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International Journal of Bioprinting Printing organoids in peptide matrices

and high), FIB (low), LAM (low and high), Matrigel, and were observed. Cells grown in 2D control, parent (high),
2D control reported increasing CRC cell proliferation LAM (high), and FIB-LAM have more lumens in one
rate each day. In contrast, FIB (high) and FIB-LAM colony. Single-lumen organoids are found primarily on
demonstrated a decrease in proliferation rate from day 1 to the peptide hydrogels of parent (low) and FIB (low). These
4 that was recovered on day 7. two peptide matrices better mimic the morphology of the
These results corroborate that our peptide matrices lumen as those in Matrigel compared to other peptide
are biocompatible and do not induce dysregulation of matrices. Notably, cells grown in peptide hydrogels present
the proliferative potential of the cells to be fabricated cells that are misaligned and sometimes stacked in two
into organoids. Moreover, we presume that the change or more layers but still pertain to a single lumen. This
in organoid morphology may be a result of the changes is contrary to the uniform, single-stacked morphology
in stiffness between parent peptides, FIB-containing observed in Matrigel. We observed that colonies cultured
peptides, LAM-containing peptides, and Matrigel; it is in parent (low) and FIB (low) peptides resulted in fewer
known that cell morphology will vary according to the colonies that presented a multi-layer shape, whereas
stiffness of the 3D scaffold in which they are embedded. LAM-containing hydrogels presented a higher degree of
55
Moreover, matrix relaxation and changes in the ECM multilayering and misalignment.
force facilitate crypt formation, epithelial-mesenchymal Since the architecture and polarity of the lumen is
56
transition, and differentiation. Further studies could a significant part of CRC organoid, cell–cell junctions
42
57
develop a correlation between the morphological state of and apical proteins were labeled by immunofluorescence
the SW1222-derived organoids and their gene expression staining on days 4 and 7. Cell–cell junctions were
profile, 58,59 and elucidate dysregulated pathways when distributed primarily on the lateral side of all cells within
cells are cultured in hydrogels with various mechanical each colony, as observed in the Matrigel control (Figure
properties. However, given the positive results of the 4). Expression of pan-cadherin cell–cell junction protein
viability assay, we postulate that the presented peptide in apical and basolateral membranes points toward
hydrogels must be within a suitable range of stiffness disorientation of the cell polarization, as seen in the parent
for cell growth. Various studies pointed towards using (low) peptide at day 7. Interestingly, colonies cultured in
matrices within the ranges of 300–1000 Pa for the culture parent (high) peptide do not exhibit such expressions even
of human adult colorectal organoids 42–44 and up to 5.5 kPa on day 7, suggesting the inhibition of self-organization of
for patient-derived xenografts (PDX) of CRC organoids. 60 cells into an organoid. Decorated peptides can induce pan-
After determining that the presence of biofunctional cadherin expression starting at day 4. However, the radial
sites in the matrix may play a role in the organoid cell arrangement in the LAM-containing hydrogels is less
formation process, we tested the organoid-forming prominent by day 7. Polarization of the colonies was also
capacity of all peptide combinations. In this regard, we characterized by the observation of apical markers ZO-1
developed a personalized segmentation model based on and ezrin (Figures 5 and 6). On day 4, all biofunctionalized
Cellpose 2.0 and performed morphology analysis. Initially, peptides, FIB (low), LAM (high), and FIB-LAM, have
we analyzed the percentage of colonies that have a lumen ezrin and ZO-1 expressed at the lumen apical epithelium,
and the circularity of the lumen (Figures 3, S5, and S6, consistent with Matrigel’s organoids. However, not until
Supporting Information). Matrigel reported the highest day 7 did cells in parent (low) express the apical protein
colony circularity index and percentage of colonies with biomarkers. Neither cells in the 2D control nor parent
a lumen. Therefore, it is considered a positive control for (high) have a fluorescent signal, displaying the clear edge
this analysis. of the lumen for ezrin or ZO-1. The results indicate that
only CRC cells in matrices with biofunctionalized peptide
After the efficiency analysis of organoid formation, FI
(low) and LAM (high) were selected as potential peptide synchronously express apical protein biomarkers with the
cells in Matrigel.
matrices for CRC organoid formation. These two peptides
and the mixture FIB-LAM were further evaluated to Next, we characterized the gene expression levels of
elucidate the effect of each biofunctionalization and their organoids cultured in the selected matrices to elucidate some
combination. Cytoskeleton and nuclei staining were used cell differentiation patterns. We evaluated the expression of
as fluorescent markers to better visualize the internal cell genes LGR5 (stemness), EpCAM (epithelial cells), CHGA
organization and lumen development (Figure 4). CRC (enteroendocrine cells), and MUC2 (goblet cells) and
cells embedded in Matrigel are considered the positive normalized its expression to the expression in Matrigel.
biological control, where a large, single lumen in the As observed in Figure 7a, significant overexpression of the
center, and cells aligned at the periphery around the center LGR5 gene is observed in all peptide hydrogels except in

Volume 10 Issue 5 (2024) 350 doi: 10.36922/ijb.3033