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International Journal of Bioprinting Printing organoids in peptide matrices

obtained by a standard rheological time sweep using at cultures were used as controls. Round cell colonies can be
least six replicates in five repetitions (Figures S2 and S3, observed in parent (low) and all biofunctionalized peptides,
Supporting Information). With these measurements, we either with FIB, LAM, or both (Figure 2a). These colonies
aimed to investigate the stiffness of the hydrogels. We were located in a different layer from the well plate bottom,
observed a tenfold change in the hydrogel’s moduli when where cells grew in a 2D fashion. This was evident by the
the concentration of IIFK increased from 1 to 3 mg/mL co-presence of the peptide background autofluorescence
(Figure 1d). Yet, the storage modulus is roughly maintained on the same focal plane as the organoids and surrounding
at 1 kPa using IIFK at 1.0 and 1.5 mg/mL. It has been them in all directions (not displayed). In addition,
reported that organoid growth is enhanced at stiffness the presence of round colonies with a blurred outline
values within the range of 0.2–1.3 kPa. 42,51,52 Therefore, IIFK corroborated their location being in a different focal plane
concentration was kept at 1.0 mg/mL for subsequent studies. than the well bottom. Furthermore, we observed colonies
Additionally, these results suggest that incorporating the with either round (e.g., colonies found in FIB [high] on day
FIB peptide (0.25–1.0 mg/mL) could reduce the storage 7) or ellipsoidal shapes (e.g., colonies found in FIB-LAM
modulus values of the parent peptide (1.0 mg/mL). on day 7), as well as shapes that do not fit under these two
In comparison, mixing the LAM peptide with the parent categories (e.g., colonies found in parent [high] on day 7)
peptide resulted in a hydrogel with similar mechanical in all the layers.
properties to IIFK when the content of LAM is less than We presume this change was due to the stiffness of the
the concentration of the parent peptide (1.0 mg/mL) while material (5 kPa; a fivefold increase compared to the parent
exceeding the concentration of parent peptide with LAM [low]). At day 7 of organoid culture, higher organoid
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peptide still formed a hydrogel and resulted in a hydrogel density was observed in Matrigel compared to peptide
with approximately three times the stiffness, i.e., 2224 kPa culture (Figure 2a). However, round organoids were still
for LAM (high) (Figure 1f). We also noticed a shift in the loss observed in the peptide culture, indicating the potential use
modulus after reaching the apparent critical concentration of these peptide hydrogels for CRC organoid formation.
of the mixture. We observed that the loss modulus values We also observed the growth of cells in a 2D control
remained constant for both FIB and LAM mixtures before conformation in peptide hydrogels, indicating that cells
reaching the 1 mg/mL concentration. We presumed the can precipitate into the bottom of the wells and grow in the
change in assembly dynamics may drive this phenomenon. interphase between the bottom and the peptide hydrogel.
Based on copolymerization models, one can also infer Consequently, a viability assay was performed on
that the kinetic constant of dimer formation will define all peptide concentrations to determine whether the
the coaggregation of different peptides in a mixture. Thus, biofunctionalized peptide hydrogels were cytotoxic. We
the relative concentration of the parent and biofunctional noticed that SW1222 cells seeded in all of the peptide
peptides will result in fibers with different compositions. matrices have a large viable population on all days of
This may also explain such counterintuitive behavior.
culturing (Figure 2b). Moreover, these viable populations
We evaluated the gelation time using an inverted vial seem to have a comparable density to those cultured in
test (Figure S1, Supporting Information). The parent Matrigel and the 2D control. No cytotoxic effect was detected
peptide gelated at 1.5 and 2.0 mg/mL after 3 and 1 h of throughout the culture time. These results demonstrate that
initiating gelation, respectively. We had to increase the total all peptide hydrogels supported CRC organoid viability up
concentration of the mixture peptides up to 3 mg/mL for to 7 days of culture. Besides viability, we also monitored the
FIB (low) and LAM (low) mixtures to form a hydrogel and proliferation of SW1222 cells over time (Figures 2c and S4,
up to 4 mg/mL for FIB (high) and LAM (high) to form a Supporting Information). The proliferation assay displayed
hydrogel (Figure S1, Supporting Information). Given the a significantly increased signal on day 7 compared to day 1
slight differences between loss and storage modulus, we for all peptide combinations and controls (Matrigel and 2D
presumed that the resultant gel was not stable enough to hold control). On day 1, FIB (high), FIB-LAM, and LAM (low)
itself within the inverted glass vial. Notably, the conditions had significantly higher proliferation rates than Matrigel.
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in which these hydrogels exist are very close to the critical On day 4, only the peptide matrices with LAM had the
concentration, whereby the parent peptide can form a gel same level of proliferation rate as Matrigel. A higher
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after several hours (Figure S1, Supporting Information). proliferation rate was observed in cells cultured in LAM
(high) compared with Matrigel on day 7. Although the
3.2. Culture of colorectal cancer organoids in other peptides did not have a higher fluorescence intensity
peptide combinations than Matrigel, all the CRC cells encapsulated in different
Organoids were obtained from the culture of SW1222 cells peptides exhibited higher proliferation on day 7 than on
in the peptide combinations, and Matrigel and 2D control day 1. Among all culture conditions, parent peptide (low

Volume 10 Issue 5 (2024) 348 doi: 10.36922/ijb.3033