This work was financially supported by King Abdullah University of Science and Technology. The authors acknowledge KAUST&#39;s Seed Fund Grant and KAUST&#39;s Innovation Fund awarded by KAUST&#39;s Innovation and Economic Development. The authors would like to acknowledge KAUST&#39;s Bioscience and Imaging Core Labs for supporting the biological characterization and microscopy analyses.

1. Buffer and solution preparation
NOTE: All stated concentrations are final concentrations.
<ol>
	<li>Add fetal bovine serum (FBS) and penicillin-streptomycin at a final concentration of 10% and 1%, respectively, to prepare complete Iscove&#39;s Modified Dulbecco&#39;s Medium (IMDM). Store in the dark at 4 &#176;C for up to 1 month.</li>
	<li>Mix MgCl2 (3 mM), sucrose (300 mM), and Triton X-100 (0.5%) in phosphate buffer saline (PBS) to prepare the permeabilizati...

Evaluating Biofunctional Self-Assembling Peptides for Cell Adhesion and Morphology

The vast potential of SAPs in biomedical research is underscored by their malleability and adaptability through biofunctionalization. With such an extensive array of permutations, the challenge arises in efficiently testing and determining the most promising configurations for specific applications, especially in organoid research. A fast, cost-effective solution is paramount. The SW1222 cell line, derived from human colorectal adenocarcinoma, emerges as a pivotal candidate for such intensive evaluation. Here, we present...

In recent years, self-assembling peptides (SAPs) have emerged as promising biomaterials for tissue engineering applications. SAPs possess unique properties, including spontaneous formation of nanofibers, biocompatibility, biodegradability, and non-immunogenicity, making them attractive candidates for scaffold development1. SAPs have been previously used together with various types of cells, and notably, reported ultrashort SAPs have facilitated the encapsulation of stem cells while upholding their pluripotency over prolonged periods encompassing more than 30 passages and with minimal incidence of chromosomal aberrations2,3,4. Hence, adapting SAPs for their use in organoid culture constituted a rational subsequent step.
Organoids are complex three-dimensional structures that arise from single pluripotent cells. These structures give rise to various cell types, which then self-organize to replicate the developmental processes of embryonic and tissue growth in vitro5. This innovative framework has evolved into a formidable tool for in vitro investigations, efficiently conserving the genetic, phenotypic, and behavioral attributes characteristic of in vivo organs6. Nevertheless, a principal impediment in the bench-to-bedside transition of organoid-based findings is the need for reproducibility7. This variation in results is primarily ascribed to the difficulty in fully differentiating human pluripotent stem cells into specialized cell lineages, compounded by the absence of authentic tissue architecture and the inherent complexity encountered in organoid models. Given the rise in popularity of stem-cell- and organoid-based studies8, there has been an escalated demand for biomaterials with dynamic mechanical properties and integrin-like adhesion sites or scaffolds with controlled degradability7,9. These attributes can be effectively tuned through the strategic utilization of biofunctionalized SAPs.
SAPs are short sequences of amino acids with the inherent ability to spontaneously organize into well-defined structures10. These peptides often contain alternating hydrophobic and hydrophilic residues, which drive their self-assembly through non-covalent interactions, including hydrogen bonding, electrostatic interactions, and hydrophobic effects11,12. The self-assembly process of these peptides is primarily driven by the need to minimize the system&#39;s free energy. When in an aqueous environment, the hydrophobic residues tend to cluster together to minimize their exposure to water, while the hydrophilic residues interact with the surrounding water molecules. This phenomenon leads to the formation of various nanostructures. In this case, ultrashort self-assembling peptides form nanofibers with characteristics that depend on the peptide sequence and environmental conditions2,12,13,14,15,16. These peptides adopt a &#946;-turn conformation, where individual peptide strands align parallel or antiparallel to each other, stabilized by hydrogen bonds (Figure 1). The presence of positive ions accelerates the self-assembling processes that lead to nanofiber formation.
Peptide nanofibers have been identified as possessing an innate ability to entrap significant volumes of water. This characteristic facilitates the generation of a hydrogel, which subsequently manifests as a biocompatible and biodegradable three-dimensional space conducive to cellular proliferation and growth. These self-assembling peptide nanofibers intricately emulate the topographical features inherent to the natural extracellular matrix (ECM) environment1. This resemblance endows cells with an environment that imitates their physiological habitat, further promoting optimal cellular activities17. Furthermore, the versatility inherent to these peptides permits a considerable degree of tunability4. Such adaptability enables changes to the matrix&#39;s properties, such as stiffness, gelation kinetics, and porosity. These adaptations are achieved through modifications to the peptide sequence. Consequently, self-assembling peptides (SAPs) have emerged as pivotal components in contemporary biomaterial science, extensively used as scaffolds for tissue regeneration and cellular culture13,17.
One of the critical advantages of self-assembling peptides is their ability to be easily synthesized and modified at the molecular level. This advantage allows for the incorporation of specific functional groups or bioactive motifs into the peptide sequence, enabling the design of peptides with tailored properties and functionalities18,19,20 (Figure 1B). For example, biofunctionalized SAPs can be designed to mimic the ECM and promote differentiation using the RGD peptide19,21. Peptide Ben-IKVAV has also been reported to significantly increase the expression of neuronal-specific markers due to its ligand-specific motiety22. These peptides can also be engineered to display bioactive molecules, such as integrin-binding peptides, to enhance cell survival23. Finally, other biofunctionalized SAPs have been developed to promote angiogenesis by including the IKVAV and YIGSR motifs in their structures24.
Biofunctionalized SAPs reported in an earlier publication show that self-assembling can be further modified by including the na&#239;ve peptide from which the biofunctionalized SAP was derived25. These SAP mixtures can also provide a wide array of SAP formulations that vary in their biochemical activity and physicochemical properties. For instance, the amphiphilic peptide IIFK is an ultrashort SAP that can withstand biofunctionalization with various motifs, exemplified by the incorporation of the IKVAV motif (Figure 1B). Both peptides can form nanofibers, both alone and combined. Each formulation results in hydrogels with varying physical properties (Figure 2)
However, because of the extensive array of potential permutations and alternatives obtained from the biofunctionalization of SAPs, there is a need to provide a fast and cost-effective option for organoid testing. One candidate for such intensive and cost-effective organoid evaluation is the SW1222 cell line, derived from human colorectal adenocarcinoma26,27. SW1222 cells possess characteristics that enable their aggregation into 3D structures resembling organoids, making them an ideal model for studying tissue development and regenerative medicine applications. SW1222 cells have been identified as capable of generating organoids owing to their intrinsic overexpression of the LGR5 gene27. The creation of organoids from individual LGR5+ stem cells has been convincingly exhibited before, as well as the propensity of SW1222 cells to achieve morphological characteristics of a colorectal cancer organoid28.
In this methods paper, we present detailed protocols for evaluating and characterizing the effects of various biofunctional peptides on cell adhesion and lumen formation using SW1222 cells (Figure 3). By providing step-by-step procedures and imaging analysis methods, we hope to offer valuable insights into the biofabrication of organoids and the evaluation of simplistic SAP matrices for organoid culture.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1x PBS</td>
			<td>Gibco</td>
			<td>14190144</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well plate, tissue culture treated</td>
			<td>Corning</td>
			<td>07-200-83</td>
			<td></td>
		</tr>
		<tr>
			<td>10x PBS, no calcium, no magnesium</td>
			<td>Gibco</td>
			<td>70011044</td>
			<td></td>
		</tr>
		<tr>
			<td>16% Formaldehyde (w/v), Methanol-free</td>
			<td>Thermo Scientific</td>
			<td>28906</td>
			<td></td>
		</tr>
		<tr>
			<td>24-well plate, tissue culture treated</td>
			<td>Corning</td>
			<td>09-761-146</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well black plate, tissue culture treated</td>
			<td>Corning</td>
			<td>07-200-565</td>
			<td></td>
		</tr>
		<tr>
			<td>alamarBlue Cell Viability Reagent</td>
			<td>Invitrogen</td>
			<td>DAL1025</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Ezrin antibody, rabbit monoclonal</td>
			<td>Abcam</td>
			<td>ab40839</td>
			<td>Secondary used: anti-rabbit Dylight 633</td>
		</tr>
		<tr>
			<td>Anti-pan Cadherin antibody, rabbit polyclonal</td>
			<td>Abcam</td>
			<td>ab16505</td>
			<td>Secondary used: anti-rabbit Alexa 488</td>
		</tr>
		<tr>
			<td>Anti-ZO1 tight junction antibody, goat polyclonal</td>
			<td>Abcam</td>
			<td>ab190085</td>
			<td>Secondary used: anti-goat Alexa 488</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma-Aldrich</td>
			<td>A9418</td>
			<td></td>
		</tr>
		<tr>
			<td>Cellpose 2.0</td>
			<td>NA</td>
			<td>NA</td>
			<td>Obtained from https://github.com/MouseLand/cellpose</td>
		</tr>
		<tr>
			<td>Confocal Laser Scanning Microscope with Airyscan</td>
			<td>ZEISS</td>
			<td>LSM 880</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Invitrogen</td>
			<td>D1306</td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey anti-Goat IgG (H+L) Secondary Antibody, Alexa Fluor 488</td>
			<td>Invitrogen</td>
			<td>A-11055</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Cytiva</td>
			<td>GE17-1323-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor 488</td>
			<td>Invitrogen</td>
			<td>A-11008</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-Rabbit IgG (H+L) Secondary Antibody, DyLight 633</td>
			<td>Invitrogen</td>
			<td>35562</td>
			<td></td>
		</tr>
		<tr>
			<td>Heat Inactivated Fetal Bovine Serum (HI FBS)</td>
			<td>Gibco</td>
			<td>16140071</td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ 1.54f</td>
			<td>NIH</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>IMDM</td>
			<td>Gibco</td>
			<td>12440079</td>
			<td></td>
		</tr>
		<tr>
			<td>LIVE/DEAD Viability/Cytotoxicity Kit, for mammalian cells</td>
			<td>Invitrogen</td>
			<td>L3224</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium Chloride, hexahydrate (MgCl2 6 H2O)</td>
			<td>Sigma-Aldrich</td>
			<td>M2393</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel for Organoid Culture, phenol-red free</td>
			<td>Corning</td>
			<td>356255</td>
			<td>Refered in the manuscript as Matrigel or basement membrane matrix.</td>
		</tr>
		<tr>
			<td>Microscope, brightfield</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope, EVOS</td>
			<td>Thermo Scientific</td>
			<td>EVOS M7000</td>
			<td></td>
		</tr>
		<tr>
			<td>OriginPro 2023 (64-bit) 10.0.0.154</td>
			<td>OriginLab Corp</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Peptide P (Ac,Ile,Ile,Phe,Lys,NH2)</td>
			<td>Lab-made</td>
			<td>NA</td>
			<td>Can be custom-made by peptide manufacturers such as Bachem.</td>
		</tr>
		<tr>
			<td>Peptide P1 (Ac, Ile, Ile, Phe, Lys, Gly, Gly, Gly, Arg, Gly, Asp, Ser, NH2)</td>
			<td>Lab-made</td>
			<td>NA</td>
			<td>Can be custom-made by peptide manufacturers such as Bachem.</td>
		</tr>
		<tr>
			<td>Peptide P2 (Ac, Ile,Ile,Phe,Lys,Gly,Gly,Gly,Ile,Lys,Val,Ala,Val,NH2)</td>
			<td>Lab-made</td>
			<td>NA</td>
			<td>Can be custom-made by peptide manufacturers such as Bachem.</td>
		</tr>
		<tr>
			<td>Rhodamine Phalloidin</td>
			<td>Invitrogen</td>
			<td>R415</td>
			<td></td>
		</tr>
		<tr>
			<td>Round Cover Slip, 10 mm diameter</td>
			<td>VWR</td>
			<td>631-0170</td>
			<td></td>
		</tr>
		<tr>
			<td>Scanning Electron Microscope</td>
			<td>Thermo Fisher - FEI</td>
			<td>TENEO VS</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile 30 &#956;m strainer</td>
			<td>Sysmex</td>
			<td>04-004-2326</td>
			<td></td>
		</tr>
		<tr>
			<td>sucrose</td>
			<td>Sigma-Aldrich</td>
			<td>S1888</td>
			<td></td>
		</tr>
		<tr>
			<td>SW1222 cell line</td>
			<td>ECACC</td>
			<td>12022910</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton x-100</td>
			<td>Thermo Scientific</td>
			<td>85111</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA 0.25%</td>
			<td>Gibco</td>
			<td>25200056</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Sigma-Aldrich</td>
			<td>P1379</td>
			<td></td>
		</tr>
		<tr>
			<td>UltraPure water</td>
			<td>Invitrogen</td>
			<td>10977015</td>
			<td></td>
		</tr>
	</tbody>
</table>

fabrication and characterization of colorectal cancer organoids from sw1222 cell line in ultrashort self&#45;assembling peptide matrix

Ultrashort self-assembling peptides (SAPs) can spontaneously form nanofibers that resemble the extracellular matrix. These fibers allow the formation of hydrogels that are biocompatible, biodegradable, and non-immunogenic. We have previously proven that SAPs, when biofunctionalized with protein-derived motifs, can mimic the extracellular matrix characteristics that support colorectal organoid formation. These biofunctional peptide hydrogels retain the original parent peptide's mechanical properties, tunability, and printability while incorporating cues that allow cell-matrix interactions to increase cell adhesion. This paper presents the protocols needed to evaluate and characterize the effects of various biofunctional peptide hydrogels on cell adhesion and lumen formation using an adenocarcinoma cancer cell line able to form colorectal cancer organoids cost-effectively. These protocols will help evaluate biofunctional peptide hydrogel effects on cell adhesion and luminal formation using immunostaining and fluorescence image analysis. The cell line used in this study has been previously utilized for generating organoids in animal-derived matrices.

Author Spotlight: Optimization of Ultrashort Peptide Matrices for Colorectal Cancer Organoids

Fabrication and Characterization of Colorectal Cancer Organoids from SW1222 Cell Line in Ultrashort Self&#45;Assembling Peptide Matrix

Our research aims to evaluate ultrashort self-assembling peptides as matrices for Colorectal Cancer Organoid Cultures. We address questions about organoid morphology, for viability, proliferation, and the adhesion and the impact of biofunctionalization. The goal is to optimize peptide matrix compositions for effective organoid growth, and to contribute to regenerative medicine.Cutting-edge technologies in our field involve 3D bioprinting for precise organic fabrication, microfluidic platforms for dynamic culture environments, and advanced imaging tools like atomic force microscopy and multiphoton microscopy. CRISPR-Cas9 gene editing refines genetic modification, while single cell RNA sequencing provides in-depth molecular insights. Current experimental challenges include enhancing organoid reproducability, refining vascularization for larger constructs, and mimicking complex tissue architecture.Overcoming these limitations in long-term culture and incorporating immune components into models are active areas. Achieving standardized protocols and addressing ethical concerns opposing ongoing challenges in this field. Significant findings in our field include demonstrating the suitability of ultrashort self-assembling peptides for organoid cultures.We have shown the versatility, reproducability, and stability of these peptides, providing a platform for fine tuning micro-environments. This contributes to advancing organoid-based studies, regenerative medicine, and biofunctionalized hydrogel research. Our protocol bridges and research gap, whereby providing a systematic approach to evaluating organoids in ultrashort self-assembling peptide matrices.It addresses the need for standardized methods in assessing cell behavior within these matrices, aiding in durational design of peptide-based hydrogels for optimal organoid growth and advancing regenerative medicine studies.

First, we evaluated the cells grown in a 24-well plate for 7 days using brightfield imaging. We identified small clusters of cells assembling into organoids during the week, as seen in Figure 4. A controlled scan method can follow the mobility of the cells and the organoids between different days. In general, we looked at the evolution of the morphology of the cells during the whole week. SW1222-derived organoids should have a round morphology and a light appearance. A darker appearance indi...

Read this Scientific Article Protocol about Author Spotlight:  Ultrashort Peptide Matrices Optimization for Colorectal Cancer Organoids at JoVE.com

This protocol aims to evaluate biofunctional self-assembling peptides for cell adhesion, organoid morphology, and gene expression by immunostaining. We will use a colorectal cancer cell line to provide a cost-effective way of obtaining organoids for intensive testing.

Ultrashort self-assembling peptides (SAPs) can spontaneously form nanofibers that resemble the extracellular matrix. These fibers allow the formation of ...

Research

JoVE Journal

Bioengineering

SW1222 Cell Line&#45;Derived Colorectal Cancer Organoid Fabrication in Ultrashort Self-Assembling Peptide Matrix

Immunostaining of Colorectal Cancer&#45;Derived Organoids in Peptide Hydrogel

Imaging Immunostained Colorectal Cancer&#45;Derived Organoids in Peptide Hydrogel for Evaluating the Effects of Self&#45;Assembling Peptides on Cell Adhesion