Published: April 30th, 2021
We describe detailed protocols to use patient-derived organoids for medium-throughput therapy sensitivity screenings. Therapies tested include chemotherapy, radiotherapy, and chemo-radiotherapy. Adenosine triphosphate levels are used as a functional readout.
Patient-derived organoid (PDO) models allow for long-term expansion and maintenance of primary epithelial cells grown in three dimensions and a near-native state. When derived from resected or biopsied tumor tissue, organoids closely recapitulate in vivo tumor morphology and can be used to study therapy response in vitro. Biobanks of tumor organoids reflect the vast variety of clinical tumors and patients and therefore hold great promise for preclinical and clinical applications. This paper presents a method for medium-throughput drug screening using head and neck squamous cell carcinoma and colorectal adenocarcinoma organoids. This approach can easily be adopted for use with any tissue-derived organoid model, both normal and diseased. Methods are described for in vitro exposure of organoids to chemo- and radiotherapy (either as single-treatment modality or in combination). Cell survival after 5 days of drug exposure is assessed by measuring adenosine triphosphate (ATP) levels. Drug sensitivity is measured by the half-maximal inhibitory concentration (IC50), area under the curve (AUC), and growth rate (GR) metrics. These parameters can provide insight into whether an organoid culture is deemed sensitive or resistant to a particular treatment.
Organoid models established from adult stem cells and grown in a three-dimensional (3D) extracellular matrix (ECM) and a specific growth factor cocktail (also known as HUB Organoids) are gaining traction as preclinical oncological screening platforms. Patient-derived organoid (PDO) cultures can be established from both normal and diseased tissue biopsies within 1-2 weeks and can be expanded for a minimum of 1-2 months up to unlimited timespans. Cryopreservation allows for long-term usage of well-characterized cultures. Unlike traditional two-dimensional cell line models that are clonally derived, PDO models closely recapitulate the original tumor tissue, both phenotyp....
NOTE: Before using this protocol, please ensure that the guidelines of the institution's human research ethics committee are followed. Collection of patient tissue and data described in this protocol has been performed following EUREC guidelines and following European, national and local law. All organoids were derived from consenting patients, and consent can be withdrawn at any time.
1. Prior to screening
The aim of this experiment was to examine the sensitivity of HNSCC organoids to chemotherapy and radiotherapy as single agents. We also tested the reproducibility of the results by executing the experiment multiple times with a week's interval, resulting in several biological replicates (experiments 1-3) (Figure 2). Following the protocol, on day 0, HNSCC PDOs were harvested from 6 wells of a 6-well plate and enzymatically and mechanically sheared to single cells (or small organoids <.......
This article and video describe how to perform medium-throughput drug screening using PDOs. This protocol can, with optimization, be adopted to screen organoids derived from different tissue types from those described here. Determining the ideal passage timeframe prior to the screen is important as this will vary for individual organoid cultures and depend on the tissue type. The density and size of organoids seeded per well is an important factor to optimize as faster growing models will require more space within the we.......
|384-well bioluminescence platereader; e.g. Tecan Spark 10M plate reader
|Brightfield microscope with large field of view lens (2.5x)
|Digital dispenser; e.g. Tecan D300e
|6 MV photon beam irradiator
|Elekta model Synergy, Elekta Sweden
|Liquid handler with large nozzle (“standard tube”) cassettes;
|e.g. Multidrop Combi Reagent Dispenser
|Plastic container with plate holder insert for radiotherapy
|Spark control method editor software
|Standard tissue culturing equipment (LAF cabinet, incubator, centrifuge, waterbath, etc)
|1.5 mL plastic tubes
|15- and 50-mL plastic tubes
|5, 10- and 25-mL sterile plastic pipets
|6-well cell culture plates
|Black 384-well ultra-low-attachment clear-bottom plate; e.g.. Corning 384 flat black
|Breathe-Easy sealing membrane
|pre-cut polyurethane medical-grade membrane with acrylic adhesive
|10-chambered slide with hemocytometer grid
|Multidrop Combi Reagent Dispenser standard tube dispensing cassette
|Plugged Pasteur’s pipet of which the tip has been tightened in a flame
|Reversible 20/40/70/100 µm filters: PluriStrainer
|Sterile P1000, P200, P20 and P2 pipet tips and low-retention filter tips ( e.g. Sapphire tips)
|T8 Plus and D4 Plus casettes
|100 x Glutamax
|1 M HEPES
|30% (v/v) Tween-20 diluted in PBS
|CellTiter-Glo 3D cell viability assay
|Compounds to test screen, including Staurosporin or other positive control
|ECM for CRC: growth-factor reduced Matrigel, phenol-free
|ECM for HNSCC PDOs: BME, Cultrex RGF Basement membrane extract, Type R1
|Expansion growth medium (specific for each organoid type)
|Organoid growth factors (specific for each organoid type)
|Pen/Strep (100 U/mL)
|ROCK inhibitor: Y-27632
|Required Software Packages:
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