Fabrication and Characterization of Colorectal Cancer Organoids from SW1222 Cell Line in Ultrashort Self-Assembling Peptide Matrix

Summary

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.

Abstract

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.

Introduction

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 aberrations

Protocol

1. Buffer and solution preparation

NOTE: All stated concentrations are final concentrations.

1. Add fetal bovine serum (FBS) and penicillin-streptomycin at a final concentration of 10% and 1%, respectively, to prepare complete Iscove's Modified Dulbecco's Medium (IMDM). Store in the dark at 4 °C for up to 1 month.
2. Mix MgCl₂ (3 mM), sucrose (300 mM), and Triton X-100 (0.5%) in phosphate buffer saline (PBS) to prepare the permeabilization buffer. Store this solution at 4 °C for up to 2 months.
3. Combine bovine serum albumin (BSA, 1%), glycine (0.3 M), and Tween-20 (0.1%)....

Results

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.......

Discussion

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.......

Materials

List of materials used in this article

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