Multilayer nanofilms as substrates for hepatocellular applications

Abstract

Multilayer nanofilms, formed by the layer-by-layer (LbL) adsorption of positively and negatively charged polyelectrolytes, are promising substrates for tissue engineering. We investigate here the attachment and function of hepatic cells on multilayer films in terms of film composition, terminal layer, rigidity, charge, and presence of biofunctional species. Human hepatocellular carcinoma (HepG2) cells, adult rat hepatocytes (ARH), and human fetal hepatoblasts (HFHb) are studied on films composed of the polysaccharides chitosan (CHI) and alginate (ALG), the polypeptides poly(l-lysine) (PLL) and poly(l-glutamic acid) (PGA), and the synthetic polymers poly(allylamine hydrochloride) (PAH) and poly(styrene sulfonate) (PSS). The influence of chemical cross-linking following LbL assembly is also investigated. We find HepG2 to reach confluence after 7 days of culture on only 2 of 18 candidate multilayer systems: (PAH-PSS)(n) (i.e. nPAH-PSS bilayers) and cross-linked (PLL-ALG)(n)-PLL. Cross-linked PLL-ALG and PLL-PGA films support attachment and function of ARH, independently of the terminal layer, provided collagen is adsorbed to the top of the film. (PAH-PSS)(n), cross-linked (PLL-ALG)(n), and cross-linked (PLL-PGA)(n)-PLL films all support attachment, layer confluence, and function of HFHb, with the latter film promoting the greatest level of function at 8 days. Overall, film composition, terminal layer, and rigidity are key variables in promoting attachment and function of hepatic cells, while film charge and biofunctionality are somewhat less important. These studies reveal optimal candidate multilayer biomaterials for human liver tissue engineering applications.

Fig. 1

Photomicrographs of HepG2 taken after 3 and 7 days culture on (PLL-ALG)₃-PLL, (PLL-ALG)₃-PLL-X, (PAH-PSS)₄, (PAH-PSS)₃-PAH, and the uncoated culture well.

Fig. 2

Albumin secretion rate for ARH seeded on various multilayer films and on a rat collagen I coated plate (Biocoat).

Fig. 3

Albumin secretion versus time for human fetal hepatoblasts seeded on various multilayer film biomaterials. (a) confluent systems in the absence of collagen, (b) PAH-PSS systems, (c) PLL-PGA systems, (d) PLL-ALG systems, and (e) confluent systems in the presence of collagen not shown in (b)–(d).

Fig. 3

Albumin secretion versus time for human fetal hepatoblasts seeded on various multilayer film biomaterials. (a) confluent systems in the absence of collagen, (b) PAH-PSS systems, (c) PLL-PGA systems, (d) PLL-ALG systems, and (e) confluent systems in the presence of collagen not shown in (b)–(d).

Fig. 3

Albumin secretion versus time for human fetal hepatoblasts seeded on various multilayer film biomaterials. (a) confluent systems in the absence of collagen, (b) PAH-PSS systems, (c) PLL-PGA systems, (d) PLL-ALG systems, and (e) confluent systems in the presence of collagen not shown in (b)–(d).

Fig. 4

Mass (a) and thickness (b) of PAH-PSS, PLL-ALG, and PLL-PGA films during LbL assembly, as measured by OWLS.

Fig. 5

Shear modulus (a), viscosity (b), and thickness (c) of PAH-PSS, PLL-ALG, and PLL-PGA films during LbL assembly, as measured by QCMD. Data points along right vertical axis are taken following film exposure to chemical cross-linking agents (see Section 2.3).

Fig. 5

Shear modulus (a), viscosity (b), and thickness (c) of PAH-PSS, PLL-ALG, and PLL-PGA films during LbL assembly, as measured by QCMD. Data points along right vertical axis are taken following film exposure to chemical cross-linking agents (see Section 2.3).