Scaffold fabrication in a perfusion culture microchamber array chip by O(2) plasma bonding of poly(dimethylsiloxane) protected by a physical mask

Abstract

Extracellular matrix (ECM) proteins are required for cell culture. In this paper, we report the use of O(2) plasma bonding to fabricate a perfusion culture microchamber array chip with identical-size ECM spots in the isolated microchambers. The chip was fabricated by assembly of two poly(dimethylsiloxane) (PDMS) layers, a microfluidic network layer, and an ECM array layer, which were aligned and then bonded by O(2) plasma oxidation with protection of the ECM microarray with a physical mask made from PDMS. We successfully cultivated Chinese hamster ovary K1 cells in the microchambers with fibronectin. In the fibronectin microchambers, the cells adhered and extended after 12 h of static culture and then grew over the course of 1 d of perfusion culture.

Figure 1

Structure of the perfusion culture microchamber array chip. (a) Overview of the microfluidic network on the chip. (b) Detail of a microchamber: microchamber (dark gray; depth: 271.2 μm, diameter: 1530 μm); microchannels and terrace (gray; depth: 52.5 μm, width: 100 μm); and microchannel (light gray; depth: 4.5 μm, width, 40 μm).

Figure 2

Fabrication of the perfusion culture microchamber array chip. (a) Masking of the ECM microarray. (b) Oxidation of the adhered surfaces with O₂ plasma for bonding. (c) Bonding of the microfluidic network layer and the ECM array layer.

Figure 3

Protection of the ECM from O₂ plasma oxidation with the physical mask. (a) Fluorescence image before oxidation. Note that the homogeneous fluorescence image indicates homogeneous immobilization of ECMs on the PDMS surface. (b) Fluorescence image after oxidation (output power: 100 W, oxidation time: 10 s).

Figure 4

Micrographs of the perfusion culture microchamber array chip. (a) Fluorescence micrograph of immobilized ECMs on the ECM array layer. (b) Fluorescence micrograph of ECM spots on the bottom of the microchambers of the chip. (c) Bright field image of the microchamber array. (d) Overlaid bright field and fluorescence micrographs of the microchamber array and immobilized ECMs.

Figure 5

Culture of CHO-K1 cells in the perfusion culture microchamber array chip. Micrographs of CHO-K1 cells after static culture for 12 h in (a) a fibronectin microchamber, (b) an unmodified PDMS microchamber, and (c) a collagen microchamber. Micrographs of CHO-K1 cells after perfusion culture for 1 d in (d) a fibronectin microchamber, (e) an unmodified PDMS microchamber, and (f) a collagen microchamber. (a^′)–(f^′) are enlargement in each microchamber.