functions such as urea secretion, and CYP1A1 and CYP3A2 metabolic activity. We propose the use of the CS culture as an improved culture platform to current hepatocyte spheroid-based culture systems.
    Full-text · Article · Dec 2015 · Biomaterials
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    • "Biomaterials xxx (2014) 1e10 Please cite this article in press as: Huang Y-J, Hsu S-h, Acquisition of epithelialemesenchymal transition and cancer stem-like phenotypes within chitosan-hyaluronan membrane-derived 3D tumor spheroids, Biomaterials (2014), form rigid aggregates [9]. These factors greatly affect the precise evaluation of biological or biochemical endpoints in drug screening [10]. Hence, a simple and well-characterized platform for rapid generation of tumor spheroids is necessary to improve the limitations of tumor spheroid culture system in drug screening processes. "
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    ABSTRACT: Cancer drug development has to go through rigorous testing and evaluation processes during pre-clinical in vitro studies. However, the conventional two-dimensional (2D) in vitro culture is often discounted by the insufficiency to present a more typical tumor microenvironment. The multicellular tumor spheroids have been a valuable model to provide more comprehensive assessment of tumor in response to therapeutic strategies. Here, we applied chitosan-hyaluronan (HA) membranes as a platform to promote three-dimensional (3D) tumor spheroid formation. The biological features of tumor spheroids of human non-small cell lung cancer (NSCLC) cells on chitosan-HA membranes were compared to those of 2D cultured cells in vitro. The cells in tumor spheroids cultured on chitosan-HA membranes showed higher levels of stem-like properties and epithelial-mesenchymal transition (EMT) markers, such as NANOG, SOX2, CD44, CD133, N-cadherin, and vimentin, than 2D cultured cells. Moreover, they exhibited enhanced invasive activities and multidrug resistance by the upregulation of MMP2, MMP9, BCRC5, BCL2, MDR1, and ABCG2 as compared with 2D cultured cells. The grafting densities of HA affected the tumor sphere size and mRNA levels of genes on the substrates. These evidences suggest that chitosan-HA membranes may offer a simple and valuable biomaterial platform for rapid generation of tumor spheroids in vitro as well as for further applications in cancer stem cell research and cancer drug screening.
    Full-text · Article · Oct 2014 · Biomaterials
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    • "Compared with 2-D substrata, 3-D scaffolds provide the cells an environment closer to the normal tissue environment, therefore should be a better choice for spheroid formation. Indeed, it was reported that hepatocytes organized into spheroids when cultured in some hydrogel scaffolds, even in the absence of galactose ligands [22] [23] [24]. Although a lot of galactosylated 3-D scaffolds have been reported , it is usually difficult to harvest the hepatocyte spheroids from the 3-D matrix, which is a prerequisite for their biological analysis and also for their further applications, e.g. as building blocks in organ printing [5]. "
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    ABSTRACT: Various galactosylated scaffolds have been developed for hepatocyte culture because galactose ligands help maintain cell viability, facilitate the formation of multicellular spheroids, and help maintain a high level of liver-specific functions. However it is difficult to harvest the cell spheroids generated inside the 3D scaffolds for their further biological analysis and applications. Here we developed a new galactosylated hydrogel scaffold which solidifies in situ upon heating to physiological temperature, and liquefies again upon cooling back to room temperature. The new scaffold is composed of poly(N-isopropylacrylamide) (PNIPAM) microgel and poly(ethylene glycol) (PEG). Because of the thermosensitivity of PNIPAM microgel, the mixed dispersions gel upon heating and liquefy upon cooling. PEG was added to reduce the shrinkage of the gels. Part of the PNIPAM microgel was replaced with galactosylated one to afford a series of blend gels with various content of galactose ligands. HepG2 cells, a human hepatocarcinoma cell line, were encapsulated in the in situ-formed gels. As expected, the cell viability increases with increasing content of galactose ligands. In addition, compact multicellular spheroids were obtained in gels containing galactose ligands, while loose spheroids formed in gel without galactose ligands. The cells cultured in galactose-containing gels also exhibit higher level of liver-specific functions, in terms of both albumin secretion and urea synthesis, than those cultured in gel without these ligands. The new galactosylated scaffold not only promotes the formation of hepatocyte spheroids, but also allows for their harvest. By cooling back to room temperature to liquefy the gel, the hepatocyte spheroids can be facilely harvested from the scaffold. The reversible galactosylated scaffold developed here may be used for large scale fabrication of hepatocyte spheroids.
    Full-text · Article · Dec 2013 · Acta biomaterialia
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