Tailor-Made and Biodegradable Vascular Scaffolds - Microsurgery: Advances, Simulations, and Applications - page 290

with trypsin/EDTA. These cells were seeded on scaffolds and
maintained with the same media.

8.7.6.2 Cell seeding and culture on scaffolds
Sheet-like PLCL scaffolds were developed in the same manner as
described using the MSP. After oxidization and washing with 70%
ethanol, the scaffolds were dried. During drying, HuMedia EG2,
carbonic acid buffer and HEPES, were added to neutralize the 0.5%
type I collagen solution. The dried scaffolds were coated with neutral
collagen under decompression for 5 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 six-
well plate. Over 4 days, HUVECs were cultured in wells with scaffolds.
(Cells were seeded on the irst and third day. Number of cells: 2.67 ×
10 5 /well, 5% CO 2 )

8.7.6.3 Observation of viable cells on scaffolds

An inverted optical microscope (IX 71, Olympus Co. Ltd., Japan) was
used to observe HUVECs on scaffolds with a conventional luorescent
system. 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: 10

×

). SYTO 13 Green-luorescent nucleic acid stains was used to
observe cellular status on the scaffolds. The excitation and emission
wavelengths of SYTO 13 dye in the presence of DNA were 488 nm
and 509 nm, respectively.
Figure 8.27 shows a luorescent micrograph of nucleic acids of
HUVECs on a scaffold. White sites indicate nucleic acids of HUVECs.
Because the sheet-like scaffold has 3D porous structure, some cells
were observed out of the focal plane. Although not as clear as SEM
images, contours of pores can also be observed from this image.
Black sites around the center of the igure indicate residual ferrite
particles. We believe these ferrite particles have little damage
on cell growth and morphology because some groups have been
encapsulating ferrite particles inside living cells [40, 45]. From this
image showing coherent and viable HUVECs all over the scaffold,
at least we can say that that scaffolds developed by our fabrication
method is safe for cell culture.