Biomedical Engineering Reference
In-Depth Information
Using this system, Toner and colleagues showed that primary hepatocytes from rat liver in serum-
free medium attach selectively to the collagen areas and do not spread onto the albumin areas for
the irst 12 to 24 hours (
Figure 2.33b
and
d
). On the other hand, 3T3-J2 ibroblasts did not respect
the collagen/albumin template and attached nonpreferentially to all collagen and albumin-coated
areas (
Figure 2.33c
and
e
), presumably because ibroblasts can attach to their own, abundant ECM
protein secretions. Hence, a micropatterned coculture of hepatocytes and ibroblasts can be achieved
by seeding ibroblasts
ater
full attachment and spreading of the hepatocytes.
Obviously, the method is not restricted to patterning cocultures only—the nonadhesive
background may remain bare of cells. Using a litof process similar to that of Toner's group's,
Helen Buettner and colleagues at Rutgers University made micropatterned stripes of laminin
(chemisorbed to an aminosilane SAM via a glutaraldehyde linkage) on a physisorbed albumin
background to study nerve growth cone dynamics. he mean outgrowth length along 20- or
30-μm-wide laminin stripes was observed to be smaller than that on uniform laminin surfaces,
whereas outgrowth direction was strongly biased in the direction parallel to the stripes.
he litof, solvent-based patterning strategy outlined in this section deserves further com-
mentary. In essence, it is based on the removal of photoresist with a strong organic solvent while
exposing the protein pattern to the solvent. his raises two important concerns. First, many poly-
meric materials used in tissue culture (e.g., polystyrene) are attacked by organic solvents and
must therefore be ruled out as the substrate. Second, proteins undergo partial denaturation when
exposed to most solvents. Notwithstanding, the work reviewed previously demonstrates that
cells attach, spread, grow, and function on denatured ECM proteins, presumably because the
degree of denaturation does not mar integrin binding to the ECM peptide fragments (see Section
2.1.3). his is not surprising in view of the fact that such denatured micropatterns, which are rou-
tinely visualized by secondary immunoluorescence, feature ainity binding with their natural
antibody. However, other long-term functions, more sensitive to the state of denaturation of the
underlying ECM protein or to the presence of residuals from the acetone-stripped material, might
be impaired by this culture technique. Also, the method cannot be combined with other biomol-
ecules such as antibodies that are irreversibly damaged by the solvent. In 1997, A. H. Bates and
coworkers from the Western Regional Research Center reported a creative solution to this prob-
lem: the biomolecular layer was irst immobilized over the whole substrate, covered with a thin
layer of sucrose (a well-known stabilizer of the tertiary structure of proteins on drying) to protect
it from subsequent processing, and dried. hus, a layer of sucrose-covered immobilized antibod-
ies could be later coated with photoresist without damage; the photoresist was then patterned by
UV exposure and dissolved on the exposed areas, leaving the sucrose-protected layer exposed.
Ater the sucrose was dissolved in water, the exposed antibody could be removed by a brief oxygen
plasma etch. he photoresist/sucrose-protected antibody could be nondestructively uncovered by
dissolving the photoresist in acetone and the sucrose in water. Finally, the antibody micropattern
was shown to retain its full immunoreactivity. No cellular applications were reported.
Most interestingly, it appears that the chemisorption procedure may be skipped altogether, at
least for certain conditions and cell types. What happens if one performs the protein immobi-
lization procedure devised by Toner's group but, against conventional wisdom, skips the silane
derivatization and the glutaraldehyde steps and exposes the (now physisorbed) protein to solvent
to lit of the photoresist? his is exactly what Bruce Wheeler and colleagues. hen at the University
of Illinois at Urbana-Champaign did, with remarkable success. hey created PDL patterns on glass
by blocking designated areas with a photoresist pattern and physisorbing PDL on the exposed
areas; interestingly, removal of the photoresist by sonication in acetone did not result in complete
removal of the physisorbed PDL nor compromised its cellular attachment function (
Figure 2.34
).
hey seeded B104 neuroblastoma cells, a cell line shown to possess neuron-like properties such as
electrical excitability, bipolarity, and neurotransmitter production, and to be induced into difer-
entiation by dibutyryl-cyclicAMP, an agent which also stops their proliferation. he cytophobicity
of glass was found to be superior than that of a phenyltrichlorosilane (PCTS) SAM; whereas com-
pliance of somata attachment and neurite growth to the PDL pattern was overall poorer than that
