Page 172 - IJB-9-1

International Journal of Bioprinting Lumen-forming colorectal cancer organoids

pumps’ system allows for a specific flow rate for each solution, and therefore a suitable environment for cellular growth.
thus resulting in a more consistent printed structure. The Extracellular matrix is characterized by the presence of
DOBOT Magician robotic arm with a modified head was collagen, which forms an intricate network of fibers that
used to mount the in-house-designed and -fabricated nozzle. support cell growth [18,28,29] . Both the IIFK and IKVAV
The nozzle used was custom designed to have three inlets motifs are amphiphilic in nature and have been reported
and one outlet with a 0.5 mm inner diameter. Two of the to be able to form fibers in solution [31,32] . Interestingly,
inlets are for the peptide solution and 1× PBS to merge and longer IDP peptide displays self-assembling characteristics
form the hydrogel, and the third is for cell dispensing. Flow into nanofibers as well, which may be attributed to the
rates of 50 µL/min, 25 µL/min, and 15 µL/min were used amphiphilic nature of IKVAV, along with the gradient of
for the peptide solution, PBS, and cells, respectively. Printing hydrophobicity of peptide IIFK.
files were designed with SOLIDWORKS and transferred to In addition, we investigated the secondary structure
Repetier-Host to obtain the G-code for 3D printing with the of the two peptides and the peptide mixture by using CD
robotic arm. spectroscopy (Figure 2E, Figure S3). The CD spectra of IIFK
2.14. Statistical analysis showed a gradual structural transition from a disordered
Statistical analysis was performed by using the one-way to a partially ordered structure at low concentrations. The
analysis of variance (ANOVA) and two-way ANOVA spectrum at 1 mg/mL showed a negative peak around 195
tests. Significance was evaluated using Fisher’s LSD and nm, which is characteristic of random coil structure, while
considered to be statistically relevant below P = 0.05 (*) and the spectrum at 4 mg/mL showed a positive peak around
P = 0.01 (**) using OriginPro (Origin 2022b version 9.9). 195 nm and a negative peak around 220 nm, which is
typical of β-sheet. A transition between the two structures
3. Results is evident at 3 mg/mL. A gradual structural transition from
a disordered to an ordered structure was observed for IDP
3.1. Physicochemical characterization of laminin- at 1 mg/mL, 2 mg/mL, and 3 mg/mL; a similar transition
like biofunctional peptide was observed for IIFK:IDP (1:1 molarity ratio) at 1 mg/mL,
The self-assembling peptide IIFK was considered for use in 3 mg/mL, and 4 mg/mL. The observed transition suggests
this study based on the previously reported positive effect a structural transformation in the peptide from random
on cell expansion . IIFK is composed of a hydrophobic coil to β-sheet since the peak that is around 195 nm (π
[14]
tail from nonpolar amino acids (Ile, I and Phe, F) and a π ) is characteristic of random coil structure, while the
*
positively charged amino acid (Lys, K) at the C-terminal, maximum around 200 nm (π π ) and the minimum around
*
which gives this peptide its amphiphilic nature. This 220 nm (n π ), seen in both IDP and peptide mixture at the
*
amphiphilic property enables the self-assembly of IIFK highest concentrations, are typical of antiparallel β-sheet .
[33]
into ordered supramolecular nanofibers. A combination of Therefore, we can appreciate how the combination of IIFK
the tetrapeptide IIFK and the IKVAV motif with a linker and IDP provides a different secondary structure milieu
was rationally designed and investigated for its gelation compared to individual elements.
capability (chemical structures in Figure 2A). Inverted
vial tests were performed to monitor the critical gelation We proceeded with the analysis of the rheological
concentration (CGC) and gelation time for IIFK, IKVAV, properties of these materials. Based on the time sweep test
and the new IKVAV-derived peptide (IDP), respectively performed, we confirmed that these peptides are able to
(Figure 2B). The gelation results (Figure 2B, Figure S2) form gels with tunable mechanical properties. The stiffness
showed that IDP has a CGC of 2 mg/mL, while in of different concentrations of IIFK and the combination
combination with IDP (1:1), it increases to 3 mg/mL. The of IIFK and IDP was examined by measuring the storage
gelation time of IDP at CGC is 20 s, while the mixture modulus (G’). The mechanical stiffness of these two
decreases its gelation time to 10 min. hydrogels increases as the final concentration increases
(Figure 2F, Figure S4). When compared with each other,
Furthermore, SEM micrographs and AFM topography IIFK by itself had higher G’ values than when it is combined
performed on the three peptides showed a nanofibrous with the modified IIFK. The rigidity of both hydrogels
morphology (Figure 2C and D). Table 1 shows the was within the rigidity range that supports continuing
characteristic AFM lengths of the fibers formed by IIFK, multipotency maintenance [15,32] and bioprinting .
[14]
G3, and the mixture of the two. We can observe how the
morphology of the fibers from IIFK and IIFK:IDP (1:1) 3.2. Cell viability and proliferation of CRC cells in
Mix resembles each other more compared to the fibers biofunctional hydrogel scaffold
from IDP. This sort of self-assembly into fibrous structure A screen study was performed to investigate the
is advantageous as it has the ability to form hydrogels biocompatibility of the different peptides and their effect

Volume 9 Issue 1 (2023)olume 9 Issue 1 (2023) 164 https://doi.org/10.18063/ijb.v9i1.633
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