Protocol to profile the bioenergetics of organoids using Seahorse (2024)

Addressing bioenergetics is key to evaluate the impact of metabolism on the regulation of biological processes and its alteration in disease. Organoids are in vitro grown self-organizing structures derived from healthy and diseased tissue that recapitulate with high fidelity the tissue of origin. Bioenergetics is commonly analyzed by Seahorse XF analysis. However, its application to organoid studies is technically challenging. Here, we share our in-house optimized protocols to examine organoid bioenergetics in response to drugs, gene knockdown, or to characterize the metabolism of specific cell types.

For complete details on the use and execution of this protocol, please refer to Ludikhuize etal. (2020).

• •

  Analysis of bioenergetics in organoids is technically challenging

• •

  We share a detailed protocol to overcome the pitfalls of Seahorse analysis in organoids

• •

  Accurate timing of organoid re-plating improves the robustness of Seahorse analysis

• •

  DNA-based normalization overcomes organoid-related limitations for normalization

Table 1

Different applications for profiling bioenergetics in organoids

Seahorse XF analyzer

Agilent provides multiple Seahorse XF Analyzers (8 well, 24 well, or 96 well systems). Our protocol is optimized for the Seahorse XFe24 system (24 wells). General manufacturer’s instructions for performing a mitochondrial or glycolysis stress test can be respectively found at https://www.agilent.com/cs/library/usermanuals/public/XF_Cell_Mito_Stress_Test_Kit_User_Guide.pdf and https://www.agilent.com/cs/library/usermanuals/public/XF_Glycolysis_Stress_Test_Kit_User_Guide.pdf.

Alternatives: Seahorse XFe96 (96 wells) or Seahorse XF HS mini (8 wells) Analyzers can be alternatively used.

Preparation of assay medium

In this protocol, we prepare assay medium with base medium with phenol red (Agilent, #102353-100) and we buffer it to pH 7.4 with NaOH. This medium can be stored for 24h at 4°C.

Alternatives: Assay medium can be prepared based on different types of media. For Agilent’s recommendation on alternative media visit: https://www.agilent.com/cs/library/selectionguide/public/5991-7878EN.pdf

Preparation of stocks of substrates and inhibitors

Stocks of substrates and inhibitors can be prepared in advance by dissolving them as depicted below. Glucose can be stored at 4°C. All other stocks should be stored at −20°C. All stocks can be stored for at least six months.

For mitochondrial stress tests, prepare the following stocks:

• 2.5mM Oligomycin in DMSO

• 2.5mM FCCP in DMSO (prepare in fume hood)

• 2.5mM Rotenone in DMSO

• 2.5mM Antimycin A in DMSO

For glycolysis stress tests, prepare the following stocks:

• 2.5mM Oligomycin in DMSO

• 1M glucose in H₂O

DNA-based normalization

For normalization, we isolate and quantify organoid DNA by using the QIAamp DNA Micro Kit. Although alternative DNA extraction methods could be applied, the expected amounts of purified DNA are low in concentration, hence the method needs to be able to account for that.

Culturing organoids

Timing: 1–2weeks

Organoid growth rates vary depending on the tissue of origin, differentiation rates, and genetic mutations. To achieve an accurate and reliable assay, the number of organoids per well needs to be comparable within and across conditions. To better control for differences in growth rates, we first expand our organoids in normal culture plates and re-plate the organoids in Seahorse Cell Culture microplates plates 24–72h prior to analysis. This re-plating step is meant to better control for the differences in number and size of the organoids between the conditions to be compared on the day of the Seahorse analysis. Thus, this timing should be adjusted depending on the growth rate of the organoid type. For mouse small intestine-, human colon- or colorectal tumor-derived organoids, 36–48h prior to analysis is an adequate re-plating time.

When long treatments are needed, for instance for organoid differentiation protocols, the treatments can be started during normal culture and continued after re-plating into Seahorse microplates. Here, we introduce our culturing methods for organoids derived from mouse small intestine, human colon, and colorectal tumors.

For mouse intestinal organoids:

• 1.

  Disrupt organoids by mechanical shearing and plate them in a 24-well plate.

  • a.

    Collect organoids in 1mL of ice cold DMEM F12 advanced medium supplemented with penicillin/streptomycin, 10mM HEPES, and 20mM glutamax (+++ medium).

    Note: Collecting 100μL of organoids in Matrigel at the density depicted in Figure2Ci is sufficient.

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    Figure2

    Organoid culturing

    (A) 1,000μL tip on a 1,000μL pipet.

    (B) 200μL tip on top of a 1,000μL tip.

    (C) Mouse small intestinal organoids before splitting, after splitting, and before re-plating.

    (D) P16t colorectal tumor-derived organoids before trypsinization, after trypsinization, and before re-plating.

  • b.

    Disrupt organoids by pipetting up and down with a 1000μL pipet.

    • i.

      First pipet up and down with a single 1,000μL tip (Figure2A).

    • ii.

      When organoids are not disrupted yet, pipet up and down with a 200μL pipet tip on top of a 1,000μL pipet tip (Figure2B) until organoids are broken into small crypts. The grade of mechanical shearing of the organoids should be monitored and visualized in between steps using a microscope.

      Note: To prevent organoids from sticking to the pipet tips, pre-wet the pipet tips by pipetting op and down with cold+++ medium before starting the mechanical shearing.

    • iii.

      Add 5mL of ice cold+++ medium to the organoids.

      See Also

      Seahorse XF Assay Journey | AgilentSeahorse Assays Normalisation | BMG LABTECHMeasurement of mitochondrial respiration in adherent cells by Seahorse XF96 Cell Mito Stress Test

  • c.

    Centrifuge at 140–220× g at 4°C for 5min (Thermo Scientific SL40R centrifuge with Thermo Scientific 75003607 rotor, 800–1,000rpm).

  • d.

    Re-suspend the pellet in 200–300μL of Matrigel to reach the following density (Figure2Cii)

    • i.

      200μL of organoids in Matrigel is sufficient for the later re-plating step in the Seahorse cell culture microplate.

  • e.

    Plate organoids in a 24-well plate and culture for 36–48 h.

    • i.

      Plate 50μL Matrigel per well divided in 3–4 droplets.

  • f.

    Incubate the plate upside down at 37°C for 30min.

  • g.

    Add culture medium.

• 2.

  Culture organoids for 36–48h prior to re-plating them in a Seahorse Cell Culture microplate (Figure2Ciii).

Note: The time of culturing before re-plating organoids in Seahorse microplate largely relies on the growth rate of the organoids. As an example of this, medium composition favoring organoid expansion by preventing differentiation, such as Wnt addition to mouse small intestinal organoids (Rodríguez-Colman etal., 2017), requires a shorter culture time from splitting to re-plating. In addition to that, the selection of the culture timing should be adjusted to properly address the research question.

For colorectal cancer or colon organoids:

• 3.

  Disrupt organoids by trypsinization and plate in a 24-well plate.

  • a.

    Collect organoids in 10mL of ice cold+++ medium.

    Note: Collecting 50μL of organoids in Matrigel in the following density (Figure2Di) will be sufficient.

  • b.

    Centrifuge at 140–220× g at 4°C for 5min (Thermo Scientific SL40R centrifuge with Thermo Scientific 75003607 rotor, 800–1,000rpm).

  • c.

    Trypsinize the organoids in 2mL of trypsin or trypLE solution until organoids are disrupted (single cells or small clumps of cells depending on the organoid line).

  • d.

    Add 10mL of ice cold+++ medium.

  • e.

    Centrifuge at 220–320× g at 4°C for 5min (1,000–1,200rpm).

  • f.

    Remove supernatant and add 10mL of ice cold+++ medium.

  • g.

    Centrifuge at 220–320× g at 4°C for 5min (1,000–1,200rpm).

  • h.

    Re-suspend the pellet in 150–300μL Matrigel to reach the following density (Figure2Dii).

    • i.

      200μL of organoids in Matrigel is sufficient for the later re-plating step in the Seahorse cell culture microplate.

  • i.

    Plate organoids in a 24-well plate

    • i.

      Plate 50μL Matrigel per well, divided in 3–4 droplets.

  • j.

    Incubate the plate upside down at 37°C for 30min.

  • k.

    Add culture medium.

• 4.

  Culture organoids until the desired size prior to re-plating them in a Seahorse Cell Culture microplate (Figure2Diii). Check Note in step 2 for a comment on this.

Plate organoids for analysis

Timing: 1–3days

Seahorse analysis has to be performed by plating organoids in Seahorse XF24 V28 PS cell culture microplates (Agilent, #100882-004) (Figure3A). Below we describe step-by-step how to accurately re-plate organoids to guarantee reliable measurements.

• 5.

  At least 24h before plating, incubate the Seahorse XF24 V28 PS cell culture microplate to be used and a flask filled with water (Figure3B) at 37°C. This step enhances the adhesion of the Matrigel drop to the bottom of the plate.

• 6.

  Collect the previously cultured organoids in 1mL of ice cold+++ medium.

Note: It is important not to disrupt the integrity of the organoids while collecting. For mouse small intestinal organoids cultured for 36h on medium supplemented with EGF, Noggin, and R-spondin, it is recommended to collect 4 wells of a 24-well plate (50μL Matrigel each) to fill 20 wells of a Seahorse microplate. For colon- or colorectal tumor-derived organoids cultured for 7days after trypsinization, it is recommended to collect 1 well of a 24-well plate to fill 20 wells of a Seahorse microplate.

• 7.

  Centrifuge at 140–220× g at 4°C for 5min (800–1,000rpm).

Note: Depending on the organoid type, centrifugation can affect organoid integrity. Choose the gravitational force that efficiently pulls down the organoids, but does not cause organoid disruption.

• 8.

  Re-suspend the organoid pellet in a volume of Matrigel that leads to the appropriate organoid density (steps b–d).

  • a.

    It is important to reduce the variability in organoid density across and within experiments.

  • b.

    For mouse small intestine- and colon- or colorectal tumor-derived organoids, 20–40 organoids per well should be plated to measure OCR and ECAR within the range of sensitivity of the method (Figures 4A and 4B). Organoid density can be further adjusted to the specific requirements of the organoid type, under the consideration that the obtained measurements are within the range of sensitivity of the method (see CRITICAL).

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    Figure4

    Intestine-derived organoids in a Seahorse cell culture microplate and DNA quantification

    (A and B) Organoids plated in a Seahorse cell culture plate. Organoid densities have been adjusted to fit the requirements of the organoid type. Mouse intestinal organoids undergo crypt formation-based morphological changes and are plated at a lower density than wild-type colon and colorectal tumor organoids. The depicted densities result in OCR and ECAR measurements within the sensitive range. (A) Mouse small intestinal shFoxo1/3 organoids treated with doxycycline or Mdivi-1 for 36 h. (B) Wild-type colon and colorectal tumor-derived organoids P9t, P16t, and P19bt. In (A) and (B), yellow crosses indicate control wells.

    (C) DNA quantification of mouse small intestine- and human colon- and colorectal tumor-derived organoids in (A) and (B).

  • c.

    As a guideline for mouse small intestinal organoids, collect 4 wells of a 24-plate, start by re-suspending the organoids in 100μL of Matrigel and dilute further when necessary.

  • d.

    As a guideline for colon- or colorectal tumor-derived organoids, collect 1 well of a 24-plate, start by re-suspending the organoids in 150μL of Matrigel and dilute further when necessary.

CRITICAL: Small volumes of Matrigel rapidly polymerize at room temperature. Prevent this by keeping the organoid-containing tubes on ice at all possible times. The density of organoids in Matrigel droplet needs attention: if the density of the organoids is too low or too high, the sensitivity of the method may limit the reliability of the measurements. ECAR can be reliably measured between 20 and 200 mpH/min. OCR can be reliably measured between 100 and 1,000 pmol O₂/min (higher OCR measurements could be considered upon validation).

• 9.

  Plate organoids in a Seahorse Cell Culture Microplate [Troubleshooting 1] [Troubleshooting 2].

  • a.

    Place the Seahorse microplate on top of the pre-warmed water-filled flask (Figure3B).

    Note: Placing the microplate on top of a warm surface facilitates plating 3μL Matrigel droplets in the middle of Seahorse Cell Culture as the warmer temperature enhances the stability of the droplet, as such, and accelerates its polymerization.

  • b.

    Plate 3μL of Matrigel (without organoids) in 4 control wells (Figures 4A and 4B).

    Note: OCR and ECAR measurements are sensitive to the presence of Matrigel. Therefore, in control wells Matrigel should also be plated in the same volume.

  • c.

    Plate droplets of 3μL Matrigel with organoids in the other wells (Figures 3C, ​C,4A,4A, and 4B).

    Note: For an accurate assessment of the bioenergetic parameters, we suggest to have four to five wells per condition.

  • d.

    After plating, place the Seahorse microplate plate back in the incubator for 10–15min at 37°C.

    CRITICAL: Normal plating of organoids involves turning the plate upside down. Here that step should be avoided to prevent disruption of the Matrigel droplets. As small volumes of Matrigel polymerize fast, do not leave the plate for longer than the stated time to prevent drying out of the Matrigel droplet.

  • e.

    Add 200μL of culture medium in each well.

    CRITICAL: Due to the limited plating area, organoids should be plated in the middle of the well and in between the three nodes (Figure3C). Inaccurate plating leads to its adherence to the walls of the well and consequently to inaccurate measurements.

• 10.

  When required add the treatment of interest to the selected wells.

• 11.

  Culture organoids in the Seahorse microplate for 1–3days until a mitochondrial or glycolysis stress test is performed.

Note: As described in the “culturing organoids” section, this time frame is dependent on the growth rates of the organoids and the desired effect of the optional treatment.

Cartridge hydration

Timing: 4–72 h

Each probe tip of the sensor cartridge contains sensor material that detects changes in O₂ concentration and pH (Figure3D). For correct functioning, the probe tips of the sensor cartridge have to be hydrated in XF Calibrant for a minimum of 4h and a maximum of 72 h. For practical reasons we recommend to hydrate the cartridge for 12 to 24 h.

• 12.

  Lift the sensor cartridge from the Utility plate, and pipet 1mL of XF Calibrant into each well of the Utility plate (Figure3D).

• 13.

  Place the sensor cartridge back on the Utility plate with the Hydro Booster in between, in such a way that the probe tips of the Sensor Cartridge are submerged in XF Calibrant.

• 14.

  Place the Sensor Cartridge in a non-CO₂ 37°C humidified incubator for at least 4 h, but at maximum for 72 h.

XF mitochondrial stress test or glycolysis stress test

Timing: 3 h

Agilent offers multiple assays to analyze different bioenergetic parameters. We have optimized the assay medium composition, the concentrations of substrates and inhibitors and the measurement settings for the XF Analyzer for performing mitochondrial and glycolysis stress tests in mouse small intestine, colon, and colorectal tumor-derived organoids.

• 15.

  Take pictures of the organoids in all wells of the Seahorse microplate (Figures 3C, ​C,4A,4A, and 4B) by a bright-field microscope.

Note: Imaging the organoids helps to control for viability and to compare differences in organoid growth and morphology across conditions and among experiments.

• 16.

  Prepare 50mL assay medium.

  • a.

    In this protocol, we prepare assay medium with base medium with phenol red (Agilent, #102353-100) and buffer it to pH 7.4 with NaOH. Assay medium needs to be supplemented with different metabolic substrates as indicated in Tables 2 and ​and3,3, and can be stored for 24h at 4°C.

    Table 2

    Assay medium mitochondrial stress test

    Reagent	Default assay medium compositiona	Optimized assay medium composition for intestine-derived organoidsb	Volumes to prepare 50mL optimized assay medium
    Glucose (1 M)	10mM	10mM	0.5mL
    Pyruvate (100mM)	1mM	5mM	2.5mL
    Glutamine (200mM)	2mM	2mM	0.5mL
    NaOH (1 M)	Adjust to pH 7.4	Adjust to pH 7.4	25μL
    Base medium	n/a	n/a	46.5mL
    Total	n/a	n/a	50mL

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    ^aDefault assay medium composition suggested by Agilent.

    ^bAssay medium composition optimized for mouse small intestine- and human colon- or colorectal tumor-derived organoids.

    Table 3

    Assay medium glycolysis stress test

    Reagent	Default assay medium compositiona	Optimized assay medium composition for intestine-derived organoidsb	Volumes to prepare 50mL optimized assay medium
    Glutamine (200mM)	2mM	2mM	0.5mL
    NaOH (1 M)	Adjust to pH 7.4	Adjust to pH 7.4	25μL
    Base medium	n/a	n/a	49.5mL
    Total	n/a	n/a	50mL

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    ^aDefault assay medium composition suggested by Agilent.

    ^bAssay medium composition optimized for mouse small intestine- and human colon- or colorectal tumor-derived organoids.

  • b.

    For a mitochondrial stress test, prepare assay medium according to Table 2.

  • c.

    For a glycolysis stress test, prepare assay medium according to Table 3.

Note: Following Agilent’s recommendations, substrates should be provided in saturating concentrations to ensure that they will not be limiting OCR and ECAR measurements. In Tables 2 and ​and33 we have listed the saturating substrate concentrations adjusted for both, mouse small intestine- and human colon- or colorectal tumor-derived organoid analysis. Note that these concentrations are organoid type-specific and need to be determined empirically when applied to other organoid types.

• 17.

  Prepare organoids for the XF analysis.

  • a.

    Remove the culture medium from the organoids.

  • b.

    Carefully wash organoids twice with 500μL assay medium.

  • c.

    Add 525μL assay medium into each well.

  • d.

    Incubate organoids in an incubator without CO₂ control at 37°C for at least 30min, but at maximum 1 h.

Optional: When analyzing the effect of drugs/treatment on bioenergetics, it could be necessary to maintain the treatment during the assay. This needs to be decided depending on the mechanism of action of the drug and the research question. Note that the assay medium is not buffered. Therefore, depending on the compound type, it can be required to readjust the pH of the compound’s stock.

• 18.

  During the incubation time of organoids in assay medium, prepare the injection solutions. Injectionsshould be prepared in assay medium and 75μL of these injections should be pipetted in the injection ports (Figure3D). The device will inject the drugs in the order of port A to port D.

  • a.

    For a standard mitochondrial stress test, prepare Oligomycin, FCCP and Rotenone & Antimycin A and inject 75μL of each in port A, B, and C, respectively (Table 4 and Figure3D).

    Table 4

    Mitochondrial stress test injections optimized for mouse small intestine-, human colon- and colorectal tumor-derived organoids

    Injection port	Drug	Final concentration in Seahorse plate	Injection concentration	To add from 2.5mM stock (μL)	XF assay medium (μL)
    Port A	Oligomycin	5μM	40μM	32	1,968
    Port B	FCCP	2μM	18μM	14.4	1,985.6
    Port C	Rotenone and Antimycin A	2μM+ 2μM	20μM	16 (each)	1.68
    Port D

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  • b.

    For a standard glycolysis stress test, prepare glucose, Oligomycin and 2-deoxyglucose (2-DG) and inject 75μL of each in port A, B, and C, respectively (Table 5 and Figure3D).

    Table 5

    Glycolysis stress test injections optimized for mouse small intestine-, human colon- and colorectal tumor-derived organoids

    Injection port	Drug	Final concentration in Seahorse plate	Injection concentration	To add from stock	XF assay medium (μL)
    Port A	Glucose	10mM	80mM	160μL	1,840
    Port B	Oligomycin	5μM	45μM	36μL	1,964
    Port C	2-DGa	100mM	1 M	333mg	2,000
    Port D

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    ^a2-Deoxyglucose solutions are not stable over time and should be dissolved on the day of performing the glycolysis stress test

  • c.

    Upon completion of preparation of the injection ports, place the sensor cartridge hold in the Utility plate back into the incubator.

Note: When other organoid types are used, optimal substrate and inhibitor concentrations have to be determined. We recommend to start with titrating Oligomycin, followed by respectively FCCP and Rotenone and Antimycin A. The lowest concentration that causes maximal inhibition of every drug should be selected. For an indication of inhibitor concentrations used for cell lines, check the Seahorse XF Analyzer guides: https://www.agilent.com/en/support/cell-analysis/seahorse-assay-guides-templates

Optional: For analysis of the immediate metabolic response to a certain drug, drugs can be injected during the mitochondrial or glycolysis stress test via the injection ports (Figures 1B and 1C). For such a bioenergetics assay, use injection port A for the drug of interest and move the injections corresponding to the mitochondrial or glycolysis stress test to port B, C and D respectively (Figure3D). An example of such an application in a mitochondrial stress test, can be found in our recent study (Ludikhuize etal., 2020). Note that the concentrations of the inhibitors in injection ports need to be adjusted according to the change in the volume resulting from adding preceding injection step in A. Based on the research question, the order of the substrates and inhibitors can also be re-arranged. Then, the experimental settings can be updated in the Wave software accordingly.

• 19.

  Prepare settings of the XF Analyzer.

  • a.

    Open the Wave software.

  • b.

    Open a mitochondrial or glycolysis stress test template, and fill in the assay details. A detailed explanation on how to create and customize assay template files using the Wave software can be found in section 2.4 in: https://www.agilent.com/en/product/cell-analysis/how-to-run-an-assay.

  • c.

    Make sure you adjust the XF analysis settings in the “Instrument Protocol” section.

    • i.

      For optimized mitochondrial stress test settings for mouse small intestine- and colon- or colorectal tumor-derived organoids, see Table 6.

      Table 6

      Settings mitochondrial stress test

      Settings			Defaulta	Intestine-derived organoidsb	Your organoids?
      Basal	3 cycles	mix	3min	4min
      		wait	2min	0min
      		measure	3min	2min
      Oligomycin	1 cycle	mix	3min	5min
      		wait	2min	10min
      		measure	3min	2min
      	2 cycles	mix	3min	4min
      		wait	2min	0min
      		measure	3min	2min
      FCCP	3 cycles	mix	3min	4min
      		wait	2min	0min
      		measure	3min	2min
      Rotenone+ Antimycin A	3 cycles	mix	3min	4min
      		wait	2min	0min
      		measure	3min	2min

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      ^aSuggested by Agilent.

      ^bThese settings can be applied to mouse small intestine- and human colon- or colorectal tumor-derived organoids.

    • ii.

      For optimized glycolysis stress test settings for mouse small intestine- and colon- or colorectal tumor-derived organoids, see Table 7.

      Table 7

      Settings glycolysis stress test

      Settings			Defaulta	Intestine-derived organoidsb	Your organoids?
      Basal	3 cycles	Mix	3min	4min
      		Wait	2min	0min
      		Measure	3min	2min
      Glucose	3 cycles	Mix	3min	4min
      		Wait	2min	1min
      		Measure	3min	2min
      Oligomycin	1 cycles	mix	3min	5min
      		wait	2min	10min
      		measure	3min	2min
      	2 cycles	mix	3min	4min
      		wait	2min	0min
      		measure	3min	2min
      2-DG	3 cycles	mix	3min	4min
      		wait	2min	0min
      		measure	3min	2min

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      ^aSuggested by Agilent.

      ^bThese settings can be applied to mouse small intestine- and human colon- or colorectal tumor-derived organoids.

  • d.

    Save your template.

CRITICAL: compared to cell lines, organoids show a slower response to Oligomycin, possibly due to the presence of Matrigel. Therefore, we adjusted the settings by applying a 10-min waiting step right after the injection of the inhibitor in both, the mitochondrial stress test and the glycolysis stress test (Tables 6 and ​and77).

• 20.

  Run the mitochondrial or glycolysis stress test [Troubleshooting 3].

  • a.

    Upon clicking on “Run,” the Wave software will ask to insert the sensor cartridge in the XF Analyzer.

  • b.

    Remove the Hydro Booster and the lid (Figure3D), and place the sensor cartridge hold in the Utility plate in the XF Analyzer.

  • c.

    After the calibration step which takes approximately 15min, the Wave software will request to replace the Utility plate by the Seahorse Cell Culture microplate containing the organoids.

  • d.

    The XF analysis will automatically begin.

    Note: Once the experiment has finished, the OCR and ECAR measurements will be displayed on the XF Analyzer screen. A first inspection of the results can be done at this point. When high standard deviations within one condition are observed, switching from “group” view to “well” view, allows to detect possible outliers (Figure5), which can result, for instance, from inaccurate plating or differential organoid viability. Pictures taken at step 15 can be used to judge the cause of large standard deviations. Excluding outliers from the analysis can be considered (Figures 5A–5C) [Troubleshooting 4].

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    Figure5

    Detection of possible outliers in Seahorse XF Wave software

    OCR measurements of an exemplary mitochondrial stress test. In (A), the measurements are displayed as “group view” and in (B) as “well view.” In(B),the line in bold indicates the outlier measurement. (C) “Group view” of the OCR measurements displayed in (A) upon exclusion of the outlier depicted in (B).

• 21.

  After the experiment, carefully remove medium from the wells.

CRITICAL: The plate should be removed immediately after the experiment has finished in order to prevent loosing organoid viability as a result of the presence of the inhibitors used in the assay.

Pause point: When required, after medium aspiration, the Seahorse Cell Culture Microplate can be stored at −20°C for at least 2weeks to later proceed with the DNA quantification step.

DNA quantification

Timing: 2–3 h

Calculation of bioenergetic parameters requires normalization to correct for differences in numbers of cells, organoids, or amount of biological material. Standard normalization methods are based on the determination of cell numbers or protein content. Due to the presence of Matrigel, normalization by protein content is not adequate when using organoids. Quantification of cell numbers in organoids is technically challenging due to their 3D structure and the limited number of cells. A reliable alternative for normalization of OCR and ECAR measurements from organoids is normalization by the DNA content. Below we describe how to collect and isolate DNA from organoids cultured on Seahorse Cell Culture microplates.

• 22.

  Add 100μL ice cold Cell Recovery Solution (Corning, #354253) to each well of the Seahorse Cell Culture microplate.

Note: Upon decision to remove a certain outlier from analysis in step 20, the organoids from this well should not be collected for DNA quantification.

• 23.

  Incubate the plate at 4°C for 30–60min.

Note: Incubation in Cell Recovery Solution facilitates organoid collection and removal of the Matrigel.

• 24.

  Collect the organoids and pool the organoids belonging to one condition in an Eppendorf tube.

• 25.

  Centrifuge at 10,620× g at 4°C for 5min (Eppendorf 5417R centrifuge with an Eppendorf F45-30-11 rotor, 10 000rpm).

• 26.

  Remove supernatant while leaving the pellet undisrupted.

• 27.

  Isolate genomic DNA by using the QIAamp DNA micro kit according to the manufacturer’s instructions.

  • a.

    In order to maximize the DNA yield, add 30μL elution buffer let it stand for 2–3min to maximize DNA elution.

• 28.

  Measure genomic DNA by Nanodrop.

CRITICAL: Aiming for similar organoid densities across conditions improves the robustness of the analysis. To be noticed, normalization by pooled DNA corrects for variability across conditions but does not correct for differences within conditions. Therefore, the variation in organoid density within conditions should be kept low.

Note: In these protocols, we apply DNA normalization; this does not exclude the application of other normalization methods. Importantly, RNA quantification could be considered when differences in DNA amount are expected across compared conditions. Importantly, some organoid types could have a high rate of shedding dead cells. This requires attention, as damaged cells can be metabolically low/inactive but they still contribute to total DNA quantification.

Problem 1

The Matrigel droplets do not stay in the middle of the Seahorse microplate well after re-plating (step9).

Potential solution

Incubate the Seahorse microplate at 37°C for at least 24h and put the plate on top of a pre-warmed water-filled flask. When this is not sufficient, reduce the amount of medium that is mixed with Matrigel. Diluted Matrigel will hardly form droplets. Alternatively, reducing the volume of the Matrigel droplets from 3 to 2.5μL can help.

Problem 2

The organoids are disrupted or broken or their appearance indicates low fitness or viability in the Seahorse cell culture microplate (step 9).

Potential solution

Some organoid lines can be particularly sensitive to mechanical shearing compromising their fitness and viability. To minimize this, try to reduce the pipetting movements while re-plating the organoids. Alternatively, try to adjust the timing of the re-plating to when the organoids have a size that is less sensitive to mechanical shearing. Depending of the organoid type, this size can be either larger or smaller. For intestinal organoids, re-plating the organoids when they are rather small (less than 120μm diameter, Figure2C), protects them from mechanical disruption.

Problem 3

The organoids do not or only mildly respond to the injections (step 20).

Potential solution

First of all, it is essential to control the stocks of the inhibitors and replace by new fresh stocks when the organoid response to the drugs decreases over time.

When the low response occurs with fresh and correctly prepared drug stocks, it should be checked whether the unresponsiveness of the organoids is a technical artifact or the actual phenotype of the organoids. For instance, when Oligomycin does not increase or only mildly increases the ECAR (Figure6C), it should be analyzed whether Oligomycin was functional. In this experiment, Oligomycin did reduce the OCR (Figure6D), indicating that the amount of Oligomycin that was used was correct. Furthermore, it should be examined whether the setup of the experiment was optimal. For instance, it should be checked whether increasing glucose concentrations will enhance the glycolytic capacity, in order to check that glucose availability was not limited in the experiment.

When the ineffectiveness of the drugs cannot be attributed to technical problems, the density of the organoids should be checked. When the density of the organoids is high, the drugs can possibly be less effective. In that case, try to reduce the number of organoids plated in the Seahorse Cell Culture microplate wells. When the organoids are plated in the proper density, titrate the concentrations of the injection reagents. When this does not work, this can be repeated in presence of more or less substrates in the XF assay medium.

Problem 4

There is high variability in OCR and ECAR measurement within technical replicates of an experiment (step 20).

Potential solution

This observation can result from uneven organoid numbers in the different wells. To avoid that, carefully re-suspend the organoids in the Matrigel and plate them immediately in the Seahorse cell culture microplate. Alternatively, inaccurate plating can lead to Matrigel drops fusing to the wall of the plate instead of being centered in the well (Figure3C). This also leads to variable measurements of OCR and ECAR. Follow the steps concerning microplate incubation at 37°C and the use of a warm water-filled flask (step 9). Plating accuracy can be checked by microscope inspection after plating. Consider to exclude “failed wells” from the analysis. When doing so, restrain to collect that well for DNA extraction.

Problem 5

There is high variability in OCR and ECAR measurement between different experiments (Section Quantification & statistical analysis, step 5).

Potential solution

Make sure that the timing of the experiments is always similar. Reduce variability in culture time after splitting or trypsinizing the organoids and plating organoids in the Seahorse cell culture microplate. Furthermore, always plate the same number of organoids per well in every experiment.

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Maria J. Rodríguez Colman (M.J.RodriguezColman@umcutrecht.nl).

Materials availability

This study did not generate new unique reagents, plasmids, or organoid lines.

Data and code availability

This study did not generate new datasets or codes.

This work was financially supported by Dutch Cancer Society (KWF 2016-I 10471, KWF 2017-II 11315).

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