The cell-coated mesh nanoES was then gently lifted from the SiO 2 substrate using
fine forceps, rolled onto a polystyrene or glass tubular support 1.5 mm in diameter,
then maintained in incubator for another 2 weeks for maturation of the vascular
structure (Fig.4.4e). For pH sensing experiment, the temporary tubular support was
removed, and segments of polystyrene tubing were connected to the open ends of
the vascular construct (Fig.4.4f), and a PDMSfluidic chamber with input/output
tubing and Ag/AgCl as shown in Fig.4.4h.
4.2.4 Staining
Immunostaining: First, cells werefixed with 4% paraformaldehyde, pre-blocked by
1% bovine serum albumin (BSA) in PBS and permeabilized by 0.2–0.25% Triton
X-100 and 1% bovine serum albumin (BSA) in PBS. Second, cells were incubated
with primary antibodies in 1% BSA in 1X PBS with 0.1% (v/v) Tween 20 (PBST)
overnight at 4 °C. Finally, cells were incubated with the secondary antibodies with
fluorophores. Hoechst 34580 was used for staining nuclei. Neuronal class III
b-Tubulin (TUJ1) mouse monoclonal antibody, anti-a-actinin mouse monoclonal
antibody, anti-smooth musclea-actin rabbit polyclonal antibody and corresponding
secondary antibody were used for staining neuron, cardiomyocytes and HASMC.
Labelling nanoES/tissue scaffold: SU-8 resist for nanoES fabrication was
doped with rhodamine 6G before deposition and patterning. PLGA electrospun
Fig. 4.4 Schematic of vascular nanoES construct preparation and pH sensing.aA free-standing
mesh-like nanoES.bIndividual devices were wire-bonded to PCB connecters.cA modified
petri-dish wasfixed over the scaffold with silicone elastomer.dThe hybrid scaffold was sterilized
with UV-light illumination for 1 h and soaking in 70% ethanol solution for 0.5 h, coated with
fibronectin/gelatin solution overnight and seeded with HASMCs.eAfter 7–14 days in culture, the
HASMC-seeded nanoES (d) was rolled against a tubular support and cultivated for further days.
fThe tubular support was removed and tubing was connected to the ends of the lumen of the
HASMC construct.gThe medium was removed while keeping the construct moist.hA PDMS
chamber was assembled around the construct, attached to tubing to bathe the outside of the
construct and Ag/AgCl electrodes to measure pH in the bathingfluid
4.2 Experimental 45