Increased stability of glycol-terminated self-assembled monolayers for long-term patterned cell culture

Abstract

Self-assembled monolayers (SAMs) are widely used to confine proteins and cells to a pattern to study cellular processes and behavior. To fully explore some of these phenomena, it is necessary to control cell growth and confinement for several weeks. Here, we present a simple method by which protein and cellular confinement to a pattern can be maintained for more than 35 days. This represents a significant increase in pattern stability compared to previous monolayer systems and is achieved using an amide-linked glycol monomer on 50 Å titanium/100 Å gold-coated glass coverslips. In addition, this study provides insight into the method of SAM degradation and excludes interfacial mixing of the monomers and blooming of the adlayer as major mechanisms for SAM degradation.

Figure 1

Protein and cell resistant SAMs were created from a) ether-linked glycol thiol (1), b) ester-linked glycol thiol (2), and c) amide-linked glycol thiol (3)

Figure 2

Live-cell phase-contrast images acquired weekly for CHO-K1 cells grown on a 95 μm circles pattern with an ether-linked glycol (1) mono-layer background on varying gold thicknesses. Pattern stability is maintained for 14 days on 50 Å and 100 Å gold substrates.

Figure 3

Live-cell phase-contrast images acquired weekly for CHO-K1 cells grown on a 95 μm circles pattern with an ester-linked glycol (2) mono-layer background on varying gold thicknesses. Pattern stability is maintained for 28 days on 100 Å gold substrates.

Figure 4

Live-cell phase-contrast images acquired weekly for CHO-K1 cells grown on a 95 μm circles pattern with an amide-linked glycol (3) mono-layer background on varying gold thicknesses. Pattern stability is maintained for 35 days on 100 Å gold substrates.

Figure 5

Cell attachment as a function of monomer linkage, gold thickness, and time. Spread cells are indicative of loss of pattern stability (A, C, E). Round cells are often unattached or weakly attached to the substrate (B, D, F). Dashed lines represent confluent substrates and complete loss of pattern.

Figure 6

Patterned substrate adhesion measured using QNM SPM. The wider lines are the glycol-terminated areas whereas the thinner lines are alkane-terminated. The ether-linked at day 1 (A) and day 21 (B), ester-linked at day 1 (C) and day 21 (D), and amide-linked at day 1 (E) and day 21 (F) do not show significant blurring of the pattern, indicating that interfacial mixing has not occurred.

Figure 7

Scanning probe microscopy height images of gold substrates obtained in peak force tapping mode.

Scheme 1

Synthetic scheme for the synthesis of the ester-linked glycol-terminated thiol (2).

Scheme 2

Synthetic scheme for the synthesis of the amide-linked glycol-terminated thiol (3).