Patient-derived organoid cultures of pancreatic ductal adenocarcinoma are a rapidly established 3-dimensional model that represent epithelial tumor cell compartments with high fidelity, enabling translational research into this lethal malignancy. Here, we provide detailed methods to establish and propagate organoids as well as to perform relevant biological assays using these models.
Pancreatic ductal adenocarcinoma (PDAC) is amongst the most lethal malignancies. Recently, next-generation organoid culture methods enabling the 3-dimensional (3D) modeling of this disease have been described. Patient-derived organoid (PDO) models can be isolated from both surgical specimens as well as small biopsies and form rapidly in culture. Importantly, organoid models preserve the pathogenic genetic alterations detected in the patient's tumor and are predictive of the patient's treatment response, thus enabling translational studies. Here, we provide comprehensive protocols for adapting tissue culture workflow to study 3D, matrix embedded, organoid models. We detail methods and considerations for isolating and propagating primary PDAC organoids. Furthermore, we describe how bespoke organoid media is prepared and quality controlled in the laboratory. Finally, we describe assays for downstream characterization of the organoid models such as isolation of nucleic acids (DNA and RNA), and drug testing. Importantly we provide critical considerations for implementing organoid methodology in a research laboratory.
Pancreatic ductal adenocarcinoma (PDAC) is a lethal disease characterized by late diagnosis in most patients, a lack of effective therapies, and a resultant low 5-year overall survival rate that remains less than 10%1. Only 20% of patients are diagnosed with a localized disease suitable for curative surgical intervention2,3. The remaining patients are typically treated with a combination of chemotherapeutic agents that are effective in a minority of patients4,5. To address these pressing clinical needs, researchers are actively working on early detection strategies and the development of more effective therapies. To accelerate clinical translation of important discoveries, scientists are employing genetically engineered mouse models, patient derived xenografts, monolayer cells lines, and, most recently, organoid models6.
Three-dimensional epithelial organoid culture using growth factor and Wnt-ligand rich conditions to stimulate proliferation of untransformed progenitor cells were first described for the mouse intestine7 and were quickly adapted to normal human pancreatic tissue8. In addition to normal ductal tissue, organoid methodology allows for the isolation, expansion, and study of human PDAC8. Importantly, the method supports the establishment of organoids from surgical specimens, as well as fine and core needle biopsies, allowing researchers to study all stages of the disease9,10. Interestingly, patient-derived organoids recapitulate well-described tumor transcriptomic subtypes and may enable development of precision medicine platforms9,11.
Current organoid protocols for PDAC enable the successful expansion of more than 70% of patient samples from chemo-naïve patients9. Here we present the standard methods employed by our laboratory to isolate, expand, and characterize patient-derived PDAC organoids. Other PDAC organoid methodologies have been described12,13 but no comparison of these method has been thoroughly performed. As this technology is relatively new and advancing quickly, we expect that these protocols will continue to evolve and improve; however the principles of tissue handling and organoid culture will continue to be useful.
All human tissue collection for research use was reviewed and approved by our Internal Review Board (IRB). All of the following protocols are performed under aseptic conditions in a mammalian tissue culture laboratory environment.
1. Media Preparation
2. Isolation of PDAC Organoids
NOTE: Thaw the basement membrane extract (BME) solution (growth factor reduced; see Table of Materials) on ice in a 4 °C environment (fridge or cold room) for at least 12 h prior to use. Incubate the tissue culture plates for organoid culture in a 37 °C incubator for at least 12 h prior to use.
3. Passaging of PDAC Organoids
4. Freezing and Thawing of PDAC Organoids
5. Characterization of PDAC Organoids
NOTE: The characterization of the organoids should be performed on an established culture after several passages to diminish the risk of contamination from non-epithelial cell types such as fibroblasts and immune cells.
To illustrate the challenges associated with isolating organoids from PDAC, we show the establishment of a patient derived organoid culture from a small hypocellular tumor sample. After initial plating, only a few organoids were visible per well, as shown in Figure 1. Organoids were allowed to grow larger over the span of 2 week and were passaged according to our protocol to establish a more robust culture, as shown in the early and late passage 1 representative pictures (Figure 1). It is important to note that the larger cystic organoids observed in the late primary isolate were easily broken down into smaller fragments during the mixing of organoids with ice-cold BME, as described in step 2.13.
To demonstrate the outcome of the pharmacotyping protocol, we prepared single cells from an established and fast growing representative PDAC organoid as described in this protocol. 1,000 viable cells were plated per well and allowed to recover over 24 h before cytotoxic chemotherapeutic agents, Gemcitabine and Paclitaxel, were dosed. We performed a 9-point dose response assay in triplicate starting with a low dose of 100 pM and ending with a high dose of 2 µM. After 5 day treatment, representative pictures were taken for vehicle (DMSO), 2 µM Gemcitabine, and 2 µM Paclitaxel treated wells (Figure 2). Immediately after taking the pictures, cell viability was assessed using luminescence cell viability reagent and plotted using graphing software (Figure 2).
Figure 1: Representative pictures are shown for a PDAC organoid isolation as well as after the first passage. Both early (1–3 days) and late (7–10 days) time points are shown to illustrate organoid growth over time. Scale bar = 200 µm. Please click here to view a larger version of this figure.
Figure 2: Dose response analysis. (Left) Representative dose response analysis obtained using an established PDAC organoid culture, with standard deviation of triplicates shown as error bars. (Right) Pictures illustrating the effect at the end of the assay of vehicle (DMSO) treatment as well as 2 µM Gemcitabine and 2 µM Paclitaxel. Scale bar = 100 µm. Please click here to view a larger version of this figure.
Here, we present current protocols for isolating, expanding and characterizing patient-derived PDAC organoids. Our current success rate of establishing organoid culture is over 70%; therefore, these methods have not yet been perfected and are expected to improve and evolve over time. Important consideration should be given to sample size, as PDAC has a low neoplastic cellularity. Consequently, small specimens will contain few tumor cells, and will only generate a handful of organoids. Additionally, many patients receive chemotherapy and/or chemoradiation-based neoadjuvant treatment prior to surgical intervention15. If the treatment is effective for a particular patient, the tumor tissue may be devoid of viable cells. Acquisition of chemo-naive patient samples is preferred for initial optimization of these methods, but this is not always possible. Interestingly we have found that ischemia time following surgical removal of the tumor tissue is not a major criterion for successful organoid isolation, as long as the sample is processed within 24 h.
Pancreatic ductal adenocarcinoma is a disease characterized by a strong desmoplastic reaction and deposition of a dense stromal matrix. While organoids are an excellent tool for the rapid isolation and expansion of the epithelial compartment, the model does not recapitulate the complex stroma of PDAC. Other methodologies such as patient derived xenografts16 or air liquid interface culture17 allow for a stromal compartment, however they may be challenging to expand quickly. When choosing a model system, the researcher should carefully consider the strengths and weaknesses of each6.
The heterogeneous biology of this disease impacts organoid establishment as some patient-derived organoids grow extremely well in our conditions while other are much slower by comparison. The protocols above describe a Wnt ligand rich condition to isolate and expand all patient-derived organoids, yet others have shown that some patient's tumors are able to grow in the absence of Wnt conditioned media11,12. Further testing will be required to determine if using a range of media conditions enhances the successful establishment of organoids, as was recently demonstrated for ovarian cancer organoids18. This multiplex approach is however limited by the low number of tumor cells that can be isolated from small patient samples. Additionally, normal untransformed ductal organoids can arise from an organoid isolation, particularly if the tumor tissue is adjacent to normal tissue9. To reduce the risk of normal organoid contamination, larger tissue samples can be subdivided into smaller independent fragments using morphological differences such as well vascularized (blood is visible) versus hypovascular regions, and hard nodules versus soft tissue.
The methods and protocols described here are the current standard approaches used in our laboratory for organoid isolation and they should be tested and adapted for each laboratory environment. For instance, the enzymatic dissociation (steps 2.6 to 2.9) of the tumor tissue is particularly important to optimize. Small equipment differences (nutator vs rotator mixer) can lead to significantly different timing for this step. Furthermore, the tissue dissociation can be fine-tuned by increasing or reducing the concentration of the Collagenase/Hyaluronidase mixture. Care must be taken to not treat all samples in the same manner. For example, in some cases organoids can be isolated from ascites fluid from advanced PDAC patients without mechanical or enzymatic dissociation.
DNA sequencing is the current gold standard to determine the presence or absence of tumor organoids as PDAC is driven by frequent mutations in KRAS, TP53, SMAD4 and CDKN2A. Transcriptomic analysis can reveal different tumor subtypes while pharmacotyping can uncover patient-specific therapeutic vulnerabilities9. These protocols enable PDAC researchers to develop their own library of patient-derived organoids and to profile the biology of these models.
The authors have nothing to disclose.
We are grateful for the support of the UC San Diego Moores Cancer Center Biorepository and Tissue Technology Shared Resource, members of the Lowy laboratory, and the UC San Diego Department of Surgery, Division of Surgical Oncology. AML is generously supported by NIH CA155620, a SU2C CRUK Lustgarten Foundation Pancreatic Cancer Dream Team Award (SU2C-AACR-DT-20-16), and donors to the Fund to Cure Pancreatic Cancer.
12 channel pipette (p20, p100, or p200) with tips | |||
12 well plates | Olympus | 25-106 | |
15 ml LoBind conical tubes | Eppendorf | EP0030122208 | |
15 ml tube Rotator and/or nutator | |||
37 °C CO2 incubator | |||
37 °C water bath | |||
384 well plates | Corning | 4588 | Ultra low attachment, black and optically clear |
A 83-01 | TOCRIS | 2939 | |
ADV DMEM | ThermoFisher | 12634010 | |
Animal-Free Recombinant Human EGF | Peprotech | AF-100-15 | |
Automated cell counter | |||
B27 supplement | ThermoFisher | 17504044 | |
Cell Recovery Solution | Corning | 354253 | Reagent that depolymerizes the Basement Membrane Extract at 4 °C |
CellTiterGlow | Promega | G7570 | Luminescence cell viability reagent |
Chloroform | Sigma | C2432 | |
Computer | |||
CryoStor CS10 | StemCELL Tech | 07930 | Cell Freezing Solution |
Cultrex R-spondin1 (Rspo1) Cells | Trevigen | 3710-001-K | |
DMEM | ATCC | 30-2002 | |
DNase I | Sigma | D5025 | |
Drug printer | Tecan | D300e | This is the drug printer we use in our laboratory |
Excel | For data analysis | ||
Extra Fine Graefe Forceps | Fine Science Tools | 11150-10 | |
FBS | ThermoFisher | 16000044 | |
G-418 | ThermoFisher | 10131035 | |
Gastrin I (human) | TOCRIS | 3006 | |
Gentle Collagenase/hyaluronidase | STEMCELL Tech | 7919 | |
GlutaMAX | ThermoFisher | 35050061 | Glutamine solution |
GraphPad Prism | For data analysis | ||
HEPES | ThermoFisher | 15140122 | |
Laminar flow tissue culture hood | |||
Luminometer | |||
L-Wnt-3A expressing cells | ATCC | CRL-2647 | |
MACS Tissue Storage Solution | Miltenyi biotec | 130-100-008 | |
Matrigel Matrix | Corning | 356230 | Basement Membrane Extract (BME), growth factor reduced |
Mr. Frosty Freezing Container | ThermoFisher | 5100-0001 | |
N-Acetylcysteine | Sigma | A9165 | |
Nicotinamide | Sigma | N0636 | |
p1000 pipette with tips | |||
p200 pipette with tips | |||
PBS | ThermoFisher | 10010049 | |
Penicillin/Streptomycin | ThermoFisher | 15630080 | |
primocin | InvivoGen | ant-pm-2 | |
Rapid-Flow Filter Units (0.2 µm) | ThermoFisher | 121-0020 | |
Recombinant Human FGF-10 | Peprotech | 100-26 | |
Recombinant Murine Noggin | Peprotech | 250-38 | |
Sterile Disposable Scalpels, #10 Blade | VWR | 89176-380 | |
Tissue culture centrifuge | |||
Tissue Culture Dishes 10 cm | Olympus | 25-202 | |
TRIZol | ThermoFisher | 15596018 | Acid Phenol solution |
TrypLE Express | ThermoFisher | 12605010 | |
Y-27632 | Sigma | Y0503 | |
Zeocin | ThermoFisher | R25001 |