Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

Abstract

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates. 1 While microcontact printing can be employed to directly print a large number of molecules, including proteins, 2 DNA, 3 and silanes, 4 the formation of self-assembled monolayers (SAMs) from long chain alkane thiols on gold provides a simple way to confine proteins and cells to specific patterns containing adhesive and resistant regions. This confinement can be used to control cell morphology and is useful for examining a variety of questions in protein and cell biology. Here, we describe a general method for the creation of well-defined protein patterns for cellular studies. 5 This process involves three steps: the production of a patterned master using photolithography, the creation of a PDMS stamp, and microcontact printing of a gold-coated substrate. Once patterned, these cell culture substrates are capable of confining proteins and/or cells (primary cells or cell lines) to the pattern.The use of self-assembled monolayer chemistry allows for precise control over the patterned protein/cell adhesive regions and non-adhesive regions; this cannot be achieved using direct protein stamping. Hexadecanethiol, the long chain alkane thiol used in the microcontact printing step, produces a hydrophobic surface that readily adsorbs protein from solution. The glycol-terminated thiol, used for backfilling the non-printed regions of the substrate, creates a monolayer that is resistant to protein adsorption and therefore cell growth. 6 These thiol monomers produce highly structured monolayers that precisely define regions of the substrate that can support protein adsorption and cell growth. As a result, these substrates are useful for a wide variety of applications from the study of intercellular behavior 7 to the creation of microelectronics. 8 While other types of monolayer chemistry have been used for cell culture studies, including work from our group using trichlorosilanes to create patterns directly on glass substrates, 9 patterned monolayers formed from alkane thiols on gold are straight-forward to prepare. Moreover, the monomers used for monolayer preparation are commercially available, stable, and do not require storage or handling under inert atmosphere. Patterned substrates prepared from alkane thiols can also be recycled and reused several times, maintaining cell confinement. 10 Video LinkThe video component of this article can be found at http://www.jove.com/details.php?id=3164 Protocol Preparation of the Patterned Master (Figure 1)Note: For best results, photopatterning should be carried out in a cleanroom environment. Table 1. The acetone will evaporate during the second step of the spin program leaving a clean, dry wafer. 2. Apply approximately 1 mL AZ9245 photoresist/in (in diameter) to the wafer and spin-coat using the conditions described in Table 1. 3. Soft-bake the photoresist-coated wafer at 110°C for 2 m using a high-uniformity hotplate. 4. Photopattern the substrate using...

Increased Stability of Glycol-Terminated Self-Assembled Monolayers for Long-Term Patterned Cell Culture

“…24 Gold thicknesses ranging from 50 Å to 250 Å with a 50 Å titanium adhesion layer in all cases were examined. These thicknesses were compatible with inverted live-cell phase-contrast microscopy.…”

Cell Co-culture Patterning Using Aqueous Two-phase Systems

“…Our lab has demonstrated this capability by printing arrays of cells to study cell migration in a wound healing assay and to examine the effects of juxtacrine and paracrine signaling in the differentiation of embryonic cells 16,17,20 . Other methods, including patterning of extracellular matrix 28 , inkjet printing 29 , and patterning by laminar flow in microfluidic devices 25,26 have also been used to localize cells. These other methods are effective approaches to achieving well-defined patterns of cells and can often achieve single-cell precision.…”

Spatial confinement instigates environmental determination of neuronal polarity

“…Bare regions of the gold substrate were backfilled with an amide-linked glycol-terminated alkanethiol. 8 The background glycol-terminated alkanethiol monolayer is well established to resist non-specific protein adsorption and is capable of confining cells to well-defined patterns. 7,9 Laminin, a common extracellular matrix protein found in vivo and used in neuronal culture because it promotes neurite outgrowth in a non-directive manner, was non-specifically adsorbed to the hexadecanethiol to produce a series of laminincoated starburst patterns.…”