Stable immobilization of rat hepatocytes as hemispheroids onto collagen‐conjugated poly‐dimethylsiloxane (PDMS) surfaces: Importance of direct oxygenation through PDMS for both formation and function

Abstract

The highly oxygen-permeable material, poly-dimethylsiloxane (PDMS), has the potential to be applied to cell culture microdevices, but cell detachment from PDMS has been a major problem. In this study, we demonstrate that a combination of collagen covalently immobilized PDMS and an adequate oxygen supply enables the establishment of a stable, attached spheroid (hemispheroid) culture of rat hepatocytes. The bottom PDMS surfaces were first treated with oxygen plasma, then coupled with aminosilane followed by a photoreactive crosslinker, and they were finally reacted with a collagen solution. X-ray photoelectron spectroscopy (XPS) and contact angle measurements showed that the covalent immobilization of collagen on the surface occurred only where the crosslinker had been introduced. On the collagen-conjugated PDMS surface, rat hepatocytes organized themselves into hemispheroids and maintained the viability and a remarkably high albumin production at least for 2 weeks of culture. In contrast, hepatocytes on the other types of PDMS surfaces formed suspended spheroids that had low albumin production. In addition, we showed that blocking the oxygen supply through the bottom PDMS surface inhibited the formation of hemispheroids and the augmentation of hepatocellular function. These results show that appropriate surface modification of PDMS is a promising approach towards the development of liver tissue microdevices.

Spontaneous formation of stably-attached and 3D-organized hepatocyte aggregates on oxygen-permeable polydimethylsiloxane membranes having 3D microstructures

“…4 Hematoxylin and eosin staining of vertical crosssections of primary rat hepatocyte cultures on the different PDMS 3D substrates under O 2 + conditions (Day 14). Scale bars indicate 50 µm spheroid formation on PDMS membranes with covalentlyimmobilized collagen (Nishikawa et al 2008a). In the present study, although we just applied a simple collagen coating, it appeared that topographical features such as PDMS macropillars together with a high oxygen environment also promoted stably-attached 3D multicellular aggregate formation.…”

“…By partly focusing on this property, our group has recently reported the static culture of liver cells (human hepatoma Hep G2 cell line or primary-cultured rat hepatocytes) on smooth PDMS membranes in bottomless polycarbonate multi-well frames (Evenou et al 2010;Nishikawa et al 2008a, b). In the case of primary rat hepatocyte culture, we showed that direct oxygenation through the PDMS membrane together with appropriate chemical surface modification (collagen covalent binding) enabled the formation of stably-attached and functional hemispheroids (Nishikawa et al 2008a). …”

Low O2 metabolism of HepG2 cells cultured at high density in a 3D microstructured scaffold

“…2.2.1 Preparation of scaffolds for cell culture PDMS scaffolds were coated covalently with collagen (Cellmatrix type I-P, Nitta Gelatin, Japan) following protocol by Nishikawa et al (2008). Sterilization was done during the UV exposure step of this protocol.…”

Carbon nanotube-coated silicone as a flexible and electrically conductive biomedical material

“…With these unique properties, cells seeded on CNT-coated scaffolds can be organized using electricity and can be transplanted into the body. Furthermore, silicone exhibits high oxygen permeability [42], which is an advantage when it is implanted. However, in vivo experiments using this scaffold have not been performed yet.…”