Fabrication and Characterization of Colorectal Cancer Organoids from SW1222 Cell Line in Ultrashort Self-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 reproducibility, 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 are posing 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, reproducibility, 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 a research gap by 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 the rational design of peptide-based hydrogels for optimal organoid growth and advancing regenerative medicine studies. To begin, pipette 10-microliter droplets of 2x peptide solution into the centers of the wells in a 24-well plate. After incubation, pipette 10 microliters of 2x PBS into each well.

Use the tip of the pipette to mix the solution gently. Allow the gel droplets to stabilize for at least 20 minutes, ensuring that the droplets are intact. Next, pipette 2.5 milliliters of 0.0125%trypsin-EDTA into a flask containing two million SW1222 human colorectal adenocarcinoma cells.

Incubate the cells with trypsin at 37 degrees Celsius for five to 10 minutes until they detach from the flask. Add five milliliters of complete medium to inactivate the trypsin. Then filter the cell suspension using a 30-micrometer cell strainer.

With a 10 microliter micropipette, inject two microliters of the cell suspension in each peptide droplet. Seed 800 cells from the SW1222 cell line into each well for four days. Incubate the droplets for 30 minutes at 37 degrees Celsius under 5%carbon dioxide.

Then add 0.5 milliliters of supplemented medium to each well. To prepare the control, dilute the stock of the basement membrane matrix control with supplemented medium. Load the cell suspension into the diluted membrane basement solution.

Pipette 20 microliters of diluted basement membrane matrix into each well with the cells. Incubate the cells for 20 minutes at 37 degrees Celsius. After the droplets solidify, add 0.5 milliliters of complete medium.

Image the cells using a brightfield microscope on days one, four, and seven to evaluate organoid growth. One-week brightfield imaging of the cells showed that the small cell clusters were assembling into organoids. The SW1222-derived organoids cultured in peptide hydrogels had a round morphology with a light appearance.

A darker appearance indicated undesirable higher density as seen in colonies in peptide P on day seven. To begin, pipette 20-microliter droplets of peptide hydrogel into a 24-well plate. Seed 800 cells from the SW1222 cell line into each well for four days.

On day four, pipette out the medium. Wash each well two times with PBS. Then add 400 microliters of 4%formaldehyde solution into the wells.

Once incubation is complete, use a P-1000 pipette tip to aspirate the formaldehyde solution. Then wash the wells with PBS once more. Incubate the cells in one milliliter of permeabilization buffer for 30 minutes at room temperature.

After pipetting out the buffer and washing the wells with PBS, add 0.5 milliliters of blocking buffer. Now wash the wells with PBS after removing the blocking buffer. Then pipette 200 microliters of diluted primary antibody into the wells.

Incubate the plate overnight at four degrees Celsius. With a P-200 pipette tip, remove the solution. Then wash the wells with PBS solution.

Next, add phalloidin conjugate to a vial of diluted secondary antibody in the ratio of one to 40. Pipette this mixture into the wells of the plate. Then incubate the plate at room temperature for three hours in the dark.

After aspirating the solution and washing the wells with PBS, pipette 300 microliters of DAPI solution into each well. After incubation, wash the wells with PBS again after pipetting out the solution. Store the plate at four degrees Celsius for up to seven days.

Immunostaining showed that the organoids found in non-biofunctional peptide P, biofunctionalized peptide P1, and Matrigel were found localized in the apical and basolateral membranes. The non-biofunctional peptide P induced cell-cell junction expression in the basolateral membrane. To begin, image the stained SW1222-derived organoid samples with an epifluorescence microscope at 10x.

Use only the RHOD and DAPI channels. Launch the ImageJ software and click on Plugin, followed by Macros, then Run to run the imagej_roi_converter. py file from the CellPose GitHub site.

When a window appears, select the first file from the colon organoid folder and click on Open. Then choose the seg. npy file corresponding to the first file, and click Open.

Next press Select All in the ROI manager window and press Measure. Now click on File followed by Save As on the results window to save the results. Repeat image conversion for the same image in the folder for colon organoid lumen.

Then load the OriginPro software. Click on Data followed by Import File to locate Import Wizard. Select both CSV files corresponding to the organoid and lumen-shape properties for the same image.

Copy the lumen-shape properties into the colony results column to pair each lumen measurement with its corresponding colony. In a new column, divide the areas of the lumen and the colony to yield the relative lumen area of its corresponding colony. Select the column corresponding to the circularity of each colony and the lumen area.

Then sequentially click on Plot, Basic 2D, and Scatter. Right-click on the plot window and choose Plot Details. Click on the Centroid Pro tab and select the options reading Show Centroid Point for Subset, Connect to Data Points, and Show Ellipse.

The cells cultured in 2D had a very high variation in colony circularity. The cells cultured in Matrigel had a more extensive distribution for the relative size of the lumen.