Combined Effect of Cryogel Matrix and Temperature-Reversible Soluble–Insoluble Polymer for the Development of in Vitro Human Liver Tissue

Abstract

Hepatic cell culture on a three-dimensional (3D) matrix or as a hepatosphere appears to be a promising in vitro biomimetic system for liver tissue engineering applications. In this study, we have combined the concept of a 3D scaffold and a spheroid culture to develop an in vitro model to engineer liver tissue for drug screening. We have evaluated the potential of poly(ethylene glycol)-alginate-gelatin (PAG) cryogel matrix for in vitro culture of human liver cell lines. The synthesized cryogel matrix has a flow rate of 7 mL/min and water uptake capacity of 94% that enables easy nutrient transportation in the in vitro cell culture. Young's modulus of 2.4 kPa and viscoelastic property determine the soft and elastic nature of synthesized cryogel. Biocompatibility of PAG cryogel was evaluated through MTT assay of HepG2 and Huh-7 cells on matrices. The proliferation and functionality of the liver cells were enhanced by culturing hepatic cells as spheroids (hepatospheres) on the PAG cryogel using temperature-reversible soluble-insoluble polymer, poly(N-isopropylacrylamide) (PNIPAAm). Pore size of the cryogel above 100 μm modulated spheroid size that can prevent hypoxia condition within the spheroid culture. Both the hepatic cells have shown a significant difference (P < 0.05) in terms of cell number and functionality when cultured with PNIPAAm. After 10 days of culture using 0.05% PNIPAAm, the cell number increased by 11- and 7-fold in case of HepG2 and Huh-7 cells, respectively. Similarly, after 10 days of hepatic spheroids culture on PAG cryogel, the albumin production, urea secretion, and CYP450 activity were significantly higher in case of culture with PNIPAAm. The developed tissue mass on the PAG cryogel in the presence of PNIPAAm possess polarity, which was confirmed using F-actin staining and by presence of intercellular bile canalicular lumen. The developed cryogel matrix supports liver cells proliferation and functionality and therefore can be used for in vitro and in vivo drug testing.

A collagen based cryogel bioscaffold coated with nanostructured polydopamine as a platform for mesenchymal stem cell therapy

“…Studies have shown that the pore size of bioscaffolds is crucial for determining their function, with smaller pores providing better nutrient and oxygen transfer to facilitate cell growth and proliferation, while large pores provide a high surface area to accommodate cells. 48 Bioscaffolds with a larger mean pore sizes (i.e., 300 mm) have also been shown to be associated with a significantly higher cell viability and proliferation relative to bioscaffolds with a smaller mean pore size. 49 In keeping with these studies, structural analysis of our collagen bioscaffold demonstrated it to have a mean pore size of 300 6 100 mm, which we have shown facilitates AD-MSCs viability and proliferation, especially when compared to AD-MSCs cultured on conventional 2D culture plates.…”

“…50,51 Our bioscaffold also has a high degree of porosity (75% ± 3%) with a corresponding high swelling ratio. 48 The high porosity and interconnected macropores of our bioscaf-fold creates a physical space to facilitate cell movement and distribution throughout the bioscaffold. In turn, this is advantageous for nutrient and oxygen transfer to all cells seeded into the bioscaffold while also preventing cell loss from cellular overcrowding, which commonly occurs when using traditional cell culture plates.…”

An oxygen plasma treated poly(dimethylsiloxane) bioscaffold coated with polydopamine for stem cell therapy

“…Hence, PDMS provides a suitable “base-biomaterial” for the fabrication of porous 3D bioscaffolds. Studies have shown that the pore size of 3D bioscaffolds is crucial for determining their function—small pores provide better nutrient and oxygen transfer to facilitate cell growth and proliferation and large pores (i.e., ≥ 300 μm) provide a high surface area to accommodate cells while also facilitating their viability and rate of proliferation [38, 39]. Structural analysis of our PDMS bioscaffold demonstrated it to have a mean pore size of 300 ± 100 μm with a corresponding increase in AD-MSC viability and proliferation compared to AD-MSCs cultured on conventional 2D culture plates.…”

Reconstruction of structure and function in tissue engineering of solid organs: Toward simulation of natural development based on decellularization

“…Kumari et al used cryogel matrix combined with temperature‐reversible soluble–insoluble polymer as the scaffold for in vitro culture of liver cells. The developed spheroid structure displayed liver‐specific functions as well as morphological features such as intercellular bile canalicular lumen (Kumari, Karande, & Kumar, ). Takebe et al produced 3D liver buds by self‐organization of human iPSCs‐derived hepatocytes and transplanted the liver buds into mice, which displayed functional vasculature connecting to the host.…”