Page 171 - IJB-9-1

International Journal of Bioprinting Lumen-forming colorectal cancer organoids

voltage of 3.00 kV. The SEM sample was prepared by density of 40,000 cells/cm and left to incubate at 37ºC, under
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transferring a cell-laden peptide hydrogel onto a glass 5% CO . After 90 min, half of the well plates were fixed with
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coverslip. The gel was then dehydrated by immersing it in formaldehyde 4% for 30 min. The other half was exposed
a series of ethanol solutions of increasing concentrations. to mechanical stress to induce cell detachment. Cells were
The dehydrated gels were then dried in the Automated fixed as mentioned earlier, and all well plates were stained
Critical Point Dryer. The dried samples were sputter coated for 10 min with 4´,6-diamidino-2-phenylindole (DAPI) for
with 5 nm iridium (Ir) before imaging. cell quantification under the microscope .
[30]
2.7. Circular dichroism spectroscopy 2.10. Cell viability and proliferation assessment
Circular dichroism (CD) spectra were acquired at ambient At each time point of culture, the viability of colorectal
temperature (25°C) using the Aviv 430 spectrometer (Aviv cancer (CRC) cells in the 3D hydrogel scaffolds was
Biomedical Inc., Lakewood, NJ, USA). Nitrogen gas was determined by LIVE/DEAD Viability/Cytotoxicity Kit
used to keep the oxygen level around 5 ppm. Rectangular (Invitrogen, Thermo Fisher). The staining solution was
quartz cuvettes with optical path length of 0.1 mm (0.01 prepared with 8 µM ethidium homodimer-1 (EthD-1) and
mm when the dynode value was above 500 V) were used. 8 µM calcein AM in 1× PBS. The cell samples were washed
Data acquisition was performed at 1 nm steps, scanning 3 times with 1× PBS and then incubated in the staining
the wavelength (λ) from 300 nm to 190 nm with a spectral solution for 45 min. After staining, the staining solution
bandwidth of 1.0 nm. The CD signals were further normalized was removed, and the samples were washed 3 times with 1×
to the molar ellipticity value. Aviv 35 CD instrument and PBS. ZEISS Axio Vert fluorescence microscope was used
OriginPro 2017 64-bit software were used for data collection to view the cells. In order to determine the proliferation
and processing, respectively. The CD spectra were smoothed rate of cells, the CellTiter-Glo luminescent viability assay
to remove the background noise, without changing the was used. One hundred microliter (equivalent to culture
signal. The samples were freshly synthesized and prepared medium used with samples) of CellTiter-Glo reagent
by dissolving different concentrations in Milli-Q water. was added into each well and mixed thoroughly. The
samples were incubated for 25 min at room temperature.
2.8. Culture of colorectal cancer stem cells and cell A PHERAstar FS plate reader was used to read the plates.
seeding in 3D hydrogel
Commercial colorectal carcinoma-derived cell line SW1222 2.11. Cytoskeletal staining
was used. Cells were maintained in a fresh medium, Immunostaining was performed using Rhodamine
comprising of Iscove’s Modified Dulbecco’s Medium phalloidin (Invitrogen, Thermo Fisher) for F-actin staining.
(IMDM) (Gibco), supplemented with 1% penicillin and After fixing the cells with 4% paraformaldehyde (PFA)
10% fetal bovine serum (FBS), in a carbon dioxide (CO ) for 30 min, cells were permeabilized with 0.2% Triton
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incubator at 37°C with 5% CO . The culture media were X-100 in 1× PBS for 1 h. The permeabilized cells were
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changed every 3 days. Cells at passages 8 to 10 were used. then incubated in a blocking buffer solution, containing
Cells were split using trypsin, and single cells were obtained 1% bovine serum albumin in 1× PBS, and left overnight.
by running the cells through a 40 µm strainer. Cells were Rhodamine phalloidin (1:40) was added to the cells and
mixed with PBS and loaded to the pre-gel peptide solutions. left for 24 h. Thereafter, the cells were washed with 1× PBS,
Before seeding the cells, the peptides were sterilized by incubated in DAPI for 10 min, and then washed with 1×
UV for 1 h for 3D hydrogel culture. For cell viability and PBS. Finally, the cells were imaged using a laser scanning
proliferation assays, 96-well plate was used to form 100 μL confocal microscope (Zeiss LSM 710 inverted confocal
of 3D hydrogel from the peptide solution (8 mM, 10 mM, microscope).
and 12 mM of IIFK, IDP, and IKVAV each, and a mix of
IIFK with IDP at a 5:1 ratio) with 7000 cells suspended in 2× 2.12. Image processing
PBS. In preparing for cytoskeletal staining, the same method By measuring the diameter of the spots, and assuming
was used to form a droplet construct in a 24-well plate, with that the shape is circular for comparison, ImageJ image
a total volume of 15 μL and 1000 cells. Matrigel and 2D cell analysis software was used to measure the lumen size.
culture were used as positive controls. Media were added to 2.13. 3D bioprinting and construct assessment
each well on culture plates. An in-house-developed printing system was used to test

2.9. Adhesion assays the printability of each peptide hydrogel. The robotic 3D
Upon adding 50 μL of peptide and 50 μL of 2× PBS, a thin printing system consists of microfluidic pumps, a robotic
layer of hydrogel was formed on the surface of a 6-well plate. arm with five degrees of freedom, and a dual-coaxial nozzle.
The peptide was left to incubate at room temperature for Three microfluidic pumps were used in this system: one for
15 min. SW1222 cells were seeded on top of the wells at a the peptide, one for PBS, and the other for the cells. The

Volume 9 Issue 1 (2023)olume 9 Issue 1 (2023) 163 https://doi.org/10.18063/ijb.v9i1.633
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