Protocol to calculate the synergy of drug combinations in organoids and primary cells from murine tumors

Summary

Evaluating the synergy of drug combinations is crucial in advancing treatment regimens. Here, we present a protocol to establish primary cells and organoids from murine tumors and calculate drug synergy. We describe all necessary cell culture procedures, including establishing primary cultures, setting up treatment groups, and detecting cell viability. We then outline how to calculate the synergy score based on a bioinformatical pipeline. This approach applies to any disease model in which a combination of drugs needs to be evaluated.

For complete details on the use and execution of this protocol, please refer to Ku et al.¹

Graphical abstract

Highlights

• •Protocol to establish primary cells and organoids from murine tumors
• •Procedure to measure the drug responses of cells and organoids
• •Steps to calculate drug synergy score

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Evaluating the synergy of drug combinations is crucial in advancing treatment regimens. Here, we present a protocol to establish primary cells and organoids from murine tumors and calculate drug synergy. We describe all necessary cell culture procedures, including establishing primary cultures, setting up treatment groups, and detecting cell viability. We then outline how to calculate the synergy score based on a bioinformatical pipeline. This approach applies to any disease model in which a combination of drugs needs to be evaluated.

Before you begin

A tumor bearing mouse is required to begin with this protocol. The setup of the mouse model is not covered in this protocol and needs to be established prior to proceeding. The protocol below describes all steps using mouse primary pancreatic ductal adenocarcinoma (PDAC) cells and organoids established from the tumor of a KP^fC^ER mouse.² However, we have also used this protocol to determine drug synergy in mouse and human cell lines and human PDAC organoids. In theory, this protocol is applicable to most tissues, primary cells, cell lines, organoids, and other cells kept in vitro with few adaptations. We recommend thawing fresh aliquots of all stock solutions and preparing fresh medium prior to commencing with any downstream applications. For newly established organoids, we recommend passaging and cleaning up the organoids to reduce any potential impact of contaminants or debris on the results. We suggest preparing all buffers in advance and making sure that a sufficient amount of all solutions and reagents is available. Thaw Matrigel at 4°C the day prior to establishing or passaging organoids.

Institutional permissions

All animal experiments included in this protocol were performed with the approval of the IACUC of Korea Advanced Institute of Science and Technology (KAIST) (KA2020-115).

Researchers must acquire institutional permission to work with animals from their local animal ethics committee and receive training for animal handling and basic surgery procedures prior to implementing this protocol.

Download and install R and R studio

Timing: 30 min

This protocol utilizes a bioinformatical pipeline in the R programming language. It is, thus, required to setup R and required packages prior to the execution of the code provided here. RStudio is not strictly required for successfully applying our code, however, it provides a user-friendly interface and makes the analysis more intuitive, especially for researchers with little to no prior knowledge in R. R and RStudio can be downloaded from the official websites at the links below:

R-4.3.2: https://cran.r-project.org/.

R Studio: https://rstudio.com/products/rstudio/.

Download and install required R package

Timing: 10 min

We recommend downloading and installing the required package listed in the “key resources table” prior to starting with the analysis. We further recommend installing the BiocManager package first, followed by installing the remaining package with the code below.

> if (!require("BiocManager", quietly = TRUE))

 install.packages("synergyfinder")

> BiocManager::install("synergyfinder")

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Chemicals, peptides, and recombinant proteins
Dulbecco’s modified Eagle’s medium (DMEM)	Welgene	Cat #: LM001-05
Dulbecco’s modified Eagle’s medium/nutrient mixture F-12 (DMEM/F12)	Welgene	Cat #: LM002-04
Fetal bovine serum (FBS)	Gibco	Cat #: 12483-020
HBSS	Gibco	Cat #: 14175-095
Collagenase XI	Sigma	Cat #: C7657
Dispase	STEMCELL Technologies	Cat #: 07923
TrypLE Express	Gibco	Cat #: 12604021
DNase I	Sigma	Cat #: DN25
Bovine serum albumin (BSA)	Millipore	Cat #: 821006
B27 supplement	Gibco	Cat #: 17504-04
GlutaMAX supplement	Gibco	Cat #: 35050061
HEPES	Duchefa	Cat #: H1504.0100
Penicillin/streptomycin	Gibco	Cat #: 15140-122
Nicotinamide	Sigma-Aldrich	Cat #: N0636
Recombinant human FGF10	PeproTech	Cat #: 100-26
N-acetylcysteine	Sigma-Aldrich	Cat #: A9165
Recombinant murine Noggin	PeproTech	Cat #: 250-38-100
Human epidermal growth factor (EGF)	Merck	Cat #: SRP3027
Gastrin	Sigma-Aldrich	Cat #: G9020
Primocin	InvivoGen	Cat #: ant-pm-2
ROCKi (Y-27632)	Sigma-Aldrich	Cat #: Y0503
Matrigel growth factor reduced (GFR) basement membrane matrix	Corning	Cat #: 354230
Cyto X (cell viability assay kit)	LPS Solution	Cat #: CYT3000
Trypan blue stain (0.4%)	Invitrogen	Cat #: T10282
Recovery cell culture freezing medium	Gibco	Cat #: 12648010
Dimethyl sulfoxide (DMSO)	MP Biomedicals	Cat #: 219605580
Experimental models: Cell lines
MPC-1	Ku et al.1	–
HA-R-Spondin1-Fc 293T	AMSBIO	Cat #: 3710-001-01
Software and algorithms
R (v 4.1.2)	–	https://www.R-project.org
R studio	–	https://rstudio.com
SynergyFinder (v 3.8.2)	Zheng et al.3	–
BioRender	–	https://biorender.com
Other
VersaMax microplate reader	Molecular Devices	–
Countess automated cell counter	Invitrogen	Cat #: AMQAX2000
Centrifuge 5424 R	Eppendorf	Cat #: 540400537
Thermomixer C	Eppendorf	Cat #: 5382000015
Petri dish	SPL	Cat #: 10090
100Ø dish	SPL	Cat #: 20100
6-well dish	SPL	Cat #: 30006
24-well dish	SPL	Cat #: 30024
48-well dish	SPL	Cat #: 30048
96-well dish	SPL	Cat #: 30096
40-μm cell strainer	SPL	Cat #: 93040
22-μm syringe filter	Millipore	Cat #: SLGPR33RS
15 mL conical tube	SPL	Cat #: 50015
50 mL conical tube	SPL	Cat #: 50050
10 mL serological pipette	SPL	Cat #: 91010
5 mL serological pipette	SPL	Cat #: 91005
Cryo vial	Thermo Fisher Scientific	Cat #: 374513
Countess cell counting chamber slides	Invitrogen	Cat #: C10228

Materials and equipment

Wash medium

Reagent	Stock concentration	Final concentration	Amount
DMEM	–	–	490 mL
FBS	100%	1%	5 mL
Penicillin/Streptomycin	100×	1×	5 mL
Total	–	–	500 mL

Note: Store at 4°C for upto 1 month.

2D culture medium for mouse primary cell line (MPC-1)

Reagent	Stock concentration	Final concentration	Amount
DMEM	–	–	445 mL
FBS	100%	10%	50 mL
Penicillin/Streptomycin	100×	1×	5 mL
Total	–	–	500 mL

Note: Store at 4°C for upto 1 month and pre-warm to 37°C using a water bath prior to usage.

Cell freezing medium

Reagent	Stock concentration	Final concentration	Amount
2D culture medium	–	–	0.4 mL
FBS	–	–	0.5 mL
DMSO	–	–	0.1mL
Total	–	–	1 mL

Note: Prepare fresh medium.

Basal organoid medium

Reagent	Stock concentration	Final concentration	Amount
DMEM/F12	–	–	484 mL
GlutaMAX	100×	1×	5 mL
HEPES	1 M	10 mM	5 mL
Penicillin/Streptomycin	100×	1×	5 mL
Primocin	500×	1×	1 mL
Total	–	–	500 mL

Note: Store at 4°C for up to 1 month.

3D culture medium for organoids

Reagent	Stock concentration	Final concentration	Amount
Basal organoid medium	–	–	36.686 mL
R-Spondin 1 conditioned medium	–	–	2 mL
B27 supplement	50×	1×	800 μL
Nicotinamide	1 M	10 mM	400 μL
N-acetylcysteine	1 M	1 mM	40 μL
FGF10	100 μg/mL	100 ng/mL	40 μL
EGF	100 μg/mL	50 ng/mL	20 μL
Noggin	100 μg/mL	25 ng/mL	10 μL
Gastrin	100 μM	10 nM	4 μL
Total	–	–	40 mL

Note: Store supplements at −20°C and complete medium at 4°C and pre-warm to 37°C using a water bath prior to usage. Recommended shelf life of the complete medium is about one week at 4°C.

Note: For the cryo-recovery of organoids, add ROCKi (Y-27632) to the 3D culture medium. 2–3 days post embedding remove ROCKi (Y-27632) and grow organoid in 3D culture medium.

Alternatives: R-Spondin 1 conditioned medium can be substituted with commercially available R-Spondin 1.

Step-by-step method details

Isolation and plating of mouse primary pancreatic cancer cells

Timing: 10 days

This step describes the establishment of a primary cell and organoid culture from a murine tumor.

Tumor dissociation

Timing: 4–5 h

• 1.Sacrifice the mouse, and use sterilized equipment to dissect the pancreas, and isolate the tumor. We typically use tumors around 1.0–2.0 cm³. For bigger tumors, cut the tissue to the appropriate size while avoiding potentially necrotic areas usually located in the center of the tumor.
• 2.Place in a culture dish with cold HBSS and mince the tumor with a sterilized razor blade or scalpel into ∼1–2 mm³ pieces.
• 3.Transfer the tissue pieces into a 15 mL tube in HBSS.

Note: Pre-wet the serological pipette prior to transfer; tissue pieces are sticky.

• 4.Rinse the plate with additional HBSS and transfer any remaining tissue pieces to the 15 mL tube.
• 5.Let the tissues sink to the bottom by gravity.
• 6.Remove the supernatant and wash the tissue pieces with wash medium.

Note: Pre-wet the serological pipette prior to washing; tissue pieces are sticky.

• 7.Let the tissues sink to the bottom by gravity and remove the supernatant.
• 8.Digest the tissues in 10 mL of 0.125 mg/mL Collagenase XI and 0.125 mg/mL Dispase in DMEM containing 1× GlutaMAX and 1% FBS with constant agitation for 2–4 h at 37°C.
• 9.Let the tissues settle by gravity and move the supernatant to a new 15 mL tube.
• 10.Centrifuge the supernatant from step 9 at 250 × g for 5 min at 4°C. Remove the supernatant.
• 11.Resuspend the pellet in 1 mL TrypLE and 1 μL 10 mg/mL DNase I. Troubleshooting 1.
• 12.Incubate at 37°C for 10 min.
• 13.Add 5 mL 2D culture medium.

Primary cell (2D) culture

Timing: 10 days

• 14.Take 1 mL of suspended cells and seed in 8 mL culture medium in a 100 Ø dish. Troubleshooting 2.

Note: The appropriate amount of medium and the size of the culture dish depend on the initial size of the tumor and the digestion efficiency.

• 15.Incubate at 37°C and 5% CO₂.
• 16.After two days, carefully aspirate the old culture medium and replace with fresh medium.

Note: Tumor cells should have adhered to the plate. Any free-floating tissues and cells can be safely removed with the old media.

• 17.Incubate for ∼1 week and change medium when necessary (Figures 1A and 1B).
• 18.
  Carefully monitor cell growth. After ∼1 week the cells should have spread to cover most of the plate. Once the cells have spread, add trypsin to detach the cells.

  • a.
    Aspirate the culture medium and rinse the plate with 4 mL PBS.
  • b.
    Add 1 mL trypsin to the plate and incubate at 37°C for several minutes.
  • c.
    Once the cells have detached from the plate and float, add 4 mL of 2D culture medium to deactivate the trypsin, resuspend, and transfer the cells to a 15 mL tube.

Note: Avoid too short or long incubation times with trypsin, as this will lead to a loss of cells.

• 19.Spin down the cells at 200 g for 3 min and re-seed in an appropriate number of culture dishes. We usually split the cells into 2–3 new culture dishes.
• 20.Incubate at 37°C and 5% CO₂. The cells are now ready to be expanded and used in subsequent experiments. Troubleshooting 3 and 4.

Optional: If the heterogeneity and purity of the primary culture is a concern, cells can be seeded into 96 well plates by serial dilution for single clone expansion. Alternatively, if, as in our case, the mouse model carries a fluorescent marker gene, cells can be FACS-sorted to enrich fluorescent tumor cells only.

Figure 1.

2D cell seeding

(A) Bright-field image of primary PDAC cells near confluency.

(B) Fluorescence image of primary tdTomato-positive PDAC cells. Scale bars 500 μm.

Primary organoid (3D) culture

Timing: 2–3 h

• 21.Centrifuge the remaining cells from step 13 at 200 g for 5 min at 4°C.
• 22.Remove the supernatant and resuspend the pellet in 120 μL of Matrigel.

CRITICAL: Keep Matrigel on ice at all times to avoid solidification.

Note: Residual medium can dilute Matrigel and may prevent proper formation of Matrigel domes. We suggest removing as much medium as possible.

• 23.Seed cells in 40 μL Matrigel per well in a pre-heated 24 well plate (Figures 2A and 2B).

CRITICAL: Avoid bubbles when resuspending and seeding cells.

CRITICAL: Hold the pipette in an upright position and avoid touching the bottom of the cell culture plate.

CRITICAL: Keep the cell/Matrigel mixture on ice and swiftly seed organoids to avoid solidification inside the pipette tip. Pre-cooled tips help prevent this event.

CRITICAL: Periodically resuspend the cell/Matrigel mixture to prevent the settling down of cells. This helps ensure the even distribution of cells and consistent density throughout the experiment.

Note: Preheat the 24 well plate in the incubator for at least 6 h prior to seeding organoids.

• 24.Incubate at 37°C for 0.5–1 h until the Matrigel solidifies. Troubleshooting 6.
• 25.Overlay the Matrigel domes with 500 μL 3D culture medium and return the plate to the incubator. Troubleshooting 7 and 9.

CRITICAL: Avoid direct pipetting to Matrigel domes. We recommend gently dispensing the media along the wall of each well.

Note: We recommend passaging the organoids after 5–7 days of initial seeding (day 0). If organoids are too dense or too large and start deforming and growing out of the Matrigel dome, or if the Matrigel starts dissolving, passage the organoids immediately.

Pause point: After expansion, cells and organoids can be stored in liquid nitrogen for future use. We use Recovery Cell Culture Freezing Medium for the freezing of both primary cells and organoids. However, the freezing of either does not require any special treatment and can be achieved by homemade freezing medium or other commercially available solutions.

Figure 2.

Organoid seeding

(A) Top view of freshly seeded Matrigel domes in 3 wells of a 96 well plate.

(B) Side view schematic of a Matrigel dome.

Calculating and visualizing drug synergy in primary culture systems

Timing: variable

Cell seeding

Timing: 1 h

This step describes the seeding of cells into an appropriate cell culture vessel at the required density.

• 26.
  Harvest cells as described in step 18.

  Note: The amount of PBS and trypsin given applies to a 100 mm culture dish. If other sizes of dishes or flasks are used to maintain cells, these volumes should be adjusted accordingly. The time needed for trypsin to fully disassociate the cells from the dish varies and needs to be adjusted individually.

  • a.
    Flush the cells with 4 mL complete medium and transfer the mixture to a 15 mL tube.
  • b.
    Spin down at 200 g at 4°C, remove the supernatant, and resuspend in 2 mL culture medium.
• 27.
  Count and seed cells. Troubleshooting 8.

  • a.
    Transfer 10 μL of the cell suspension to a 1.5 mL microcentrifuge tube.
  • b.
    Add 10 μL 0.4% Trypan blue stain and mix well by pipetting.
  • c.
    Transfer 10 μL of the mixture to a cell counting slide and insert the slide into the cell counter.

    Note: Avoid bubbles.

    Alternatives: Manually count the cells using a hemacytometer.

  • d.
    Calculate the number of cells per mL in the original cell suspension.
  • e.
    Seed an appropriate number of cells into a suitable number of wells of a 96 well plate in 100 μL complete medium.

    Note: In this protocol, we seeded 1000 cells per well. The exact number depends on the characteristics of each cell line and needs to be adjusted individually. We recommend seeding cells at a density where untreated cells reach near confluency at the endpoint of the experiment.

• 28.Place cells in the incubator for 24 h.

Organoid seeding

Timing: 2–3 h

This step describes the seeding of organoids into an appropriate cell culture vessel at the required density.

• 29.
  Harvest organoids.

  • a.
    Aspirate the culture medium.
  • b.
    Collect organoids by resuspending Matrigel domes with 1 mL 0.1% BSA in PBS and transferring them to a 1.5 mL tube.

    Note: To avoid contamination, sterilize 0.1% BSA in PBS with a 0.22-μm syringe filter before use. We also recommend using pre-cooled 0.1% BSA in PBS.

    Note: We recommend combining organoids from 2 - 3 wells depending on the density and number of organoids. Avoid collecting too many wells or organoids in one tube, as this can result in the insufficient removal of old Matrigel, the loss of organoids, and might negatively impact their dissociation.

    Note: Scraping and resuspending Matrigel domes with 0.1% BSA in PBS using a blunted 1000 mL tip helps separate the organoids from Matrigel.

    Note: Rinsing the wells with an additional 500 μL 0.1% BSA in PBS minimizes the loss during this process.

  • c.
    Centrifuge at 250 g for 5 min at 4°C.

    Note: Three layers will be visible; supernatant (top), Matrigel (middle), and organoids (bottom) (Figures 3A and 3B).

  • d.
    Carefully remove the supernatant and Matrigel using a pipette.

    Alternatives: The supernatant and Matrigel can be removed using a vacuum aspirator, however, we recommend using a pipette to minimize the loss of organoids.

• 30.
  Single cell dissociation of organoids. Troubleshooting 5.

  • a.
    Add 500 μL TrypLE to the tube and physically disrupt organoids through vigorous pipetting.

    Note: Pre-wet tips prior to the resuspension of organoids to prevent organoids from sticking to the inside of the tips.

    Note: Pipetting with a blunted 1000 mL tip aids with the physical disruption of large particles and the dissociation of the organoids.

  • b.
    Incubate at 37°C for 1–1.5 min.

    Note: Adjust the incubation time according to the degree of dissociation. Prolonged incubation can reduce the viability of the cells.

  • c.
    Add the same amount (500 μL) of basal medium to inactivate TrypLE.
  • d.
    Centrifuge at 250 g for 5 min at 4°C.
  • e.
    Remove the supernatant.

    Note: Repeat steps a - e if dissociation is insufficient.

• 31.
  Organoid seeding. Troubleshooting 8.

  • a.
    Count and calculate the number of cells per mL in the original cell suspension.
  • b.
    Seed the appropriate number of cells into a suitable number of wells of a 48 well plate in 20 μL Matrigel.

Note: In this protocol, we seeded 5000 cells per well. However, the exact number depends on the characteristics of the organoids and needs to be adjusted individually.

• 32.Incubate at 37°C for 0.5–1 h until the Matrigel solidifies. Troubleshooting 6.
• 33.Overlay the Matrigel domes with 200 μL 3D culture medium and return the plate to the incubator (Figures 4A and 4B). Troubleshooting 7 and 9.

Figure 3.

Organoid passaging

(A) Freshly harvested and spun down organoids showing three separate layers: medium (top), Matrigel (middle), cells (bottom).

(B) Schematic of the picture shown in A.

Figure 4.

Organoid culture

(A) Brightfield image of a fully grown PDAC organoid.

(B) Fluorescence image of a fully grown tdTomato-positive PDAC organoid. Scale bars 50 μm.

Drug treatment

Timing: 30 min

This step describes the addition of the drugs of interest to the cells or organoids.

• 34.Prepare drug dilutions in 200 μL complete medium per well (Figure 5). We recommend preparing a master mix for each treatment condition.

Note: Set up 5–6 dilution points based on the IC₅₀ value of each drug.

Note: We recommend performing triplicates for each experimental condition.

• 35.Aspirate the culture medium completely.
• 36.Add 200 μL of the diluted drugs to the corresponding wells.

Note: Avoid using the wells at the edge of the culture dish to circumvent any potential evaporation of the culture medium.

Note: For drug combinations, thoroughly mix the first drug with the culture medium before adding the second drug to ensure a homogenous solution and accurate results.

Note: We recommend using wells with properly formed Matrigel domes to maintain the quality and reproducibility of the drug response.

• 37.Place the cell culture plate in the incubator.

Note: The time of incubation depends on the characteristics of the drugs and drug combinations used. We recommend testing the optimal dose and duration of each drug and drug combination before proceeding. Organoids generally require longer incubation times and a higher dosage due to the Matrigel dome.

Figure 5.

Drug treatment in 96 well plates

Layout of a 96 well plate prepared for cell viability measurements. Drug 1 is colored in blue, drug 2 is colored in yellow. Darker colors indicate increasing drug concentrations.

Measuring cell viability

Timing: 2–3 h

This step describes the determination of cell viability of cells and organoids.

Note: In this protocol, we have used the Cyto X Cell Viability Assay kit. In theory, any commercially available kit can be used to determine cell viability.

Alternatives: Instead of using a kit, an automated live cell counting imaging system may be used. If neither is available cell viability may be determined manually by counting Trypan blue-treated cells using a hemocytometer.

• 38.Prepare the cell viability reagent mixed medium.
• 39.Completely aspirate culture medium.
• 40.Add 200 μL of the cell viability reagent mixed medium.

Note: We recommend preparing a master mix.

Note: Add medium as quickly as possible using a multichannel pipette, especially when handling a large number of wells. In this protocol, we utilize a colorimetric assay to measure cell viability, and delays may result in inaccurate results.

CRITICAL: Direct pipetting to the bottom of the plate may result in cell detachment. Direct pipetting to the Matrigel domes may disintegrate or detach them.

• 41.Incubate at 37°C for 1–2 h.

CRITICAL: For accurate cell viability calculations, incubate mixed medium in 2–3 cell-free wells.

• 42.Measure the OD at 450 nm using a plate reader. Troubleshooting 10 and 11.

Note: Transfer 100 μL of cell viability mixture from the organoid culture plates to a new 96 well plate before measuring the OD.

Note: This concludes all cell culture experiments. After this step, researchers should have recorded cell viability for each treatment condition in cells or organoids.

Pause point: After recording cell viability, data can be stored and analyzed later.

Calculating drug synergy

Timing: 1 h

This step describes how to calculate the synergy score of two drugs using the values determined experimentally in the previous steps.

• 43.Calculate relative cell viability. Troubleshooting 12.

$$\text{Relative}\text{viability}(\%)=\frac{\text{OD}\text{value}\left(\text{sample}\text{sample}\right)-\text{OD}\text{value}\left(\text{blank}\right)}{\text{OD}\text{value}\left(\text{control}\text{sample}\right)-\text{OD}\text{value}\left(\text{blank}\right)}\times 100$$

• 44.Generate input data table (Table 1).

Note: Columns of the input table should include; block_id (sample or pair name), drug1 (name of drug 1), drug2 (name of drug 2), conc1 (concentration of drug 1), conc2 (concentration of drug 2), response (% value of relative viability), and conc_unit (unit of concentration). Save the input table as a CSV file.

• 45.
  Run the code below to calculate synergy scores. Troubleshooting 13.

  • a.
    Load library and input files.

    > library(synergyfinder)

    > input <- read.csv("input data table name.csv")
  • b.
    Reshape data and calculate synergy scores.

    > result <- ReshapeData(input, data_type="viability", impute = T, impute_method=NULL, noise=T, seed=1)

    > result <-CalculateSynergy(result, method=c("HSA","Bliss","Loewe","ZIP"), correct_baseline="non")

    > result <-CalculateSensitivity(result, correct_baseline="non")

    Note: Choose appropriate synergy and baseline correction method.

  • c.
    Plot response and synergy scores.

    > plot2DrugHeatmap(result, drugs=c(1,2), plot_block ="block_id", plot_value = "response_origin")

    > plot2DrugSurface(result, drugs=c(1,2), plot_block ="block_id", plot_value = "(synergy method)_synergy")

    Note: The range of the plot can be changed by adding z_range = c(“lower limit”, “upper limit”).

    Note: The color of the synergy score plots can be changed by adding low/high_value_color = “R Color code”.

    Note: Generally, a score larger than 10 is considered synergistic, between −10 to 10 is considered additive, and less than −10 is considered antagonistic.

    Note: For complete details on synergy calculation, please refer to the user tutorial of Zheng et al.³

Table 1.

Template excel file of input data to calculate the synergy score

block_id	drug1	drug2	conc1	conc2	Response	conc_unit

Example of input data (related to step 44). block_id, name of samples (i.e., A, 1, sample1, ....); drug1, name or abbreviation of drug 1 (i.e., gemcitabine, gem, g, ...); drug2, name or abbreviation of drug 2 (i.e., paclitaxel, pac, p, ...); conc1: concentration of drug 1, without concentration unit; conc2, concentration of drug 2, without concentration unit; response, calculated viability; conc_unit, concentration unit of both drugs (mM, μM, nM, .. ), Note that unit should be consistent between drugs. Input table should be saved as .csv format.

Expected outcomes

Drug sensitivity may differ in mouse models from different genetic backgrounds, and established cell lines do not always reflect this specificity and heterogeneity. Moreover, drug synergy cannot be reliably calculated in vivo and requires in vitro cultures instead. This protocol provides a quick and easy way to establish primary cell and organoid cultures from a mouse tumor for in vitro studies (Figures 1 and 4). We also provide instructions how to perform drug synergy experiments and the computational code required to execute synergy calculations in R. Primary cultures are a powerful tool to study the biology, drug response, and resistance mechanisms of cancer cells. The cultures established following our protocol can be utilized not only for drug synergy calculations, but also drug screens, genetic experiments, and xenografts.

Limitations

This protocol describes the establishment of primary cells and organoids from murine PDAC tumors. While the basic principle of the protocol should be applicable to many other types of tumors, adjustments will be needed. For example, some cells may require coating of the culture dish prior to seeding, different types of organoids require specific medium supplements, and digestion times have to be adjusted to the characteristics of the given tissue. Furthermore, longevity of the established cultures may vary in different genetic backgrounds and culture conditions. Lastly, the appropriate range of drug concentrations will need to be determined for each cell line and organoid, owing to their individual backgrounds. While the code we provide to calculate drug synergy in R can be utilized directly, some basic knowledge of the R programming language may be required for importing and exporting data and downloading plots.

Troubleshooting

Problem 1

Cell clumps are formed in step 11.

Potential solution

We observed that adding DNase I directly to cell resuspensions and vigorous shaking may result in clump formation. We suggest carefully preparing a mix of TrypLE and DNase I in advance and resuspending the cell pellets gently.

Problem 2

The yield of primary cells is too low in step 14.

Potential solution

Use more tissue and mince more thoroughly in step 2.

Problem 3

Primary cells are highly heterogeneous or show contamination with other cell types after completing step 20.

Potential solution

After establishing the 2D primary culture and ensuring their continued growth, single clones can be expanded by serial dilutions in 96 well plates. Our mouse model included a fluorescent tdTomato reporter protein marking all mutant cells. If such a fluorescent marker is present, cells can by FACS-sorted to remove any non-fluorescent cells from the culture and obtain a pure culture of cancer cells.

Problem 4

Primary cells are not growing well after completing primary cell culture step 20.

Potential solution

Some cells may require a higher concentration of FBS, especially in the beginning of culture. FBS may be increased to up to 20% and gradually decreased once the cells adjust to the cell culture conditions.

Problem 5

Cell viability is too low after organoid dissociation in step 30.

Potential solution

Reduce the incubation time with TrypLE and minimize pipetting.

Problem 6

Matrigel domes are not properly formed in steps 24 and 32.

Potential solution

• •Matrigel domes are not fully solidified.

Incubate longer when solidifying the Matrigel. Pre-incubation of plates prior to seeding also helps with the solidification of Matrigel.

• •Medium is not properly removed before resuspending with Matrigel.

Remove residual medium as much as possible.

• •Too many cells are seeded.

Resuspend in a larger amount of Matrigel or dilute cells prior to seeding.

Problem 7

Organoids are slow to grow and expand after completing organoid culture steps 25 and 33.

Potential solution

Cancer organoids can exhibit a slower growth rate than normal organoids and sometimes are smaller and show a more disrupted morphology. For the initial seeding of single cells, it is paramount to add the Rho kinase inhibitor, ROCKi (Y-27632), to the 3D culture medium for organoids to maintain cell viability. Once organoids have formed and are routinely passaged, the inhibitor can be omitted.

Problem 8

Cell density is not consistent in steps 27 and 31.

Potential solution

We recommend performing serial dilutions of cell suspensions when handling a large number of wells. It is important to periodically resuspend cells when seeding to keep the cells from settling at the bottom of the tube.

Problem 9

Matrigel domes dissolve in culture.

Potential solution

• •Remove medium completely before resuspending the organoids with Matrigel.
• •Reduce the number of organoids per well.
• •Passage organoids before they become overgrown.
• •Reseed organoids by performing steps from 29 to 33.

Problem 10

The drug response curve or IC₅₀ value is irregular.

Potential solution

We recommend testing the drug response in several different concentrations around the known IC₅₀ value. However, the drug response or IC₅₀ value may vary depending on the context and in different cell lines and organoids. If the results remain atypical, test at broader range of concentrations and narrow down the range to find appropriate concentrations.

Problem 11

Cells or organoids are dying even at low doses of drugs in step 42.

Potential solution

It is important to identify the starting concentration for each drug for each type of cell or organoid prior to synergy calculations, as individual cultures can exhibit significantly different sensitivities to different types of drugs.

Problem 12

The differences within replicates are too large in steps 43.

Potential solution

• •Make sure cells are seeded at the same density in all wells.
• •Properly mix all reagents before adding to the wells.
• •Avoid pipetting error when adding medium.
• •To avoid evaporation, do not use the wells at the edge of plate.

Problem 13

Packages in R are not loading properly or giving an error message in step 45.

Potential solution

Make sure the suggested version of R, R Studio, and all required packages, including any dependencies, are installed. When copying bits of code, ensure to not omit or change any lines of code or individual symbols.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Dae-Sik Lim (daesiklim@kaist.ac.kr).

Technical contact

Technical questions on executing this protocol should be directed to and will be answered by the technical contacts, David Eisenbarth (deisenb@iu.edu) and Bomin Ku (gubomin@kaist.ac.kr).

Materials availability

This study did not generate new unique reagents.

Data and code availability

This study did not generate datasets.

Acknowledgments

This work was supported by a grant from the National Research Foundation of Korea (NRF), funded by the Ministry of Science and ICT (MSIT; 2020R1A3B2079551 to D.-S.L.). The graphical abstract and schematic figures were created with Biorender.com.

Author contributions

D.E. and B.K., conceptualization, methodology, validation, data curation, investigation, formal analysis, visualization, writing – original draft, and writing – review and editing. D.-S.L., writing – original draft, writing – review and editing, supervision, and funding acquisition.

Declaration of interests

The authors declare no competing interests.