Biomedical Engineering Reference
In-Depth Information
minutes. The collagen-coated scaffolds were kept at 37 ° C for 1 hour
to initiate the formation of collagen gel, after which scaffolds were
ixed onto the wells of a 24-well plate. Over 4 days, HUVECs were
seeded in wells with and without scaffold (on the irst and third days,
number of cells: 0.5 × 10 4 /well at each seeding, 5% CO 2 ).
8.6.3
Observation of HUVECs on Scaffolds
An optical microscope (BX62, Olympus Co. Ltd., Japan) was used to
observe HUVECs on scaffolds with a luorescent system including two
mirror units (blue and green excitation, Olympus), a conventional
broadband illumination source, and an electron multiplying CCD
(Luca EM , Andor Technology Plc., USA). The scaffolds with seeded
HUVECs were stained after being cultured for 4 days. After cell
staining, the scaffolds were exposed to excitation light under the
luorescent microscopic system (objective lens: 40 × ). SYTO 13
Green-luorescent nucleic acid stains (Molecular Probes, Inc., USA)
and phalloidin-rohdamine X (Wako Pure Chemical Industries,
Ltd., Japan) conjugate were used to observe cellular status on the
scaffolds. In general, SYTO dyes stain nucleic acids while phalloidin
conjugates stain structures with F-actin behavior such as actin ibers.
The excitation and emission wavelengths of SYTO 13 dye in the
presence of DNA were 488 and 509 nm, respectively. The excitation
and emission wavelengths of the phalloidin-rohdamine X conjugate
in the presence of F-actin were 556 and 574
nm, respectively
(Fig. 8.12).
Figure 8.12 Fluorescent micrograph of HUVECs on a scaffold. The nucleic
acids and F-actin were stained by SYTO 13 and phalloidin-
rohdamine X conjugate, respectively, after cell culture (4 days).
Green sites indicate nucleic acids of HUVECs and red sites
indicate F-actin.
 
Search WWH ::




Custom Search