Induction of hepatic tissues in multicellular spheroids composed of murine fetal hepatic cells and embedded hydrogel beads

Abstract

Introduction Three-dimensional (3D) multicellular spheroids are useful tools for simulation of cellular functions in vitro . However, it is difficult to culture certain epithelial cell types under 3D spheroid conditions because these cells cannot resist autonomous cell death, triggered by disordered cell polarity. The objective of this study was to find a method that enables spheroid culture of such epithelial cells utilizing hydrogel beads without cell death. Methods We used murine E14.5 fetal hepatic cells for the spheroid composition because they are sensitive to disorganized structures. Spheroids were formed by injecting 1-μl fresh medium containing 1000 fetal hepatic cells and the same number of alginate hydrogel beads (20 μm in diameter) into a 3% methylcellulose medium in the presence of dexamethasone and oncostatin M to induce hepatic differentiation. After 7 days of culture, microstructures were observed using hematoxylin and eosin staining and immunostaining using anti-CK8/18 antibody. Albumin secretion rate was determined by the enzyme-linked immunosorbent assay method. In addition, polarity-related proteins, E-cadherin, ezrin, and MRP2 were observed with immunostaining. Results Control spheroids without the use of alginate hydrogel beads showed extensive internal lack of epithelial hepatic cells. The spheroids containing alginate hydrogel beads exhibited sheet- or cord-like structures of epithelial hepatic cells, and it was clear that cell death of epithelial cells had been prevented. Albumin secretion data also supported the improvement of epithelial hepatic cell viability when alginate hydrogel beads were used. Localization of polarity-related proteins indicated the partial reconstitution of cell polarity in the spheroids using alginate hydrogel beads. Conclusion Based on these data, we concluded that the application of alginate hydrogel beads was effective in improving the epithelial hepatic cell culture of multicellular spheroids.

Enhancement and Maintenance of Hepatic Metabolic Functions by Controlling Three-Dimensional Aggregation of Cryo-Preserved Human iPS Cells Derived Hepatocyte-Like Cells

“…In addition, small molecule strategy is also reported to promote further hepatic differentiation from HLC 22,23 . Our MC spheroid formation method also has an advantage to form an "inner-manipulated" spheroid by adding biomolecules 24 or hydrogel beads 25,26 . For example, if we established HLC spheroid blended with hydrogel beads homogenously, differentiation of HLC sphere might be promoted by bringing out the differentiation factors effectively and evenly in the spheroid.…”

A review of manufacturing capabilities of cell spheroid generation technologies and future development

“…The release of the spheroids can be easily realized by washing the two‐phase system with fresh medium (Han et al, 2015). In another study, 3% methylcellulose (MC) has been used (Motoyama et al, 2016). Medium regulation based approach has special requirement for the materials, which poses concerns over the material effects on cell growth and behavior.…”

“…The release of the spheroids can be easily realized by washing the two-phase system with fresh medium (Han et al, 2015). In another study, 3% methylcellulose (MC) has been used (Motoyama et al, 2016).…”

Improved Oxygen Supply to Multicellular Spheroids Using A Gas-permeable Plate and Embedded Hydrogel Beads

“…We also developed a method to fabricate 3D hybrid spheroids comprised of cells and with 20 µm-diameter hydrogel beads. When we mixed an equal number of cells and beads, the beads formed microchannel-like structures in the spheroids [14], which enhanced protein secretion by hepatocytes and pancreatic beta cells [14,15,16]. These effects were thought to be derived from improved oxygen and nutrient supply or waste product removal.…”