Preparation of a sponge-like biocomposite agarose–chitosan scaffold with primary hepatocytes for establishing an in vitro 3D liver tissue model

Abstract

Designing a three-dimensional (3D) macroporous scaffold with desired bio-functional properties is an important aspect for fabricating an in vitro liver tissue model with applications in pre-clinical therapeutics testing. In the present study a bio-polymeric composite scaffold of agarose-chitosan (AG-CH) was synthesized at optimized sub-zero temperature and evaluated for its suitability in in vitro liver tissue engineering. The scaffold showed high porosity (83 AE 2%) with interconnected pores (average pore diameter 40-70 mm). High swelling kinetics on account of the hydrophilic pore channels in the AG-CH scaffold allows unhindered migration of cells and gaseous exchange. At neutral pH, the negative charge on the surface of the AG-CH scaffold ensures increased cell-to-cell interfacial interaction followed by colonization of hepatocytes. Rheological studies of the hydrated scaffold demonstrate its high spongelike visco-elastic behavior without any fracture deformation up to 34 AE 1 N, which insinuates its applicability for soft-tissue engineering. The AG-CH scaffold showed $15% degradation in a span of four weeks in sterile PBS at physiological pH, which could help to maintain the structural integrity of neotissue formation. In vitro primary hepatocytes proliferation in the AG-CH scaffold showed an increase in cellular metabolic activity. The hepatic functions like albumin secretion and urea synthesis were established for the primary hepatocytes in the 3D scaffold and were higher in comparison to the control.The expression of hepatic CYP450 biomarker was observed in the in vitro cultured hepatocytes immobilized in the 3D AG-CH scaffold. Thus, the AG-CH scaffold with suitable physico-chemical properties and hepatic cell compatibility present its potential for developing an in vitro liver tissue model.

Fabrication and Characterization of Heparin/Collagen Sponge for in Vitro Differentiation of Wharton’s Jelly-Derived Mesenchymal Stem Cells into Hepatocytes

“…SEM images showed that both heparinized and non-heparinized sponges had an optimal porosity for cell growth; however, the mean value of the pore size of heparinized sponges was more than that in non-heparinized ones which might consequently protect better angiogenesis after transplantation and better nutrient and waste product exchange for in vitro hepatocyte toxicological and pharmacological experiments. The average pore size with a diameter of 40 -70 µm has been suggested to be appropriate for hepatocyte migration into the sponges and their functionality (40). Cell attachment and reduction in cell aggregation have been found to be affected by the pore size of the scaffolds (39).…”

Biofunctionalized Hydrogel Microscaffolds Promote 3D Hepatic Sheet Morphology

“…With emerging advances in 3D materials fabrication, there are new opportunities to design tissue engineering platforms that overcome the shortcomings of conventional 2D substrates . As one of the most architecturally complex organs in the human body, the liver has motivated numerous 3D design strategies, including liver cell (or so‐called hepatocyte) encapsulation within matrices, spheroid culture, sandwich hydrogels, cell sheet engineering, and macroporous scaffolds . Based on the results obtained with various 3D platforms comprised of natural or synthetic biomaterials as well as with rigid 2D polymeric substrates, there is general agreement that structural and functional properties of cultured hepatocytes more faithfully mimic the in vivo phenotype in 3D platforms …”

“…[ 6,7 ] As one of the most architecturally complex organs in the human body, the liver has motivated numerous 3D design strategies, including liver cell (or so-called hepatocyte) encapsulation within matrices, [8][9][10] spheroid culture, [11][12][13][14] sandwich hydrogels, [ 15,16 ] cell sheet engineering, [17][18][19][20][21][22][23] and macroporous scaffolds. [ 24 ] Based on the results obtained with various 3D platforms comprised of natural or synthetic biomaterials as well as with rigid 2D polymeric substrates, there is general agreement that structural and functional properties of cultured hepatocytes more faithfully mimic the in vivo phenotype in 3D platforms. [ 25,26 ] One of the most promising 3D scaffolds entails a microstructured hydrogel platform that provides hexagonally packed interconnected spherical cavities in order to promote cell aggregation and the formation of a cellular spheroid in each cavity, as demonstrated for hepatocytes and other cell types.…”

Self‐assembled biogenic melanin modulated surface chemistry of biopolymers‐colloidal silica composite porous matrix for the recovery of uranium

“…AACS and MAACS matrices were examined for various physicochemical properties like porosity and internal architecture, swelling kinetics and swelling ratio, hydraulic permeability, density, degradation, zeta potential, thermogravimetic behavior, chemical attribution of functional groups by FTIR, and rheological behavior. The characterization procedures are described in our previously reported studies …”

“…The surface charge potential (zeta potential) of AACS and MAACS matrices was examined at different pH. For zeta potential analysis, processing of solid samples was performed as per the previously reported study . Briefly, matrices were dried (~50 mg dried mass) at 60°C for 4 h followed by grinding of matrices using mortar and pestle.…”