Protocol for the establishment and characterization of South African patient-derived intestinal organoids

Abstract

Patient-derived organoids are valuable for modeling disease pathogenesis, therapeutic screening, and advancing personalized medicine. Here, we present a protocol for generating and characterizing intestinal organoids from South African patients, using both healthy and cancerous tissues. We describe steps for generating organoid cultures, quantitative reverse-transcription polymerase chain reaction (RT-qPCR), immunofluorescence microscopy, and histological staining. Notably, we provide a detailed method for cholesterol profiling in organoids and tissues, offering a powerful tool for researchers exploring lipid biology in organoid systems.

Keywords: Cancer; Molecular Biology; Organoids.

Graphical abstract

Figure 2

Representative images of cancerous tissue digestion for organoid generation (A) Tissue digestion after 10 min incubation shows the presence of large cell clusters, which requires further digestion. (B) Tissue digestion after 20 min incubation shows sufficiently digested cell clusters, with the ability to pass through a 70 μm strainer. Images acquired using the Leica DmiL Inverted Light Microscope at 10× magnification with the representative scale bar indicating 50 μm.

Figure 1

Workflow of generating primary organoid cultures from fresh tissue (A) The tissue is washed. (B) The tissue subsequently undergoes mechanical and enzymatic digestion. Digestion time varies between tissue specimens. (C) Sufficient digestion is confirmed with the visualization of intestinal crypts. (D) Crypts are filtered through a 70 μM strainer and pelleted. (E and F) Red blood cell (RBC) lysis is required if the pellet is red in color (indicative of contaminating red blood cells). (G) The pellet is then resuspended in a mixture of tissue seeding media and BME and plated in a pre-warmed 24-well plate. (H) Following a maximum of 30 min, 500 μL of IntestiCult organoid media is added.

Figure 3

Insights into organoid morphology Undifferentiated organoids exhibit a thin-walled structure with a clear lumen, indicative of a proliferative state suitable for maintenance and passaging. Differentiated organoids display a thicker wall and a dark lumen, suggesting organoid maturation and suitability for downstream analysis of differentiated cell types. Images acquired using the Leica DmiL Inverted Light Microscope at 10× magnification with the representative scale bar indicating 50 μm.

Figure 5

Monitoring human intestinal organoid growth dynamics (A and B) Organoid growth dynamics. At day 0, the initial growth of the organoid presents as a single stem cell that will progress to form a cyst-like structure. The cyst-like structure is comprised of proliferating progenitor cells and a central lumen that will increase in size. Between days 3 and 4, budding of the cyst-like structure will appear, which will give rise to more cyst-like organoids. These cyst-like structures will increase in size and will reach maturity between days 7 and 10 in culture, where the structure of the organoid resembles that of the intestine. The lumen will also progress to become darker as this is the region in which the villi would develop, and dead cells are shed towards the lumen. Images acquired using the Leica DmiL Inverted Light Microscope at 10× magnification with the representative scale bar indicating 50 μm.

Figure 6

Organoid digestion to single cells (A) Organoids incubated in TrypLE for 5 min exhibit partial dissociation, with the presence of large cell clusters indicative of incomplete enzymatic digestion. (B) Continued incubation with aspiration facilitates further dissociation of organoids into smaller cell clusters. (C) Following 15 min of TrypLE incubation and aspiration, organoids are sufficiently dissociated into single cells and the reaction is terminated. Images acquired using the Leica DmiL Inverted Light Microscope at 10× magnification with the representative scale bar indicating 50 μm.

Figure 4

Timeline of organoid development over 10 days Organoids are digested down to single cells and resuspended in BME (D0). Organoids are monitored for growth and differentiation where they will increase in size as they progress through culture. At Day 7 organoids should be passaged or may be left to differentiate post Day 10 for experimental use. Organoids left for longer than 10 days without passaging will be susceptible to cell death compromising the viability of the line. Figure created using BioRender.com.

Figure 7

Representative images of organoid morphology following organoid thawing (A) Organoids on the day of thawing exhibit a slightly darkened, granular morphology. (B) Following 1-day post-thaw, organoids show an increase in size and at this point are ready for digestion to single cells. (C) Single cells give rise to many organoids and should adopt a proliferative morphology with thin walls and clear lumen. (D) Organoids progressively increase in size and should subsequently be cultured as per the standard timeline. Images acquired using the Leica DmiL Inverted Light Microscope at 10× magnification with the representative scale bar indicating 50 μm.

Figure 8

Visual representation of sufficient organoid confluency for RNA extraction (A and B) Organoids that have reached a confluency of ∼80% per 100 μL dome are suitable for RNA extraction. Representative images for appropriate confluency of (A) normal organoids that grow to form large cystic structures and (B) cancer organoids that form smaller acinar structures. Images acquired using the Leica DmiL Inverted Light Microscope at 4× and 10× magnification, respectively, with the representative scale bar indicating 50 μm.

Figure 9

Organoid processing for FFPE workflow (A) Following organoid differentiation (post day 10 in culture) organoids are fixed in 10% neutral buffered formalin (NBF) for 30 min. (B) Organoids are washed and resuspended in 2% agarose. (C) The agarose cone is subjected to ethanol dehydration and clearing with xylene. (D) Thereafter, the agarose cone is embedded in paraffin wax and allowed to set in a square mold. (E) The wax block is sectioned with a microtome, where slides are left to dry on a drying rack (∼60°C) for at least 2 h. (F) The presence of organoids on each section should be validated before beginning downstream assays.

Figure 10

Organoid morphology variation between patient samples (A) Patient B054N exhibits predominantly cystic like organoid structures. (B) Patient B055N exhibits predominantly extensively budded (acinar) like organoid morphology. (C) Patient B056N exhibits heterogeneous morphology of both cystic and budded structures. All images were captured at P1, Day 10. Images acquired using the Leica DmiL Inverted Light Microscope at 10× magnification with the representative scale bar indicating 50 μm.

Figure 11

Morphological differences in normal versus cancer organoid morphology (A) Healthy organoids tend to grow very large prior to exhibiting detailed differentiated structures while (B) cancer organoids typically form smaller acinar structures when fully differentiated. Images acquired using the Leica DmiL Inverted Light Microscope at 10× magnification with the representative scale bar indicating 50 μm.

Figure 12

H&E staining of organoids and patient-matched tissue (A) Human colon transverse section indicating crypt and villus axis (epithelial cells and goblet cells) as well colonic lumen as well as associated stroma, and patient-matched normal organoid with an accurate representation of columnar epithelial and goblet cells surrounding a central lumen. (B) Human cancerous colonic tissue displaying a highly dysmorphic structure mainly depicting cancer associated stroma, and patient-matched cancerous organoids present as aberrant crypt-like structures recapitulating the malignant crypts present in the tumor tissue. All images were acquired using the EVOS M700 Imaging System (Thermo Fisher Scientific, USA). Scale bars represent 200 μm.

Figure 13

PAS and NPS staining of organoids and patient-matched tissue (A) PAS staining of monosaccharide units to characterize goblet cell population within normal and cancer tissue and organoids. (B) NPS staining of sulfated mucopolysaccharides to characterize goblet cell population within normal and cancer tissue and organoids. Arrows indicate the presence of goblet cells. All images were acquired using the EVOS M700 Imaging System (Thermo Fisher Scientific, USA). Scale bars represent 200 μm.

Figure 14

Characterizing organoid cell composition RT-qPCR analysis of the gene expression markers utilized to characterize organoid cultures in (A) normal and (B) patient-matched cancer organoid lines. Data is represented as dCT values. Gene expression is indicated relative to β-actin (characterization validated in n=4 organoid lines (P2) at Day 10 in culture). A Student’s T-Test was performed for statistical analysis, where ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001 and ns = not significant at 95% CI. Error bars are representative of the standard deviation relative to the mean.

Figure 15

Visual representation of patient matched tissue and organoid, normal and cancer, immunofluorescence staining (A) EPCAM staining (green) is indicative of epithelial cells. Nuclei are stained blue (DAPI). (B) VIL1 staining (red) is indicative of enterocytes. Nuclei are stained blue (DAPI). (C) MUC2 staining (orange) is indicative of goblet cells. Nuclei are stained blue (DAPI). (D) CHGA staining (pink) is indicative of enteroendocrine cells. Nuclei are stained blue (DAPI). All images were acquired using the EVOS M700 Imaging System (Thermo Fisher Scientific, USA). (characterization validated in n=6 organoid lines (P2) at Day 10 in culture. Scale bars represent 200 μm.

Figure 16

Cholesterol staining of B055 organoid and patient matched tissue (A) Filipin staining (blue) indicative of free cholesterol (B) Vybrant Alexa Fluor (red) indicative of lipid raft cholesterol content and (C) BODIPY (green) staining indicative of lipid droplets present in normal and cancer tissue and patient-derived organoids respectively. All images were acquired using the EVOS M700 Imaging System (Thermo Fisher Scientific, USA) (Cholesterol profiles validated in n=6 organoid lines (P2) at Day 10 in culture). Scale bars represent 200 μm.