Establishment of Pancreatic Organoids from Normal Tissue and Tumors

Abstract

Establishment of patient-derived adult stem cell-based pancreatic (tumor) organoids was first described in 2015. Since then, multiple laboratories have demonstrated the robustness of this method. We recently described the generation of a pancreatic cancer biobank containing 30 well-characterized tumor organoid models. Here, we describe the applied methods in detail. Use of tumor-selective media prevents contamination with normal cells. Generated organoids can be cryopreserved and can serve as a living biobank of pancreatic cancer for biomarker identification and drug screening. For complete information on the generation and use of this protocol, please refer to Driehuis et al. (2019).

Keywords: Cancer; Organoids; Stem Cells.

Graphical abstract

Figure 2

Examples of the Cystic and Dense Morphologies of Pancreatic Organoid Cultures (A) Representative bright-field images of two patient-derived pancreatic tumor organoid cultures with cystic morphology. The top two panels show an organoid culture that has a cystic appearance similar to a normal culture; forming thin-walled large cysts. The bottom two panels show a cystic culture with a phenotype that is only observed in tumor cultures and not in normal (non-tumor) cultures. These cysts are thick-walled, appear dark, and often contain dead cells in the center of the structures. Although these organoids do not show an empty lumen, they are still clearly cystic, containing a (single- or multi-) layered row of epithelial cells that surround an internal compartment that does not contain epithelial cells. Scale bar left panels, 500 μm. Scale bar right panels, 100 μm. (B) Representative bright-field microscopy images of two patient-derived pancreatic tumor organoid cultures that show an overall dense morphology. In the dense structures, no lumen can be observed. However, in both cultures shown here, a fraction of the organoids of the culture showed a lumen, thereby representing a mixed phenotype. Organoids that form a lumen are indicated by an asterisk, whereas examples of dense organoids are indicated using an arrow. Such mixed phenotype cultures are commonly observed. Scale bar left panels, 500 μm. Scale bar right panels, 100 μm.

Figure 1

Bacterial and Fungal Infections in Organoid Cultures (A) Bacterial infection in BME/Matrigel drop. Representative bright-field microscopy images of bacterial colonies (indicated by arrows) that grew inside the BME/Matrigel drops. Because of their shape, the round colonies can be interpreted by some as organoids if only look at briefly when quickly scanning a culture plate. In the BME droplet shown here, an organoid was also present (indicated by an asterisk). Scale bar, 500 μm left panel, 100 μm right panel. (B) Bacterial infection in organoid culture medium. Representative bright-field microscopy images of bacteria growth in culture organoid medium. In this case, a darker “halo” can often be observed around the BME/Matrigel droplet. This is where the bacteria accumulate. The media also can appear cloudy (because of the presence of bacteria). Scale bar, 500 μm left panel, 100 μm right panel. (C) Fungal (yeast) infection in an organoid culture. Representative bright-field microscopy images of yeast cell, which is much larger than a bacterium. Often, white “colonies” can be observed by the naked eye in the BME/Matrigel drops. Here, the yeast networks (arrows) mainly grew from organoids (asterisk), which can still be seen present hidden under the yeast. Scale bar, 100 μm left panel, 50 μm right panel.

Figure 5

Technical Aspects of Passaging Organoid Cultures (A) Representative images from glass Pasteur pipettes before (indicated by an asterisk) and after (indicated by arrowheads) narrowing of the opening by a flame. The second panel shows a side view of a glass pipette before (asterisk) and after (arrowhead) narrowing by flame. (B) Representative image of a P1000 pipette with a 1,250 μL tip that holds a 10 μL pipette tip on top, which can be used as an alternative to the narrowed glass pipette to shear organoids during passaging. The smaller volume tip on top of the larger volume tips narrows the opening of the tip, allowing the shearing of organoids. (C) Representative image indicating how a 15 mL plastic tube containing the organoid culture suspension can be placed under the bright-field microscope to monitor the size of organoids during passaging.

Figure 3

Epithelial Cells Isolated from Tumor Resections Show Variable Outgrowth on the Different Tumor Organoid Media (A–C) Representative bright-field images showing the outgrowth of three samples (isolated from tumors of three different patients), when cultured in either tumor media 1 (TM1, left panels) or tumor media 2 (TM2, right panels). While sample 1 (S1) seemed to grow out equally well in both media, sample 2 (S2) showed a clear preference for TM2. Sample 3 (S3) did not grow in any of the two-tumor media. Scale bars, 100 μm. (D) Flowchart for helping decision taking when establishing organoid models using the different tumor media. S1, S2, and S3 indicated in the flowchart refer to the representative images shown in (A)–(C).

Figure 4

Passaging of Organoid Cultures (A) Representative bright-field images of two organoid cultures that were plated too densely after passaging, resulting in death of organoid structures. Death can be observed mostly in the center of the BME/Matrigel dome, most likely because of lack of nutrients (that diffuse into the droplet from the media) and oxygen. Live organoids (enriched on the side of the drops) are indicated by arrows, whereas dead organoids (enriched on the center of the drops) are indicated with asterisks. Scale bar, 500 μm. (B–D) Representative bright-field microscopy images of three different organoid cultures split for passaging. The left panels show the appearance of the cultures right after shearing (d0). The right panels show the appearance of the cultures few days later (7–8 days) and right before they will be passaged. (B) Representative bright-field image of a culture that was plated with a density that was too low, as can be seen by the limited number of organoids that grew out after 7 days in culture. Here, the organoid culture was pooled (the material was collected and replated in a smaller volume of BME) to assure that organoids were in close proximity to each other. After that, when organoids grew big, the culture could be passaged again. (C and D) Representative images of a cystic (C) and dense (D) organoid culture that were split and subsequently passaged at a correct density. This density allowed efficient outgrowth of the organoids, without the cell death shown in Figure 4A as can be observed in the right-side panels. Scale bar, 500 μm.

Figure 6

Freezing of Pancreatic Tumor Organoid Cultures Representative bright-field images showing the morphology of three different pancreatic tumor organoid cultures in the passage for freezing. The left panels depict how the cultures looked directly after performing 1:1 passaging in preparation of the freezing of the culture. The panels on the right side show the morphology of the same cultures on the day they were frozen. The day of freezing is indicated on the left corner of the image. As can be seen in these images, different cultures (with different growth speeds) require a different amount of days before they reach the right size for freezing. Scale bar left and central panels, 500 μm. Scale bar right panels, 100 μm.

Figure 7

Troubleshooting: Pancreatic Tumor Organoids (A) Representative bright-field images showing the result of culturing organoids with old media. Both panels show the growth of the same tumor organoid culture, that was either expanded for 8 days on 4-week-old medium (left panel), or expanded on freshly prepared medium (right panel). Scale bar, 500 μm. (B) When freezing organoids, the organoids should have the correct size and not be too large. Representative bright-field microscopy images show the result of thawing a culture that was frozen when the organoids reached a large size. As can be seen, even after 11 days, no new organoids grew out from the thawed cells. This culture could not be recovered and was lost. Scale bar, 500 μm. (C) Representative bright-field image of an example showing an organoid culture that recovered only poorly from thawing and was plated at a density that was too low (first panel) to result in good outgrowth of the culture (second panel). In this case, the culture could be saved by pooling the organoids that grew out at day 10 (second panel), passaging them and plating them in a decreased volume of BME/Matrigel. Scale bar, 500 μm. (D) Representative bright-field image of an example showing an organoid culture derived from breast tissue that is attaching to the bottom of the culture plate. The surface where the 2D cells are growing is indicated with arrows. Scale bar, 500 μm.