Isolation and Characterization of Patient-derived Pancreatic Ductal Adenocarcinoma Organoid Models

Summary

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.

Abstract

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.

Introduction

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 intervention^2,3. The remaining patients are typically treated with a combination of chemotherapeutic agents that are effective in a minority of patients^4,5. To address these pressing clinical needs, researchers are actively ....

Protocol

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

1. Conditioned media preparation.
   NOTE: The human pancreatic organoid media requires abundant growth factors and nutrients as well as conditioned media supplementation to provide sufficient growth stimulation for organoid expansion. Both conditioned mediums described below are prepared from commercially available cell lines (see Table of Materials). For complet....

Results

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 (

Discussion

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 .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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