Biomaterials, Biodegradables and Biomimetics Research Group

Comunications - Poster

Assessment of human liver cancer cell fate in alginate/collagen/chitosan microbeads: effects of cell seeding density on hepatocyte viability, morphology and functionality


Development of a 3D bio-engineered hepatic tissue that exhibit stable phenotype upon long-term maintenance is a major challenge in the field of liver tissue engineering. The cell encapsulation technology using hydrogels is a promising strategy applicable to generate microtissue constructs, including bioartificial microliver. These microgels provide a protective and conducive microenvironment within which the cells spread and proliferate efficiently. However, a suitable equilibrium between the stability and diffusional properties need to be maintained to promote the long-term functionality of encapsulated cells. In this study, human hepatocellular carcinoma cells (HepG2) were encapsulated within different natural-origin biomaterials: Alginate (0.5% (w/v), 1.0% (w/v), 1.5% (w/v)); Alginate (1.0% (w/v), 1.5% (w/v)) mixed with rat-tail collagen type I (1 mg/mL) (1:1) and, Alginate (1.5% (w/v)) mixed with rat-tail collagen type I (1 mg/mL) coated with medium molecular weight-chitosan (0.75% (w/v) (multilayered microbead). The goal was to improve the viability and functionality of encapsulated HepG2 cells in alginate hydrogels by blending with rat-tail collagen type-I, upon long-term culture. Moreover, to prevent the possible leakage of collagen and simultaneously increasing the stability of the microbead, a natural polymeric cross-linker, chitosan, was used to coat the alginate/collagen microbeads. The microencapsulation of cells were performed by using a syringe pump and a 27G needle gauge, followed by ionic cross-linking with CaCl2 solution. The size and morphology of the microbeads produced by the proposed system were uniform, when parameters were kept constant. The mean diameter of the microbeads was around 1.5 mm. Live/dead assay results on Day 1 showed  the cell viability dropped significantly, but within three days of culture the remaining viable cells regained vitality and started proliferating. After 7 days, for all cell seeding conditions tested (0.5, 1 and, 2 million cells/mL) cell proliferation was associated with 3D cell organization, which adopted different morphologies. Interestingly, 1.5% (w/v) alginate, 1.5% (w/v) alginate mixed with collagen type I and, the chitosan coated alginate/collagen microbeads supported rearrangement of HepG2 cells towards both hepatic cord-like structures as well as typical spheroids formation, while in the rest of the conditions cell proliferation was associated exclusively towards spheroids organization. In summary, we succeeded in reconstructing a 3D bio-artificial microliver construct with hepatic cord-like organization and spheroids formation, using HepG2 cells  and alginate hydrogel-based microencapsulation model. Acknowledgements: Authors acknowledge the financial support from Portuguese Foundation for Science and Technology R&D grant POCI-01-0145-FEDER-016715 (PTDC/BBB-ECT/4317/2014) for the project MicroLiver and European Research Council grant agreement ERC-2012-ADG 20120216-321266 for the project ComplexiTE.

The FoReCaST First Workshop
ALGINATE, Chitosan, HepG2, microbeads
Closed Access
Peer Reviewed
Year of Publication
Date Published
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