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Method Article

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

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DOI:

10.3791/66060

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May 3rd, 2024

 ,  , 
1Laboratory for Nanomedicine, Biological & Environmental Science & Engineering, King Abdullah University of Science and Technology, 2Computational Bioscience Research Center, King Abdullah University of Science and Technology, 3Red Sea Research Center, King Abdullah University of Science and Technology

In This Article

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 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'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 β-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'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 naïve 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.

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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%) in PBS to prepare a blocking buffer. Store this solution at 4 °C for up to 2 months.
  4. Dilute 10x to 2x PBS with ultrapure water under sterile conditions. Store this solution at room temperature for over 6 months.
  5. Sterilize 3 mg of the peptide powder under ultraviolet light for 30 min. Add 500 µL of ultrapure water to dissolve using a vortex mixer.
    NOTE: The final concentration must be twice the target concentration, i.e., 6 mg/mL, as the 1x concentration will be 3 mg/mL.

2. Colorectal cancer organoid fabrication from SW1222 in peptide

  1. Thaw an aliquot overnight at 4 °C and keep it in ice until needed to prepare the basement matrix control.
  2. Place a 10 µL droplet of 2x peptide solution in the center of each well in a 24-well plate. Allow the plate to incubate at room temperature for 10 min. Alternatively, deposit 8-10 droplets per well in a 6-well plate.
    NOTE: Samples meant to be used in confocal and electron microscopy are more accessible to manipulate if seeded on top of a sterile coverslip.
  3. Add 10 µL of 2x PBS. Mix the PBS solution gently using the tip. Allow the gels to stabilize for at least 20 min, ensuring the droplets remain almost intact.
    NOTE: The goal of PBS addition and gentle mixing is to maintain the integrity of the droplet and avoid spreading the hydrogel into a layer.
  4. Detach SW1222 cells using 0.125% trypsin-EDTA solution (5 mL). Incubate the cells with trypsin at 37 °C for 5-10 min until they detach from the flask. Abstain from tapping the flask to avoid cell clumping.
  5. Add 5 mL of complete medium to inactivate the trypsin. Filter the cell suspension using a 30 µm cell strainer. Alternatively, use a syringe with a 25 G needle to disrupt the aggregates.
  6. Prepare a cell suspension of 0.4 × 106 cells/mL and inject 2 µL of the cell suspension in each peptide droplet using a 10 µL micropipette.
  7. Incubate the droplets for 30 min at 37 °C, 5% CO2. Add 0.5 mL of supplemented medium to each well after incubation.
  8. While waiting, prepare the basement membrane matrix control by diluting the main stock (8-12 mg/mL, depending on the lot) to a final concentration of 3 mg/mL using the supplemented medium. Add cells to this solution (800 cells per 25 µL).
    NOTE: Basement membrane manipulation must be done on ice at all times. Tips must be rinsed in ice-cold PBS before touching the hydrogel. Otherwise, polymerization will occur inside the tips.
  9. Plate 20 µL droplets of the basement membrane solution in each well. Allow the droplets to gel by incubating for 20 min at 37 °C. After the material is gelled, add 0.5 mL of complete medium.
  10. Change the medium every 3-4 days. Observe the cells organizing and showing signals of lumen formation as early as day 4.
  11. Image cells in brightfield on day 1, day 4, and day 7 to evaluate organoid growth.

3. Viability and proliferation

  1. Prepare three black-well plates for the viability assay and three black-well plates for the proliferation assay. Add 25 µL of 2x peptide solution per well and gelate using the same volume of 2x PBS. Seed the cells as described above.
  2. Prepare three wells for each formulation of peptide hydrogel to be used as blanks in the proliferation plate. Do not seed cells on these.
  3. Remove the medium from the viability well plate and wash with 1x PBS for 3 x 5 min.
  4. Dilute Calcein-AM and ethidium homodimer to 8 µM from the viability/cytotoxicity Kit for mammalian cells (see Table of Materials) into the same light-protected conical tube. To prepare 2 mL of this solution to 2 mL of PBS in a light-protected conical tube, add 4 µL of the Calcein-AM stock solution and 8 µL of the Ethidium homodimer-1 stock solution.
  5. Add the calcein/ethidium homodimer solution prepared in step 3.4 to the 96-well plate. Add 100 µL per well. Protect from light and incubate at room temperature for 30 min.
  6. Wash for 3 x 5 min with 1x PBS. Image a minimum of three fields (center, top, bottom) when using a 4x objective or a minimum of five fields (center, top left, top right, bottom left, bottom right) when using a 10x and 20x objective under a fluorescence microscope.
  7. Remove the medium from the proliferation well plate to ensure a final volume of 90 µL.
  8. Add 10 µL of Alamar Blue solution (see Table of Materials) to reach a final concentration of 10%.
  9. Protect from light and incubate for 2-4 h at 37 °C, 5% CO2
  10. Use a well plate reader to measure fluorescence (Ex/Em = 530-560/590 nm/nm) or absorbance (570 nm). Average the values obtained for the blanks and subtract them from the values from each well that contains cells.
  11. Calculate proliferation values by averaging the signal of each condition and subtracting the average of their respective blanks. Create graphs by generating an indexed box plot of relative absorbance/fluorescence signal versus time.

4. Adherence assay of cells to matrix

  1. Prepare 100 µL of peptide hydrogels in two independent 96-well plates.
  2. Seed 20,000 cells in each well and add 100 µL of medium. Incubate the plates for 90 min at 37 °C, 5% CO2.
  3. After incubation, take only one plate and carefully resuspend the medium using a P200 micropipette for 20-30 s. Be careful not to disrupt the hydrogel by keeping the tip from the gel. Alternatively, if the hydrogel mechanical properties permit it (G' > 8,000 Pa), tap the four sides of the well plate using a moving vortex.
    NOTE: Resuspending the media (or tapping the well plate with the vortex) will remove both unattached cells and cells with tenuous adhesion to the surface of the matrix. The cellular attachment will occur within 90 min, although the strength of this attachment will vary depending on the specific peptide hydrogels and their adhesive and binding properties. Colorectal organoid growth is known to be favored in matrices with stiffness values of under 2,000 Pa. Hence, because the hydrogels presented in this protocol are soft and brittle, we make a point of using gentle mechanical stress.
  4. Remove 100 µL of medium from both well plates and add wash once with 1x PBS.
  5. Prepare a 1 µg/mL DAPI solution. Add 100 µL of the DAPI solution to all wells and incubate for 5 min at room temperature.
  6. Image a representative number of fields under a fluorescence microscope. Ensure all images are obtained using the same objective to keep the pixel/distance ratio constant.
    NOTE: Image nine fields for a 10x objective or 15 fields with a 20x objective.
  7. Load the fluorescence images on image analysis software (e.g., ImageJ) and analyze the particles on all images.
    1. From the Image menu, select Type and click on 8-bit.
    2. From the Image menu, select Adjust and click on Brightness/Contrast. Click on the Auto button | Apply.
    3. From the Image menu, select Adjust and click on Threshold. Click on the Auto button and close the window.
    4. From the Analyze menu, click on Analyze Particles. Tick the option to summarize and click OK.
    5. On the Summary window, hover over the File menu and click Save to save the summary values.
  8. Export the results for all images to standard statistics software.
  9. Calculate the percentage of area covered by cells before and after stress. Make an indexed box plot of the percentage of area against each condition.

5. Immunostaining of organoids in peptide hydrogel

  1. Seed 800 cells in 20 µL droplets of peptide hydrogel for 4 days, as described in steps 2.1-2.10.
  2. On day 4, remove the medium and wash each well 2x using 1x PBS.
  3. Add 400 µL of a 4% formaldehyde solution. Incubate the well plate for 30 min at room temperature.
  4. Remove the formaldehyde solution with a P1000 and wash once with 1x PBS.
  5. Add 1 mL of permeabilization buffer and incubate for 5 min at room temperature.
  6. Remove the permeabilization buffer with a P1000 and wash once with 1x PBS.
  7. Add 0.5 mL of blocking buffer and incubate for 30 min at room temperature.
  8. Remove the blocking buffer with a P1000 and wash with 1x PBS.
  9. Dilute the primary antibody in the blocking buffer according to the manufacturer's instructions (see Table of Materials). Add 200 µL to two wells.
  10. Incubate overnight at 4 °C.
  11. Remove the staining solution with a P200 and wash once with 1x PBS.
  12. Dilute the secondary antibody in blocking buffer, according to the manufacturer's instructions (see Table of Materials). Add the phalloidin conjugate at a ratio of 1:40 in the secondary antibody solution.
  13. Protect from light and incubate at room temperature for no more than 3 h. Remove the staining solution with a P200 and wash once with 1x PBS.
  14. Prepare a DAPI solution of 1 µg/mL in the blocking buffer. Add 300 µL of the DAPI solution to each well.
  15. Protect from light and incubate for 5 min at room temperature.
  16. Remove the staining solution with a P200 and wash once with 1x PBS.
  17. Protect from light and store at 4 °C for up to 7 days.

6. Imaging and image analysis of organoids in matrix

  1. Image the stained samples using an epifluorescence microscope at 10x. Use only the phalloidin and DAPI channels.
  2. Open the Cellopose software and press ctrl + L to load an image. As the software will have access to all the files in the location of the selected image, be sure to save all fluorescence images in the exact location without having any subfolders.
  3. Type the average diameter of the colonies in the Segmentation panel and click ENTER.
  4. Select a custom model for colon organoids and click on the Run Model button.
    NOTE: Here, we suggest using two from-scratch models in Cellpose2.029,30
  5. Press ctrl + s to save the masks as a .npy file.
  6. Use the keyboard arrows to move through the images of the folder and run the model for colon organoids on all images in the folder.
  7. Make a copy of the folder and repeat steps 6.2-6.6 using a model for the colon organoid lumen.
  8. Open ImageJ and click on Plugin | Macros | Run… to run the imagej_roi_converter.py file available from the Cellpose Github site and wait for a window to appear.
  9. Select the first file from the colon organoid folder and click Open.
  10. Select the seg.npy file corresponding to the first file and click Open.
    NOTE: ImageJ will convert the masks into regions of interest and overlap them with the corresponding fluorescence images.
  11. Click Select All in the ROI Manager window and click Measure.
  12. Click File | Save as… on the Results window and save the results.
    NOTE: This will result in a .csv file containing all the shape properties of each colony in the field.
  13. Repeat steps 6.8-6.12 for the same image in the folder for colon organoid lumen.
  14. Load OriginPro and press ctrl + 3 to open the Import Wizard. Click on the three dots button and select both .csv files corresponding to organoid and lumen shape properties for the same image.
  15. Copy and paste the results from the lumen shape properties in one column next to the results of the colony to ensure that each lumen measurement is paired with its respective colony.
  16. In a new column, divide the areas of the lumen and the colony to yield the relative lumen area of its containing colony.
  17. Select the column corresponding to the circularity of each colony and the lumen area. Click on Plot | Basic 2D | Scatter.
  18. Right-click on the plot window and select Plot details. Click on the Centroid(PRO) tab and tick the options reading Show Centroid Point for Subset, Connect to Data Points, and Show Ellipse.

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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...

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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...

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Disclosures

The authors have no conflicts of interest to declare.

Acknowledgements

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

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
1x PBSGibco14190144
6-well plate, tissue culture treatedCorning07-200-83
10x PBS, no calcium, no magnesiumGibco70011044
16% Formaldehyde (w/v), Methanol-freeThermo Scientific28906
24-well plate, tissue culture treatedCorning09-761-146
96-well black plate, tissue culture treatedCorning07-200-565
alamarBlue Cell Viability ReagentInvitrogenDAL1025
Anti-Ezrin antibody, rabbit monoclonalAbcamab40839Secondary used: anti-rabbit Dylight 633
Anti-pan Cadherin antibody, rabbit polyclonalAbcamab16505Secondary used: anti-rabbit Alexa 488
Anti-ZO1 tight junction antibody, goat polyclonalAbcamab190085Secondary used: anti-goat Alexa 488
BSASigma-AldrichA9418
Cellpose 2.0NANAObtained from https://github.com/MouseLand/cellpose
Confocal Laser Scanning Microscope with AiryscanZEISSLSM 880
DAPIInvitrogenD1306
Donkey anti-Goat IgG (H+L) Secondary Antibody, Alexa Fluor 488InvitrogenA-11055
GlycineCytivaGE17-1323-01
Goat anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor 488InvitrogenA-11008
Goat anti-Rabbit IgG (H+L) Secondary Antibody, DyLight 633Invitrogen35562
Heat Inactivated Fetal Bovine Serum (HI FBS)Gibco16140071
ImageJ 1.54fNIHNA
IMDMGibco12440079
LIVE/DEAD Viability/Cytotoxicity Kit, for mammalian cellsInvitrogenL3224
Magnesium Chloride, hexahydrate (MgCl2 6 H2O)Sigma-AldrichM2393
Matrigel for Organoid Culture, phenol-red freeCorning356255Refered in the manuscript as Matrigel or basement membrane matrix.
Microscope, brightfield
Microscope, EVOSThermo ScientificEVOS M7000
OriginPro 2023 (64-bit) 10.0.0.154OriginLab CorpNA
Penicillin-Streptomycin (10,000 U/mL)Gibco15140122
Peptide P (Ac,Ile,Ile,Phe,Lys,NH2)Lab-madeNACan 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-madeNACan 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-madeNACan be custom-made by peptide manufacturers such as Bachem.
Rhodamine PhalloidinInvitrogenR415
Round Cover Slip, 10 mm diameterVWR631-0170
Scanning Electron MicroscopeThermo Fisher - FEITENEO VS
Sterile 30 μm strainerSysmex04-004-2326
sucroseSigma-AldrichS1888
SW1222 cell lineECACC12022910
Triton x-100Thermo Scientific85111
Trypsin-EDTA 0.25%Gibco25200056
Tween 20Sigma-AldrichP1379
UltraPure waterInvitrogen10977015

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