Page 546 - IJB-10-3

International Journal of Bioprinting Engineered 3D-printed PVA vascular grafts

metallic channel served as the platform input and was (ii) ATP production assay: ATP production is measured
connected to the feeding glass via a flexible hose (Figure 8). through luminescence produced via a reaction
The larger metallic channel represented the platform between luciferin and luciferase, catalyzed by ATP.
output and was linked to a collector and a vacuum pump The CellTiter-Glo 2.0 Viability Assay (Promega, USA)
using another flexible hose (Figure 8). Both ends of the was used in this study. To measure luminescence, the
PVA graft were secured onto the metallic channels using cells were plated in solid white 96-well plates with a
acrylic resin. flat bottom (Corning, USA) and treated with graft

To initiate the water flow, the vacuum pump was extract according to the protocol described above.
activated, causing water to move from the feeding pump to Following the incubation time, the cell samples were
the collector (feeding glass-hose-platform-hose-collector). prepared according to the producer’s specifications,
The vacuum pump was set to 40 mbar to simulate normal and the luminescence was measured accordingly.
vasculature conditions (resembling small veins). The (iii) LDH release assay: Following the incubation
water collected in the collector was then used to derive the period, 50 µL of the supernatant in each well was
velocity and fluid flow. collected and added to a clean well in a new 96-
well plate to measure LDH released by cells. LDH
2.8. Biological assessment is a cytosolic enzyme that acts as a catalyst in the

2.8.1. Extract method metabolism of lactate to pyruvate. LDH is released
The extract was prepared according to ISO 10993-5:2009 in the extracellular medium due to cell membrane
(Biological evaluation of medical devices; Part 5: Tests for breakages. Hence, extracellular LDH level is a
in vitro cytotoxicity) and ISO 10993-12:2012 (Biological measure of cell death following necrosis. To measure
evaluation of medical devices; Part 12: Sample preparation released LDH following the cells’ exposure to the
and reference materials). The previously sterilized samples extract, the CyQUANT LDH Cytotoxicity Assay
were immersed in a complete culture medium at a ratio (Invitrogen, USA) was used. Samples were prepared
established according to ISO standards. The samples were according to the producer’s specifications and the
incubated for 24 h at 37°C, under orbital shaking (50 absorbance was measured at 490 nm.
rpm) inside a thermostatic bath. Following the incubation 2.8.2. Direct contact method
period, the extraction medium was separated from the Vascular graft samples were immersed overnight in
samples and filtered using a 0.22-µm pore membrane. The complete cell culture media to remove any possible
prepared medium was used for cell treatment. The cellular cytotoxic residues resulting from the collection method.
substrate was prepared by seeding 5000 cells/well inside a Following this, 100,000 cells/well were seeded in 24-well
96-well plate. The cells were incubated for 24 h to facilitate plates and incubated for 24 h to facilitate attachment.
their attachment. Following the incubation period, the A graft sample was then directly added into each well
culture medium (the supernatant) was removed from the and incubated for another 24 h in standard conditions.
wells and replaced with the extraction medium. The treated Thereafter, optical microscopy images of the cells were
cells were incubated in standard conditions for another 24 captured, and cell viability, ATP production, and LDH
h. Thereafter, optical microscopy images of the cells were release were quantified as follows:
captured, and cell viability, adenosine triphosphate (ATP)
production, and lactate dehydrogenase (LDH) release were (i) MTT viability assay: Following the incubation
quantified as follows: period, the samples and medium were removed and
replaced with MTT solution (Serva Electrophoresis
(i) MTT viability assay: Following the incubation Gmbh, Germany) in complete culture medium (10%
period, the extraction medium was removed and 5 mg/mL MTT) and incubated for 2 h in standard
replaced with MTT solution (Serva Electrophoresis conditions. Following incubation, the supernatant
Gmbh, Germany) in complete medium (10% 5 was removed, and the resultant formazan crystals
mg/mL MTT) and incubated for 2 h in standard were solubilized using DMSO. The absorbance of the
conditions. This method measures cell metabolism samples was determined at 570 nm.
following the transformation of the MTT tetrazolium
salt into formazan crystals. Cell metabolism is (ii) ATP production assay: Following the incubation
directly proportional to its viability. Following the period, the samples were removed from each well.
incubation period, the supernatant was removed, ATP production was measured using CellTiter-Glo
and the resultant formazan crystals were solubilized 2.0 Viability Assay (Promega, USA) according to the
using dimethyl sulfoxide (DMSO). The absorbance producer’s specifications, and the luminescence was
of the samples was determined at 570 nm. measured accordingly.

Volume 10 Issue 3 (2024) 538 doi: 10.36922/ijb.2193