Alginate Based Microcapsules Containing Galactosylated Chitosan as a Synthetic Matrix in 3D Culture of Hepatocytes

Background and Aim: Microencapsulation provides a suitable 3Dplatform for tissue engineering and plays an important role in developingbioartificial liver devices. Alginate-based microcapsules have beentested for the treatment of liver failure. The hepatocytes in pure alginatemicrocapsules exhibit low viability and poor liver-specific functions. Theemployment of galactosylated chitosan (GC) is one effective methodfor improvement of alginate-based microcapsules. In this work, analginate-galactosylated chitosan/chitosan (AGC/C) microcapsule hasbeen presented for the 3D culture of HepG2 cells and the microcapsulecharacteristics and liver-specific functions of cells have been evaluated.Methods: For investigation of the mechanical property of microcapsules,hydrogels were placed in a material testing machine (Zwick/Roell Z010,Germany) and compacted with a crosshead speed at 2.0 mm/min. For swelling test, the microcapsules were put in PBS solution at 37℃ for 7days and the diameter of microcapsules was measured using an opticalmicroscope (IX71 Olympus, Japan). For permeability of the microcapsules,the release of bovine serum albumin (BSA) was investigated. Finally, forcell functionalities of HepG2 cells were suspended in an alginate-basedsolution with a density of 2×106 cells/mL. For the preparation of Alg/GCsolutions with the proportion of 1%/0.5%, the GC solution was droppedinto Alg solution and mixed at a high rate. The microcapsules wereprepared by means of an electrostatic microencapsulation method (9 kv),and eventually, the AGC microcapsules were coated with 0.3% chitosansolution. MTT assay was used to measure the viability of cells. Theconcentration of glucose and lactate in the supernatant was determinedusing a kit (GOD PAD).Results: The microcapsules of AGC/C showed the faster release of BSAthan alginate/chitosan (AC) microcapsules, indicating the presence of GCin the AGC/C microcapsule improve the permeability of microcapsules.This improvement is related to the interaction of carboxyl groups ofalginates with amino acid groups of GC. In addition, the large volumeand spatial hindrance of GC in the core lead to decrease the interactionsbetween alginate and external chitosan, causing structural loosing ofthe membrane. Analysis of compression assay showed that stiffness ofAGA/C and AC microcapsules were 6.75 and 7.62 kPa, respectively. MTTassay for cell proliferation of AGC/C microcapsules revealed about 20%increase in the 5th day compared to the AC microcapsules. During thefive days of cell culture, the secretion of albumin was increased.Conclusion: The solubility of the chitosan derivatives increases byintroducing galactose groups. The increase of galactose ligands enhancesthe viability and formation of multicellular spheroids of HepG2 cellscultured in AGC/C microcapsules and improves their liver-specificfunctions than in traditional AC microcapsules. Therefore, GC because ofexcellent adhesion and spheroid formation of hepatocytes has potency asone of synthetic ECMs for liver tissue engineering.

Keywords:

HepG2 cell line , AC microcapsule , Galactosylated chitosan , Mechanical stability