Biomedical Engineering Reference
In-Depth Information
Maroudas, then at the Imperial Cancer Research Fund in London, devised an original method
to create patterns of parain on tissue culture substrates. A TEM grid wetted with a solution of
parain was placed onto a tissue culture dish and heated to 50°C to 60°C; because of thermal
shrinkage, parain retracted from the tissue culture surface and coalesced under the TEM grid.
he grid could be removed mechanically, and cells were observed to attach only to the resulting
100-μm-square “islands” of tissue culture substrate surrounded by the (nonadhesive) parain
background. Although simple, this method has the limitation that parain cannot easily be pat-
terned by photolithographic methods.
Poly(
N
-isopropylacrylamide) (poly-NIPAM) is a thermoresponsive polymer which is insol-
uble in aqueous solutions above a certain lower critical solution temperature (LCST = 32°C
in pure water and 25°C in pH 7.4 saline). It is possible to culture cells on poly-NIPAM and
then induce cell detachment by lowering the incubation temperature below the LCST point.
Detachment by temperature manipulation is appealing because it circumvents treatment with
supplements such as trypsin, which are damaging to cells. Several groups, many of them in
Japan, have focused on this strategy since the 1990s.
In 1998, Yukio Imanishi and Yoshihiro Ito's groups at Nara Institute of Science and Tech-
nology in Ikoma, Japan, synthesized a random copolymer of NIPAM and acrylic acid (20:1) and
photosensitized it by coupling the carboxylic acids with azidoaniline (a molecule which con-
tains a photosensitive phenylazide group). hus, thin layers of this photosensitized copolymer
can be attached to (and patterned onto) tissue culture polymers by UV irradiation. hey selec-
tively exposed a copolymer ilm on polystyrene to UV light through a chrome mask and washed
away the unexposed copolymer with cold water. Fibroblasts attached onto both the copolymer
patterns and the polystyrene background, but could be detached selectively from the copolymer
areas on switching below the LCST (21.5°C). he concept is ingenious because it is based on
selective
detachment
(rather than selective
attachment
) of cells, presumably mediated by the
high hydration of the substrate. However, selective detachment was observed only when the
ibroblasts were cultured in serum-free medium—it failed when the patterned surfaces were
exposed to a ibronectin solution or serum-containing medium before seeding because ibro-
nectin was able to physisorb onto both the unprotected polystyrene background and the copo-
lymer pattern. Furthermore, the detachment temperature was cell type-dependent (10°C for rat
hepatocytes and 20°C for bovine endothelial cells), which raises reliability questions. To opti-
mize the “attachability” and “detachability,” Yuichi Mori's group at the Japan Research Center
in Kanagawa cast-dried and exposed a mixture of type I collagen and poly-NIPAM solutions
and found that approximately 100% attachability and detachability of human dermal ibroblasts
seeded in 10% fetal bovine serum could be obtained if the collagen content was 4% to 5% and
if the UV exposure was 2000 J/m
2
; it was hypothesized that cross-linking of collagen to poly-
NIPAM occurs on UV irradiation. Sheets of ibroblasts were selectively detached ater 3 days of
culture, resulting in the formation of cellular spheroids, the size of which could be controlled
by changing the pattern dimensions. his work was later extended to a total of 23 cell types, but
data on long-term cultures are still unavailable.
he thickness of the poly-NIPAM layer may be a critical factor. A study in 1996 led by Françoise
Winnik at RIKEN in Saitama, Japan, reported reduced levels (60%-70%) of ibrinogen and ribo-
nuclease A physisorption on ultrathin (<100 Å) poly-NIPAM layers grated onto aminosilane- or
methacrylsilane-derivatized silicon oxide. Because the poly-NIPAM layer could be patterned
by laser ablation through a stainless steel stencil mask, and featured reduced levels (70%-90%)
of cellular attachment compared with bare glass, micropatterns of neuroblastoma-glioma cells
could be demonstrated. However, no temperature efect on protein physisorption or cell attach-
ment was observed within the range of 20°C to 40°C, which suggests that poly-NIPAM does not
undergo LCST phase change when grated to such thin layers.
In 1996, Takehisa Matsuda and Takashi Sugawara at the National Cardiovascular Center
Research Institute in Osaka, Japan, synthesized photoreactive copolymers containing phenylazide-
conjugated monomers (
Figure 2.24
). he phenylazide groups form highly reactive nitrene
