Page 168 - IJB-10-4

International Journal of Bioprinting Printing collagen type IV membrane

were in accordance with the Declaration of Helsinki. Each The printing method is more advantageous than
membrane was cut into 3 × 10 mm strips and mounted the classic mold, as it allows direct printing onto other
onto the gripping unit of the clamp. A force ramp was structures with the potential of creating multi-layered
applied at 0.5 N/min until the specimen broke (n = 3). All structures (e.g., printing Col-IV on a Col-I stroma
measurements were performed at RT. The Young’s modulus structure). Therefore, printed membranes were tested in
of the membrane is the slope of the linear elastic part of the the following experiments.
stress-strain curve.
2.9. Wound healing assays
2.6. Scanning electron microscopy analysis of Due to the limited supply of P-HCECs, this experiment
collagen type IV membranes was only conducted with B4G12. Press-to-Seal TM
The crosslinked Col-IV membrane was briefly immersed (ThermoFisher Scientific, USA) was used to adhere the
in 0.9% NaCl, transferred to 2.5% glutaraldehyde, and bioengineered Col-IV or Col-I membranes to 6-well
fixed at 4°C for 30 min. Samples were then washed in plates (each membrane/well). Cells were cultured on the
dH O for 10 min, followed by a series of dehydration steps membranes to full confluence. NETCELL PVA Eye Spear
2
with increasing ethanol concentrations: 50%, 75%, and (Network Medical, United Kingdom [UK]) was used to
90% (10 min incubation per condition), and 100% (twice, gently scratch the well to create a surface wound without
10 min each time). Finally, the sample was immersed in damaging the membranes. Wound areas were monitored
hexamethyldisilane for 30 min before air-drying in the by daily phase contrast images, using an inverted light
fume hood. After the samples were dry, they were sputter- microscope over a week. Cells cultured directly on the
coated with a layer of gold (10 nm) and subsequently wells without membranes were used as controls. At least
imaged at 7000× and 15,000× magnifications using a JSM- eight repeats were used. The images were taken daily,
7500FA LV Scanning Electron Microscope (SEM) (AIIM and Image J was used to measure the wounded area
facility, University of Wollongong, Australia). over time. Statistical analysis was performed using one-
way Analysis of Variance (ANOVA) by GraphPad Prism
2.7. Human corneal endothelial cell culture (GraphPad, USA).
An immortalized human corneal endothelial cell line,
B4G12 (i.e., a kind gift from a laboratory in Germany 2.10. Bioengineering and characterizing the corneal
[details in the Acknowledgements section]), and primary endothelium
human corneal endothelial cells (P-HCECs) were used in Human corneal endothelial cells (HCECs), B4G12,
this study. P-HCECs were isolated from human corneas were passaged from confluent flasks, and 60 μL of 1 ×
unsuitable for transplantation, obtained from NSWTB. 10 cells were seeded onto sterilized Col-IV membranes
5
All procedures were in accordance with the Declaration placed in 24-well plates. Cells were allowed to attach to
of Helsinki. Both B4G12 and P-HCECs were cultured collagen membranes for 45–50 min and maintained as
following the culturing protocol previously published. usual. P-HCECs were also seeded onto the membranes
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The maximum passage number for P-HCECs used in this and cultured as usual. Cell growth was monitored over a
study was three. week of culture through daily phase contrast images taken
using an inverted light microscope. When cells reached
2.8. Cell adhesion tests on collagen type I vs. type 80–90% confluence, human platelet lysate (hPL) was used
IV membranes to replace fetal bovine serum (FBS) in the medium. With
This test was conducted as previously published with the addition of hPL, the full confluent Col-IV membranes
20
the following modifications. Col-IV or Col-I membranes detached from the culture dish.
(molded or bioprinted) were prepared as above, then
+
+
trephined to the size of 6 mm diameter and transferred to Immunostainings of ZO-1, Na /K -ATPase, Ki67, and
96-well culture plates (Corning, Sigma, USA). Chondroitin laminin were performed on the detached B4G12-laden
sulfate (10 mg/ml) and laminin (10 µg/ml) are standard membranes. B4G12 cultured in standard conditions on
coating conditions for corneal endothelial cells and were pre-coated 35 mm petri dishes were used as controls. Due
21
used to coat wells as a comparison group. Wells without to limited cell numbers, P-HCECs on Col-IV membranes
any membrane or coatings were used as controls. B4G12 and standard culturing conditions were only labeled with
cell suspension (100 µL) with a total seeding number of ZO-1 and Na /K -ATPase. In all conditions, cells were first
+
+
48,000 was added to wells and incubated for 60 min, fixed with 10% neutral-buffered formalin (NBF) for 30 min
followed by PBS rinsing to remove loose cells. The total at RT. This was followed by three rounds of 5 min washes
cell number was quantified by lactate dehydrogenase assay in 1× PBS. The cells were then permeabilized in 0.5% Trion
(LDH) as per manufacturer instruction and previously X in PBS for 15 min at RT and then washed three times
published protocol. 20 in 1× PBS (each time for 5 min). After washing, the cells

Volume 10 Issue 4 (2024) 160 doi: 10.36922/ijb.3258