Page 177 - IJB-9-1

International Journal of Bioprinting Lumen-forming colorectal cancer organoids

area in different peptide hydrogels, assuming for simplicity IIFK when used as a scaffold for CRC cells and compared
that the lumen is circular in shape. Larger lumens were the results to when single peptides were used.
observed in peptides containing IKVAV and IKVAV The gelation and mechanical properties of the new
alone when compared to IIFK. Lumen formation in IDP peptide and its combination with the parent peptide
IIFK:IDP at different layers in 3D was also observed were analyzed using inverted vial test and rheological
(Video clips S1–4). measurements. The parent peptide (IIFK) is known to have

3.5. Characterization of 3D bioprinted construct a CGC of 1 mg/mL so variation in gelation time is expected
Based on the results of the mechanical properties, cell as the aggregation may be modified by the linker and the
viability, proliferation, and lumen formation of CRC cells IKVAV part of the IDP sequence. Interestingly, the CGC
in different self-assembling peptide hydrogels, the mixed of the IDP and IIFK (1:1) mixture and the gelation time
peptide consisting of IIFK and IDP at final concentration increased compared to non-mixed peptides. This might be
of 8 mM and 10 mM with 5 to 1 ratio was used for 3D attributed to the fact that IDP and IIFK, having two different
bioprinting. These concentrations were selected based aggregation mechanisms, tend to compete when both
on observations that a higher concentration of peptide monomers are present in the pre-gel solution.
is needed to produce a performant scaffold in 3D One key feature of this combination is its tunable
bioprinting . Cell proliferation in the printed constructs stiffness, which provides mechanically dynamic matrices .
[14]
[37]
was assessed at days 1 and 7 of culture. Effective cell We tested the effect of three different concentrations, which
proliferation was observed with both concentrations of consequently altered the stiffness of the hydrogels, cell
peptide as the ATP released increased with time of culture. proliferation, and organoid formation. We found that the
Higher proliferation rates were observed when 8 mM novel IDP peptide and its mixture with parent peptide IIFK
concentration of peptides was used and if compared to have positive outcomes, in terms of both cell viability and
Matrigel (Figure 6). proliferation. In fact, the culture of SW1222 cells within
the newly designed IDP peptide scaffold and the IIFK:IDP
4. Discussion mixtures at both concentrations (3.0 and 4.5 mg/mL) showed
Ultrashort self-assembling peptide IIFK in combination comparable viability profile and proliferation rates compared
with IKVAV was used as a hydrogel scaffold to grow to Matrigel, which is the gold standard for organoid culture.
[38]
lumen-containing colonies from CRC cells. This allows It is important to note that other research has suggested
for native ECM mimicking and offers a 3D environment the possibility of IKVAV motif inducing cellular death in
for cells. In order to manipulate the environment to meet human intestinal organoids. However, this phenomenon
the cells’ needs, physical and chemical properties can was not observed in our study based on the cell viability and
be controlled by modifying the peptide sequence to proliferation results. Finally, we used an established adhesion
[34]
[30]
include biofunctional motifs, such as IKVAV. IKVAV is assay to evaluate the effect of the biofunctional motif in the
one of the active sites present in laminin chain α1 (LMα1) IDP peptide. We demonstrated that the laminin motif was
and highly expressed in the invasive phenotype of colon readily available for cell binding in the IIFK:IDP (1:1) mixture
carcinoma . Previous studies have shown that an increase as well as the single IDP scaffold.
[25]
in concentration of IKVAV peptide or laminin-111 would Confocal fluorescence microscopy was also used
increase colonization rates of colon cancer into other to investigate cell morphology and their arrangement.
organs , presumably by the mediation of tumorigenesis Improved organoid formation and increased lumen area
[35]
by LMα1. Moreover, it has been widely accepted that the were observed across the different peptides that contained
IKVAV motif is recognized by integrins α3β1 and α6β1. the IKVAV motif. The stiffness of the hydrogels did not
Integrin α3β1 is required to maintain cell–cell interactions play a key role as no significant difference was observed
that give rise to a normal epithelial morphology . The in organoids and lumen formation in the different
[36]
design of a novel, minimalistic, laminin-like SAP scaffold concentrations tested. The morphology of organoid-like
is important as smart scaffold to provide biochemical cues colonies across the different peptides varied as polarized cell
to cellular constructs, in order to precisely recreate the configurations of organoids were detected in the modified
extracellular environment of native tissues; for instance, peptides and IKVAV. This indicates that IKVAV may have
in this case, colorectal cancer tumor tissue. These findings a positive effect on colonies development and enhancing
provide an indication of the effective preparation of a lumen volume when used in minimalistic matrices.
modified ultrashort, self-assembling peptide hydrogel
that can support CRC cells’ proliferation and organoids’ We subsequently investigated the bioprintability of this
formation. In the current research, we investigated the modified peptide hydrogel. A 3D construct was printed,
effect of IKVAV on our previously developed tetrapeptide followed by cell proliferation investigation. The printed

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Volume 9 Issue 1 (2023)olume 9 Issue 1 (2023) 169 https://doi.org/10.18063/ijb.v9i1.633