Protocol for single-cell dissociation of head and neck cancer organoids

Summary

Here, we present a protocol for dissociating head and neck squamous cell carcinoma patient-derived organoids (PDOs) into viable single-cell suspensions, suitable for use in downstream applications such as drug screening and single-cell RNA sequencing. We describe steps for maximizing single-cell yield through incubation with 0.05% trypsin and gentle mechanical dissociation. We avoid higher trypsin concentrations and extended incubation times, as these conditions promote cell aggregation and significantly reduce cell viability. Additionally, we detail procedures for the assessment of single-cell yield by automated cell counter, 24-h live-cell imaging, and flow cytometry.

Graphical abstract

Highlights

• •Steps for dissociation of three-dimensional organoids into single-cell suspensions
• •Procedures for enzymatic and mechanical digestion of patient-derived organoids
• •Guidance on assessing single-cell viability and yield

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

Here, we present a protocol for dissociating head and neck squamous cell carcinoma patient-derived organoids (PDOs) into viable single-cell suspensions, suitable for use in downstream applications such as drug screening and single-cell RNA sequencing. We describe steps for maximizing single-cell yield through incubation with 0.05% trypsin and gentle mechanical dissociation. We avoid higher trypsin concentrations and extended incubation times, as these conditions promote cell aggregation and significantly reduce cell viability. Additionally, we detail procedures for the assessment of single-cell yield by automated cell counter, 24-h live-cell imaging, and flow cytometry.

Before you begin

General considerations

This protocol details all steps in the process of dissociating aero-upper digestive (head-and-neck and esophagus) squamous cell carcinoma patient-derived organoids (PDOs) into a single cell suspension. These PDOs are initiated as a single cell suspension embedded in basement membrane matrix and established as 100–250 μm spherical structures grown in culture for 7–14 days, depending on their growth kinetics.¹ Single cell dissociation of PDOs is crucial not only for passaging in subculture, but also for ensuring accurate results from downstream applications such as single cell RNA sequencing,² flow cytometry and fluorescence-activated cell sorting,³ and drug screening for personalized medicine.⁴ This description covers enzymatic dissociation with trypsin, as well as determination of the percentage of viable single cells within the dissociated cell population by automated cell counting, 24-h live cell imaging or flow cytometry.

Institutional permissions

Utilization of this protocol requires the appropriate approval by an institutional review board (IRB) for research involving human subjects. The tissues used to generate the PDOs must be acquired in a manner that follows the IRB guidelines, which define proper recruitment, informed consent, and minimal risk collection procedures. All clinical materials were procured from informed-consented patients, according to protocols approved by the Columbia University Irving Medical Center Institutional Review Board.

Preparation for PDO passaging

Timing: 60 min

• 1.
  Prepare A83-01, TGFBR1 inhibitor.

  • a.
    Dissolve 5 mg in 2.4 ml DMSO for 5 mM stock. Store at −20°C.
• 2.
  Prepare human epidermal growth factor (hEGF).

  • a.
    Reconstitute 1 mg in 2 mL ddH₂o for 500 ng/μL stock. Store at −20°C.
• 3.
  Prepare N-acetyl-L-cysteine (NAC).

  • a.
    Dissolve 820 mg in 10 mL DPBS for 0.5 M stock. Store at −20°C.
• 4.
  Prepare soybean trypsin inhibitor (STI).

  • a.
    Dissolve 250 mg in 1 L DPBS. Store at 4°C.
• 5.
  Prepare Y-27632 stock.

  • a.
    Dissolve 10 mg in 3.2 mL DPBS. Store at −20°C.
• 6.
  Prepare DAPI stain.

  • a.
    Dissolve 5 mg DAPI in 1 mL ddH₂0 (dilute DAPI stock solution 1:5000 in DPBS to obtain working solution)
• 7.
  Prepare FACS sorting buffer.

  • a.
    Dissolve 100 mg of Bovine Serum Albumin (BSA) in 10 mL of Dulbecco’s Phosphate Buffered Saline (DPBS). Vortex until fully dissolved and store at 4°C.
• 8.Prepare basic culture media (BCM) as defined in materials and equipment setup Section.⁵

Note: For the purposes of organoid culture, these steps must be performed under sterile conditions using a standard biosafety cabinet equipped with laminar air flow. Additionally, A83-01, hEGF, NAC, FACS buffer, and STI reagents must be sterile-filtered through a 0.22 μm membrane before use.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Chemicals, peptides, and recombinant proteins
Dulbecco’s phosphate-buffered saline (DPBS)	Thermo Fisher Scientific	Cat#14190250
Matrigel matrix	Corning	Cat#354234
Y-27632 (ROCK inhibitor)	Selleck	Cat#S1049
0.25% trypsin	Thermo Fisher Scientific	Cat#25200114
0.05% trypsin	Thermo Fisher Scientific	Cat#25300120
Soybean trypsin inhibitor (STI)	Sigma-Aldrich	Cat#T9128
Advanced DMEM/F12	Thermo Fisher Scientific	Cat#12634010
N-acetyl-L-cysteine (NAC)	Sigma-Aldrich	Cat#A9165
N-2 supplement 100×	Thermo Fisher Scientific	Cat#17502048
B-27 supplement 50×	Thermo Fisher Scientific	Cat#17504044
HEPES 1 M	Thermo Fisher Scientific	Cat#15630080
Antibiotic-antimycotic 100×	Thermo Fisher Scientific	Cat#15240062
GlutaMAX 100×	Thermo Fisher Scientific	Cat#35050061
Human epidermal growth factor (hEGF)	Thermo Fisher Scientific, PeproTech	Cat#AF-100-15
A83-01 (TGFBR1 inhibitor)	Tocris	Cat#2939
R-spondin 3 and Noggin (RN) conditioned media	N/A	N/A
Bovine serum albumin (BSA)	Sigma-Aldrich	Cat#A7906
4′,6-diamidino-2-phenylindole (DAPI)	Thermo Fisher Scientific	Cat#D21490
DNase I	Thermo Fisher Scientific	Cat#90083
Critical commercial assays
Trypan blue stain (0.4%)	Thermo Fisher Scientific	Cat#T10282
Countess cell counting slide	Thermo Fisher Scientific	Cat#C10283
Experimental models: Cell lines
Human: passage 7 HSC17 PDO	Organoid and Cell Culture Core (OCCC)	HSC17
Software and algorithms
ImageJ	Schneider et al.6	https://imagej.net/ij/download.html
RStudio	Allaire et al.7	https://posit.co/downloads/
Other
Nunc 15 mL conical sterile polypropylene centrifuge tubes	Thermo Fisher Scientific	Cat#339651
Nunc 50 mL conical sterile polypropylene centrifuge tubes	Thermo Fisher Scientific	Cat#339653
Sterile 35 μm cell strainers (with 6 mL tube)	VWR	Cat#64750-25
0.2 μm sterile syringe filter (30 mm diameter)	Thermo Fisher Scientific	Cat#42225-PV
Sterile snap cap low retention microcentrifuge tubes (1.5 mL)	Thermo Fisher Scientific	Cat#3448
Cell culture microplate, 384 well, PS, F-bottom (clear bottom, sterile)	Greiner Bio-One	Cat#781976
Microplate, 384 well, PS, F-bottom, black bottom (non-binding, sterile)	Greiner Bio-One	Cat#781900
Breathe-Easy sealing membrane	Sigma-Aldrich	Cat#Z380059
Eppendorf thermomixer model 5350	Marshall Scientific	Eppendorf Thermomixer 5350 MixerMarshall Scientific https://www.marshallscientific.com › eptm50
Eppendorf centrifuge 5424 R	Marshall Scientific	Eppendorf 5424R Refrigerated CentrifugeMarshall Scientific https://www.marshallscientific.com › epp-5424r
Invitrogen Countess 3 automated cell counter	Thermo Fisher Scientific	https://www.fishersci.com/shop/products/countess-3-automated-cell-counter-6/AMQAX2000
BioTek Cytation 5 automated live-cell fluorescence imaging system	Agilent	https://www.agilent.com/en/product/cell-analysis/cell-imaging-microscopy/cell-imaging-multimode-readers/biotek-cytation-5-cell-imaging-multimode-reader-1623202
BD Influx cell sorter	BD Biosciences	https://www.bdbiosciences.com/en-us/products/instruments/flow-cytometers/research-cell-sorters/bd-influx
Varioskan LUX multimode microplate reader	Thermo Fisher Scientific	https://www.thermofisher.com/us/en/home/life-science/lab-equipment/microplate-instruments/plate-readers/models/varioskan.html

Materials and equipment

Basic cell media (BCM) Components

Reagent	Final concentration	Amount
Advanced DMEM/F12	N/A	45.9 ml
N-acetyl-L-cysteine (NAC) 0.5 M	1 mM	100 μL
RN conditioned media (RN CM)	N/A	1 mL
N-2 supplement (100×)	1×	500 μL
B-27 supplement (50×)	1×	1 mL
HEPES (1 M)	0.01 M	500 μL
Anti-anti (100×)	1×	500 μL
Glutamax (100×)	1×	500 μL
Y-27632 (10 μM)∗		50 μL
Human epidermal growth factor (hEGF) 500 ng/μL∗		5 μL
A83-01 (5 nM)∗		50 μL
Total	N/A	50 mL

Store at 4°C for up to 6 months.

CRITICAL: ∗Since these factors quickly degrade in media, add Y-27632 (for day 0 only), A83-01, and hEGF just before use to make Complete Culture Media (CCM).

Step-by-step method details

Dissociation of three-dimensional organoids into single-cell suspensions

Timing: 90 min

This step details the initial collection of established PDOs, and the optimal dissociation technique employed to produce single cell suspensions. Depending on the unique growth rate of each PDO, this step may be performed between day 7–14 of growth, when the average PDO diameter is between 100–250 μm.^1,8 This typically results in 50–150 PDOs per well.

• 1.
  Harvest three-dimensional patient derived organoids (PDOs).

  • a.
    Without disturbing the Matrigel bubble, carefully aspirate complete culture medium (CCM) from all wells.
  • b.
    Using a wide bore p1000 pipette tip, collect PDOs from 3–4 wells by resuspending Matrigel in 1 mL of pre-warmed BCM (37°C water bath) (Figure 1A).
  • c.
    Transfer cell solution to 1.5 mL microcentrifuge tubes (repeat steps 1b. and 1c. for all wells).
  • d.
    Briefly (<15 s) spin down microcentrifuge tubes in benchtop microcentrifuge to pellet PDOs.

    • i.
      If PDOs fail to form a clear pellet at the bottom of the microcentrifuge tube (Figure 1B), refer to troubleshooting problem 1 of this protocol. The pellet should be separate from the Matrigel (Figure 1C).
  • e.
    Carefully remove supernatant using a p1000 pipette, taking care not to disturb the cell pellet.
• 2.
  Chemical Dissociation of PDOs via trypsinization.

  • a.
    Using a p1000 pipette, resuspend pellets with 500 μL of pre-warmed 0.05% trypsin.

Note: For alternative dissociation techniques, refer to Figure 2A.

Note: In our experience, 0.05% trypsin is superior to 0.25% trypsin at minimizing aggregation (average 3.04% vs 19.11% multiplets, p=0.034, Figures 3A–3D), but a higher concentration of trypsin may be considered if inadequate dissociation is suspected.

• 3.
  Mechanical dissociation of PDOs via Thermomixer incubation.

  • a.
    Place tubes in the Thermomixer and incubate at 37°C and 800 rpm for 10 min.

Note: If aggregates remain, refer to troubleshootingproblem 2 of this protocol.

Note: In our experience, 10 min of dissociation in trypsin is optimal (Figures 3A–3D), though a longer incubation period (e.g. 20 min) may be considered if inadequate dissociation is suspected.

• 4.
  Trypsin inhibition and cell resuspension.

  • a.
    Add 600 μL Soybean Trypsin Inhibitor (STI) to each microcentrifuge tube, pipetting up and down to ensure proper mixing.
  • b.
    Combine all tubes pertaining to the same culture into a 15 mL conical tube.
  • c.
    Centrifuge tubes at x300 Relative Centrifugal Force (RCF) and 4°C for 5 min to pellet cells at the bottom of the tube.
  • d.
    Carefully aspirate supernatant using a p1000 pipette, taking care not to disturb the cell pellet.
  • e.
    Depending on the size of the pellet, resuspend cells in 50–500 μL of pre-warmed BCM, pipetting up and down to obtain an evenly distributed cell mixture.
• 5.
  Cell straining.

  • a.
    Place a sterile 35 μm cell strainer on a 50 ml conical and using a p1000 pipette, carefully transfer cell suspensions from tubes through cell strainer (Figure 1D).

    • i.
      Pipette cell solution in a circular manner through cell strainer.

Note: If difficulty passing cell suspension through strainer, see troubleshootingproblem 3.

Note: In our experience, the use of a cell strainer does not alter the proportion of singlets or multiplets in the cell suspension. We prefer it to filter debris, but this step may be omitted if desired.

Figure 1.

Proper handling of PDOs during passaging

(A) Collection of Matrigel bubble using 1 mL of BCM.

(B) Cell pellet with incomplete separation from Matrigel. Note cloudy haze above pellet.

(C) Clear pellet separation after centrifugation with DPBS.

(D) A 35 μm strainer is used to filter out large cell aggregations.

Figure 2.

Overview and placement of dissociation techniques A–F

(A) Comparison of dissociation techniques, with rows showing trypsin concentration, trypsin incubation time, and presence of a cell strainer.

(B) Depiction of the recommended placement of samples for the 384-well plate, used for live cell imaging.

Figure 3.

Validations of singlet yield

(A) Images showing representative Countess viability readout for dissociation technique A (left) and dissociation technique D (right). Green represents live cells while red depicts dead cells. Yellow circles indicate cell aggregates of three or more cells. Scale bar, 400 μm.

(B) Bar and line plot showing the percentage of live cells and percentage of doublet aggregation for the dissociation techniques based on Countess cell counting.

(C) Identification of live singlet cells for dissociation technique A, with P1 gate (top) excluding debris, BV421 gate (middle) excluding dead cells, and final selection of singlet/doublet cells (bottom).

(D) Bar and line plot showing the percentage of live cells and percentage of aggregation for the dissociation techniques based on FACS. Lower cell viability portends higher percents of aggregation.

Determine cell quantity, viability, and percent aggregation via cell Countess

Timing: 15 min

The following steps detail how to accurately count cells using the Countess 3 Automated Cell Counter, which outputs a live and dead cell percentage as well as percentage of cell aggregation.

• 6.Record cell suspension volume for each dissociation condition.

Note: This step is critical in determining total cell count later in the protocol. Suspension volumes may vary depending on size of cell pellet.

• 7.Again, thoroughly resuspend cell suspension before proceeding to step 8.
• 8.
  Once thoroughly resuspended, make a 10 μL 1:1 dilution of cell suspension and 0.4% Trypan Blue Stain (5 μL of each).

  • a.
    Pipette up and down to ensure solution is homogenously mixed.
• 9.Transfer 10 μL solution into cell counting chamber and insert slide into slide port.

Note: Refer to Chapter 4 of Countess 3 Automated Cell Counter User Guide⁹ for more information regarding steps 8 and 9.

Note: If an automatic cell counter is not available, refer to the following article¹⁰ for details on using a hemocytometer for manual cell counting instead.

• 10.
  The Countess will automatically display a bright-field image of cells with automated light exposure and focus.

  • a.
    If needed, manually adjust the image brightness and focus using the touchscreen slider to improve image quality and cell count accuracy.
• 11.
  Press “count” and record results (Figure 3A).

  • a.
    Instrument will capture a bright-field image and provide data such as total concentration, percentage and concentration of live and dead cells, and percentage of cell aggregates.
  • b.
    To record data, press the save button in the bottom right corner of the screen.

Note: Typical cell viability is in the range of $\ge$70%, with the percent of multiplets below 10% (See Expected Results Section).

Note: Refer to Chapters 5 and 6 of Countess 3 Automated Cell Counter User Guide⁹ for more information regarding steps 10 and 11.

384-well plate seeding

Timing: 1 h

If additional singlet validation is desired, we find that live cell imaging is helpful. This step allows for the preparation of cell suspensions to seed one 384-well plate for live cell imaging.

• 12.Handling all reagents on ice, make a 5 mL stock solution of CCM+5% Matrigel, pipetting the mixture up and down for 1 min to ensure proper mixing.

Note: Matrigel is sticky and viscous; prepare 40% more volume than needed as volume will be lost in pipette transfer steps. Avoid introducing air bubbles while mixing.

• 13.Based on live cell concentration data obtained from Countess imaging in step 11, calculate the volume of cell suspension needed for 3,000 cells.

Note: The above calculation is based on a cell seeding density of 300 cells/well. Additionally, calculations account for production of extra cell suspension volume for ease of pipetting and potential errors.

• 14.Subtract the respective cell suspension volume in step 13 from 500 μL to determine the volume of CCM+5% Matrigel required.
• 15.
  On ice, combine previously calculated cell suspension and CCM+5% Matrigel volumes into a newly labeled microcentrifuge tube.

  • a.
    Thoroughly mix cell solution by pipetting up and down for 1 min using a p1000 pipette.

    • i.
      Avoid introduction of air bubbles into cell solution.
• 16.Using a p200 pipette, dispense 50 μL of cell solution into the inner most wells of a 384-well plate, repeating to plate 6 replicates per condition (Figure 2B).

Note: Depending on downstream application, black bottom or clear bottom 384-well plates may be necessary.

• 17.Fill all remaining empty wells with 50 μL sterile DPBS using a multi-channel pipette.
• 18.
  Firmly seal plate using the Breathe Easy Sealing Membrane.

  • a.
    Peel away paper backing and use end tabs to position evenly.
  • b.
    Use a rubber roller to ensure uniform adhesion to the plate.
  • c.
    Separate the top layer of the membrane by peeling away the clear tab towards the center.
• 19.Incubate plates at 37°C, 5% CO₂, and 95% humidity.
• 20.Proceed to live cell imaging.

Note: For live cell imaging, we recommend capturing 4 images per well each hour for 24 h to capture changes in aggregation over time. For difficulty keeping cells in focus, please see troubleshootingproblem 4.

Preparation of cell solutions for flow cytometry

Timing: 15 min

Additional singlet validation may also be performed by flow cytometry. This step prepares cells for flow cytometry by ensuring adequate cell concentration and staining for live cells.

• 21.Determine the total number of cells remaining.
• 22.In a microcentrifuge, briefly spin down microcentrifuge tubes to pellet the cells.
• 23.Carefully remove the supernatant, taking care not to disturb the cell pellet.
• 24.Based on the total number of live cells remaining, resuspend cells in a volume of FACS buffer that yields a cell concentration of 5–8 million cells/mL.

Optional: If cell viability <70%, see troubleshootingproblem 5.

• 25.Immediately prior to performing flow cytometry, add 1–2 μL of 50 μg/mL DAPI solution to the cell solution to differentiate live and dead cells. As DAPI is used as an exclusion staining for dead cells, all DAPI-positive cells should be removed from analysis.
• 26.Proceed to cell sorting.

Expected outcomes

To optimize singlet yield, we tested several dissociation techniques (Figure 2A). To appropriately quantify live singlet cell populations for each dissociation technique, we utilized several different metrics, including Countess cell counts (Figures 3A and 3B), flow cytometry (Figures 3C and 3D), and live cell imaging (Figures 4A and 4B). Starting with Countess imaging, expect to see cell viability in the range of 70–99%. If cell viability falls below this range, especially for techniques C-F, please see troubleshooting problem 5. Data obtained from cell Countess and flow cytometry revealed an inverse association between live cell and aggregation percentage (Figures 3B and 3D). Additionally, aggregation did not increase in any of the dissociation techniques over the first 24 h (Figures 4 and S1A–S1F). For PDO expansion and long-term culture, PDOs were able to successfully reform following a seven day growth period, though the maturation period may vary between PDOs (Figure S2A). The organoid formation rate (OFR), or percent of live single cells that develop into PDOs,⁵ is expected to be between approximately 40% and 60% (Figure S2B).

Figure 4.

Percentage of singlet cells remain constant over 24 h

(A) Line plots showing singlet percentages by dissociation technique. The singlet percentage shown is the average over 3 h, with the x axis noting the first hour in that group (i.e. value plotted at hour 7 is the average of hours 7-9). Error bars represent the standard deviation of singlet populations within each time group.

(B) Bar plot comparing the percentage of singlets averaged over 24 h for each dissociation technique. Error bars represent the standard deviation of the mean singlet populations for each dissociation technique.

Overall, techniques using 0.25% trypsin had increased cell death and aggregation, and the use of the 35 μm cell strainer did not provide any significant additional benefit. For HNSCC PDOs, expect technique A to have the highest proportion of viable singlet cells. Moving forward, we recommend using this technique (0.05% trypsin for 10 min) as the optimal approach for yielding single cell solutions to use in downstream assays. This strategy allows for standardized plating of wells in later experiments, particularly useful for high throughput drug screening to avoid aggregates that may obfuscate results.

Quantification and statistical analysis

The following details regard the analyses of the live cell imaging data; please see the attached code for more information. Raw images captured over 24 h of live cell imaging (one image per hour) were imported into the Fiji software. Images were processed using the PHANTAST plug-in (see attached code), and the area, defined by number of pixels, of each cell in the image was output to .csv files. These files were imported to RStudio and pre-processed to ensure quality control. Wells that went out of focus during imaging, identified as frames where area values were initially present but later became absent, were excluded from the analysis. The remaining wells were grouped together by dissociation technique and averaged across three-hour time intervals (Figure 4A). Standard deviation was computed to quantify variability within each time bin. Data visualization was performed using the tidyverse package in RStudio.

Limitations

This protocol was specifically developed for use with HNSCC PDOs, and although we have had success with ESCC PDOs, more research is needed to investigate whether this same protocol may be applied to other tumor types. We validated this experiment on one HNSCC PDO in six-replicates shown in this protocol, but we have had success in several other PDOs, each with its own growth patterns and kinetics. Additionally, our study is limited to two concentrations of trypsin, which does not account for other possibilities of optimal trypsin concentrations or alternative methods of dissociation altogether. However, trypsin is a cost-efficient, well-established reagent that has proven to be effective in single cell dissociation as shown throughout this protocol. Use of alternative enzymes described in other tissue dissociation protocols (e.g., collagenase, dispase) has not been explored. Additionally, the Matrigel brand is not globally available, but other basement membrane extracts may be used as a substitute.

Troubleshooting

Problem 1

Difficulty pelleting harvested PDOs (related to Step 1d).

Centrifugation of harvested three-dimensional PDOs does not always yield a concentrated cell pellet as seen in Figure 1C. If the Matrigel is not sufficiently broken up, PDOs can remain suspended in the matrix, even after centrifugation. This may appear as a cloudy viscous layer towards the bottom of the tube with no visible cell pellet, as illustrated in Figure 1B. Incomplete pelleting of PDOs complicates the subsequent protocol step of supernatant removal and poses risk of live cell loss. To achieve a more defined cell pellet, consider implementing the following troubleshooting steps.

Potential solution

• •During PDO harvest, pipette BCM and Matrigel dome from each well up and down up to 50 times. While time consuming, this step ensures adequate matrix dissociation and homogenization of the sample.
• •After initial microcentrifuge spin down step, remove any transparent supernatant, taking care not to aspirate the cloudy, PDO containing layer. Pipette 500 μL-1 mL of pre-chilled (at 4°C), sterile DPBS up and down with the PDO containing layer 50 times to ensure sufficient matrix dissociation. Repeat the microcentrifuge spin down step and proceed with step 1e.

Problem 2

Many aggregates remain after 0.05% trypsinization for 10 min (related to step 3a).

Potential solution

• •Incubate for an additional 10 min (20 min total). This extra time at 0.05% trypsin may help the cells to separate more but comes at the risk of cell viability.
• •Pipette up and down to break up organoids and then incubate for an additional 10 min. It may help to manually break them up by pipetting during the extra incubation time.
• •Try higher concentrations of trypsin, for example 0.25% trypsin. This may, however, lead to the buildup of more dead cells.
• •Use a 35 μm cell strainer as detailed in Step 5 to filter out larger cell aggregates.

Problem 3

Difficulty passing cells through the cell strainer (related to Step 5a).

Potential solution

• •Resuspend the cell solution in a larger volume of CCM prior to straining the cells, as there may not be enough hydrostatic pressure to drive the solution through the strainer with such a low volume of suspension.
• •Apply gentle, yet sufficient pressure from the pipette tip to move the solution through the strainer.
• •Gently tap or flick the side of the 50 mL conical to clear any clogged pores in the strainer.
• •Pre-wet the strainer with media before passing the cell suspension through it.

Problem 4

Out of focus cells for live cell imaging (related to step 20). This may happen for several reasons, including varied initial plating methods and evaporation over time.

Potential solution

• •Maintain a consistent method of plating within each well of the 384-well plate. We recommend holding the pipette vertically and bringing it close to the bottom of the well before dispensing the cell suspension.
• •Be sure to fill the outside wells of the plate with an adequate amount of DPBS, as this helps to prevent evaporation that may change focus level for the image capturing.
• •Ensure the ambient temperature is not less than average room temperature since Matrigel will lose its gel-like features, making it more likely for cells to shift overtime.

Problem 5

Low (<70%) cell viability (related to Step 24). This may occur particularly for dissociation techniques C-F, where longer incubation times or a higher concentration of trypsin is likely to cause more cell membrane damage and cell death. Lower cell viability may negatively impact downstream analyses, specifically FACS, due to the accumulation of negatively charged extracellular DNA, making cells more likely to clump together.

Potential solution

• •Limit the time spent harvesting PDOs from the Matrigel bubbles. If you have several PDOs you are testing, consider leaving extra plates in the incubator and harvesting one plate at a time. Minimizing time outside of the incubator will decrease additional cell death.
• •Minimize mechanical breakdown of cells during handling and resuspension. Overly pipetting up and down, especially during the trypsin resuspension, may cause unnecessary harm to cell membranes.
• •For HNSCC PDOs, we recommend passaging on days 7–14, depending on growth kinetics of the individual PDOs. Overly extending passage length may lead to cell death, often occurring in the innermost portions of the organoid.
• •Treat single cell solutions with DNase according to standard protocols,¹¹ prior to FACS.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Anuraag S. Parikh MD (asp2145@cumc.columbia.edu).

Technical contact

Questions about the technical specifics of performing the protocol should be directed to the technical contact, Anuraag S. Parikh MD (asp2145@cumc.columbia.edu).

Materials availability

This study did not generate new or unique reagents.

Data and code availability

This published article includes all datasets and codes generated or analyzed during this study.

Acknowledgments

This research was supported by NIH K08DE033093 (A.S.P.). S.F., PhD, is supported by a Postdoctoral Fellowship, PF-23-1151788-01-DMC, from the American Cancer Society (https://doi.org/10.53354/ACS.PF-23-1151788-01-DMC.pc.gr.175436). S.F. is also supported by NIH LRP L30CA264714. This research was also supported by the Organoid and Cell Culture Core of the Columbia University Digestive and Liver Diseases Research Center (CU-DLDRC) (grant 1P30DK132710).

Author contributions

Conceptualization, M.S., I.C., A.S.P., H.N., C.M., S.F., and Y.T.; methodology, C.M., M.S., and I.C.; investigation, I.C. and M.S.; formal analysis, M.S. and I.C.; visualization, M.S. and I.C.; writing – original draft, I.C. and M.S.; writing – review and editing, M.S., I.C., A.S.P., H.N., C.M., S.F., and Y.T.; funding acquisition, A.S.P.; resources, I.C., Y.T., and C.M.; supervision, A.S.P. and H.N.

Declaration of interests

The authors declare no competing interests.