Biomedical Engineering Reference
In-Depth Information
methacrylate (poly-HEMA) by metal evaporation through a 5-μm-thick copper sten-
cil mask containing feature linewidths of approximately 20 μm. he shape of single
cells conined to spread on an island was constrained dramatically to the shape of the
island. By luorescently staining for actin and vinculin 3T3 cells spread on circle-,
triangle-, or line-shaped islands, they proved in a follow-up article in 1990 that cyto-
skeletal organization depends on island shape, for example, the focal points tended to
accumulate to the periphery of the islands, whereas the total focal adhesion area was
independent of the shape of the cell (see Section 6.2.1).
2.5.2 Cells on SAM Micropatterns
In a seminal article irst-authored by a young David Kleinfeld, Philip Hockberger's group (then
at AT&T Bell Labs in New Jersey) created the irst micropatterns of SAMs, and for the purpose
of patterning the outgrowth of neurons. As explained in Section 2.3.1, they created patterns of
alkyl-trichlorosilanes on an aminoalkyl-trimethoxysilane background [with either aminopro-
pyl (A-P), ethylenediaminepropyl (EDA-P), diethylenetriaminepropyl (DETA-P), or acetylated
EDA-P functionalities] using photolithography (
Figure 2.7
). Here, we discuss in detail their
experimental rationale, now somewhat outdated, as it provides many valuable lessons for the
BioMEMS student.
Kleinfeld et al.'s choice of aminosilanes as a cell-adhesive surface was stated as an attempt to
mimic poly-d-lysine (PDL), a poly-amino acid containing amino side groups, which is a widely
used adhesive surface coating in neuron culture as an inexpensive mimic of ECM. Surfaces
derivatized with diamines (EDA-P) and triamines (DETA-P)—but not monoamines (A-P)—
did, indeed, promote adhesion of embryonic mouse spinal cells and perinatal rat cerebellar cells,
and morphology was assessed to be very similar to that of cultures on PDL.
he goal of using methyl-terminated alkylsilanes was “to recreate the hydrophobicity of certain
surfaces which inhibit cell adhesion.” his is also arguable: as we will see, other researchers have
created cellular micropatterns in which cells attach precisely to methyl-terminated areas. his
discrepancy can probably be explained by the fact that cellular adhesiveness is highly dependent
on a number of parameters, including cell type and medium composition. Indeed, even in their
own system, Kleinfeld et al. observed that the alkylsilane-derivatized areas inhibited neuron
adhesion only if serum was present in the seeding medium. Patterns of the cerebellar cells were
preserved for at least 12 days, which allowed for the development and observation of electrical
excitability. In addition, the diferent behavior of monoamines with respect to diamines and tri-
amines cannot be evaluated without taking into account the completeness of the monolayer and
that a covalently bound layer of hexamethyldisilazane, a photoresist adhesion-promoter used in
their study, was not removed from the surface before aminosilane modiication.
Using Kleinfeld et al.'s method, Geofrey Moores' group at the University of Glasgow showed
in 1992 that ibroblast-like BHK cells attached and aligned to aminosilane patterns on a methyl-
silane background (see
Figure 2.8
). In view of the fact that cell adhesion and spreading were
greater on the aminosilane and untreated surfaces, they hypothesized that “adhesive factors
such as ibronectin” present in the medium (containing 10% calf serum) physisorbed preferen-
tially to those regions. hey also quantiied the evolution of BHK ibroblast adhesiveness con-
trast as a function of exposure to serum-containing medium, and found that it was reduced by
approximately 60% in 24 hours. In addition, it was observed that cells tended to align to and
elongate along parallel tracks of dimethyldichlorosilane on a bare glass background when the
grating period was large (>4 μm).
Hockberger's group later found that neuroblastoma cells, osteosarcoma cells, or ibroblasts
attached preferentially to the aminosilane areas (independently of the type of alkylsilane in the
background) using patterned SAMs of alkylsilanes (either
N
-octadecyldimethylchlorosilane or
dimethyldichlorosilane) and
N
-(2-aminoethyl)-3-aminopropyl-trimethoxysilane. he attachment
