Micropatterning of Substrates and Cells - Introduction to BioMEMS - page 81

Substrate
(silicon dioxide on
glass coverslip)
Spin on photoresist

I

II

UV light

Mask

Expose to UV light

III

IV

Develop photoresist

V

Deposit Poly-D-Lysine
(physisorb)

Sonicate in acetone to
remove photoresist
Result: Poly-D-Lysine pattern on
silicon dioxide substrate

100 µm

VI

FIGURE 2.34 Micropatterns. of. neuroblastoma. cells. using. liftoff. and. physisorbed. PDL.. (From.
Joseph.M..Corey,.Anna.L..Brunette,.Michael.S..Chen,.James.A..Weyhenmeyer,.Gregory.J..Brewer,.
and. Bruce. C.. Wheeler,. “Differentiated. B104. neuroblastoma. cells. are. a. high-resolution. assay. for.
micropatterned.substrates,”. J. Neurosci. Methods .75,.91-97,.1997..Reprinted.with.permission.of.
Elsevier..Figure.contributed.by.Bruce.Wheeler.)

achieved for hippocampal primary neurons on PCTS SAMs, it was improved substantially by the
addition of dibutyryl-cyclicAMP and represented a quick assay to evaluate the attachment selec-
tivity on candidate materials and to predict the substrate's suitability for primary neuron patterns.

2.6.2.2 Selective Photochemical Immobilization of Proteins
In 1996, Yukio Imanishi's group from Kyoto University pioneered the use of photoreactive growth
factors. As an example, photoreactive insulin was synthesized by coupling with azidobenzoic acid.
Insulin photoimmobilized onto tissue culture polystyrene enhanced the growth of anchorage-
dependent cells such as Chinese hamster ovary cells and mouse ibroblasts, with greater mito-
genic activity than free insulin. Micropatterns of insulin were created on PET by exposing the
substrate to UV light through a mask in the presence of a photoreactive insulin solution. he
immobilized insulin did not enhance cell attachment but transduced a growth signal to the cells.
When the medium was depleted of serum, cell growth was observed only for the cells on immo-
bilized insulin. he researchers also synthesized photoreactive polyallylamine by coupling with
N-[4-(azidobenzoyl)oxy]succinimide and grated it onto polystyrene by UV irradiation. Next,
the azidophenyl-derivatized polyallylamine was conjugated with mouse epidermal growth factor
(EGF). hus, photoreactive EGF was micropatterned onto polyallylamine-derivatized polystyrene
by UV illumination through a mask. Although cells attached everywhere across the substrate,
seeding at low densities resulted in segregated cellular stripes when the stripes were far apart (~100
μm) because cell growth was only observed in the EGF-immobilized areas. When pattern widths
smaller than the cell were used (~2 μm), patterned cell growth did not occur because all cells pro-
liferated. Regardless of the mechanism used to immobilize the proteins and the cells, Imanishi's
work introduced the novel concept of micropatterning a growth factor.

2.6.2.3 Removable Microfabricated Stencils
he abovementioned masking strategies for blocking protein chemisorption onto the back-
ground require chemical dissolution of the mask (e.g., the photoresist pattern) ater the protein