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.
"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), http://dx.doi.org/10.1016/j.biomaterials.2014.09.010 form rigid aggregates . These factors greatly affect the precise evaluation of biological or biochemical endpoints in drug screening . 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. "
[Show abstract][Hide abstract] 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.
"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   . 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 . "
[Show abstract][Hide abstract] 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.
"When smaller spheroids collide to form larger spheroids in culture, mass transfer of oxygen, nutrients, metabolites and drugs can be impeded in the inner core yielding high variability during drug safety testing . Attempts to constrain 3D spheroids in a diffusible dimension have been achieved by growing spheroids on microfabricated platforms  , polyurethane foams  , and polymeric scaffolds , but these methods do not provide the optimal chemical and mechanical microenvironments needed to maintain high level cellular functions. Furthermore , scalability in manufacturing and simplicity in operation for high-throughput, large-scale drug safety testing applications is also problematic. "
[Show abstract][Hide abstract] ABSTRACT: Hepatocyte spheroids can maintain mature differentiated functions, but collide to form bulkier structures when in extended culture. When the spheroid diameter exceeds 200 μm, cells in the inner core experience hypoxia and limited access to nutrients and drugs. Here we report the development of a thin galactosylated cellulosic sponge to culture hepatocytes in multi-well plates as 3D spheroids, and constrain them within a macroporous scaffold network to maintain spheroid size and prevent detachment. The hydrogel-based soft sponge conjugated with galactose provided suitable mechanical and chemical cues to support rapid formation of hepatocyte spheroids with a mature hepatocyte phenotype. The spheroids tethered in the sponge showed excellent maintenance of 3D cell morphology, cell-cell interaction, polarity, metabolic and transporter function and/or expression. For example, cytochrome P450 (CYP1A2, CYP2B2 and CYP3A2) activities were significantly elevated in spheroids exposed to β-naphthoflavone, phenobarbital, or pregnenolone-16α-carbonitrile, respectively. The sponge also exhibits minimal drug absorption compared to other commercially available scaffolds. As the cell seeding and culture protocols are similar to various high-throughput 2D cell-based assays, this platform is readily scalable and provides an alternative to current hepatocyte platforms used in drug safety testing applications.
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