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International Journal of Bioprinting Printing organoids in peptide matrices

to that of the cells cultured in Matrigel. Moreover, the FIB concentrations higher than 6.0 mg/mL. To satisfy the
peptide also allows cell polarization and self-organization mechanical conditions commonly attributed to colorectal
of colonies into an organoid. We also presume that organoid culture, we had to explore the lower limit of these
the decorated peptides may interact with cells, as the bioink concentrations. Hence, we proceeded to evaluate
stiffness change between the low and high conditions the printability of the bioinks at 4.5 and 6.0 mg/mL. To
for FIB and LAM peptides is negligible. Additionally, this end, we used a crescent moon shape and a criss-cross
circularity and lumen presence in each colony results grid design, as depicted in Figure 8a. As can be observed,
were significantly different among concentrations. As both shapes have a better definition using 6.0 mg/mL FIB
previously mentioned, it is also widely accepted that (low) as video 1 and video 2 demonstrate the printing of
the matrix RGD (contained in FIB) ligand is vital for the grid with FIB (low) at 6 mg/mL and LAM (high) at 4.5
organoid development. However, YIGSR (contained mg/mL, respectively.
in LAM) is a ligand known for inducing proliferation. Next, we bioprinted CRC cells and compared
In this case, we were able to detect a slight increase in the colonies’ morphology to determine organoid
proliferation rate until day 7 at FIB (high). Interestingly, characteristics. The culture was maintained for up to 8
we can observe that the percentage of colonies presenting days and was initially analyzed in terms of cell proliferation
a lumen was highly affected by the concentration of FIB and viability (Figure 8b and c). In both peptide mixtures,
peptide, while the LAM motif affected both circularity a high percentage of cell viability was maintained for up
and percentage of colonies per lumen. Given the to 8 days of culture. Single cells were observed on day 1
differences in proliferation between the LAM peptides, post-printing, which started to form the organoid colonies
we presumed that high LAM concentration would induce observed on days 4 and 8 post-printing. This indicates that
cell proliferation. Based on quantitative PCR (qPCR), the printing process did not affect cell viability or their
we can assume that the LAM peptide induces skewed potential to form organoid clusters. Similarly, a significant
differentiation into enteroendocrine cells. We can observe increase in cell proliferation was observed in both peptide
that the differentiation into goblet cells is similar to that bioinks, indicating the biocompatibility of the peptide
in Matrigel. Remarkably, stemness is higher in LAM- mixture for 3D bioprinting and its potential use for
containing peptides but reduced in RGD-containing high-throughput organoid formation for drug screening
peptides. This may be a product of our fiber system and/ applications.
or the result of adding RGD. Increased epithelial integrity
and stemness are also noted in all concentrations, but are We noticed that a higher number of colonies were
significantly higher in FIB (low), pointing further to a present in the FIB bioprinted structure, while the LAM
skewed differentiation. peptide exhibited excess debris inside the structure. We
examined these further by observing the cytoskeleton
3.3. Printability and bioprinting of FIB (high) and using confocal microscopy (Figure 8c). Interestingly,
LAM (low) most of the colonies in the LAM peptide displayed a 2D
We investigated the bioprinting capabilities of the morphology, with various small-sized lumens and no
biofunctionalized peptides FIB (low) and LAM (high). This degree of polarization. The colonies found in the FIB
systematic evaluation focused on their gelation properties peptide presented a reduced number of lumens within
and suitability for generating stable 3D structures. First, the colony and some degree of polarization. The colonies
we evaluated the gelation of a droplet of peptide extruded are also smaller in size than the 2D-like colonies observed
through our in-house bioprinting system. For this test, in the LAM peptide. These morphological characteristics
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we used the concentration of FIB (low) and LAM (high) suggest that organoid fabrication in these conditions is also
as a starting point and subsequently tested increasing feasible, proposing bioprinting as an alternative method to
concentrations. We evaluated combinations of FIB (low) manipulate and fabricate organoids in vitro.
and LAM (high) at different total concentrations, 1.5, 3.0,
and 4.5 mg/mL in the first case and 2.0, 4.0, and 6.0 mg/mL 4. Discussion
in the second case.
In this study, we designed and characterized two new
The retention of the gel is observed in FIB (low) biofunctional peptides that can form nanofibers in
starting at 1.5 mg/mL, while LAM (high) peptide forms a the presence of the parent self-assembling peptide. In
less stable hydrogel at low concentrations. For bioprinting addition, we established various mixtures of peptides and
applications, a concentration of at least 4.0 mg/mL of characterized them. We found that the physicochemical
LAM (high) is recommended. Increased gel stability is properties of the peptides vary according to their
obtained at 6.0 mg/mL. Previous projects have extensively sequence, concentration, and ratio. For instance, the
explored the use of USAPs as bioinks, 22–25 with reported gelation of FIB peptide mixtures is limited by the

Volume 10 Issue 5 (2024) 356 doi: 10.36922/ijb.3033