Protocol for functional profiling of patient-derived organoids for precision oncology

Summary Functional precision oncology—a strategy based on perturbing primary tumor cells from cancer patients—could provide a road forward for personalized treatment. Here, we present a comprehensive protocol covering generation and culture of patient-derived colorectal organoids, isolation and expansion of tumor-infiltrating lymphocytes (TILs), and isolation and culture of peripheral blood mononuclear cells (PBMCs). With this protocol, samples fulfilling the demands for performing multi-omics analysis, e.g., RNA sequencing (RNA-seq), whole-exome sequencing (WES), single-cell RNA sequencing (scRNA-seq), and (phospho-)proteomics, can be generated. For complete details on the use and execution of this protocol, please refer to Plattner et al. (2023).1


Note:
The laboratory carrying out tissue processing and cell isolation must be authorized with Biosafety Level 2 clearance and equipped with Laminar Flow cabinets.Given that primary human surgical materials may carry infectious risks in the absence of testing for all relevant pathogens (HCV, HBV, HIV, etc.), strict adherence to institutional biosafety regulations is mandatory.This includes the proper usage of personal protective equipment (e.g., lab coats, gloves), thorough decontamination of surfaces and appropriate waste disposal procedures.

Preparation for sample collection
Timing: 45 min to 1 h CRITICAL: Prepare transport box for tissue collection in the morning of the day of surgery in order to pick up the specimen as fast as possible (Figure 1).
1. Prepare all media (collection and culture media, wash media and digestion mixes) according to the tables in the 'materials and equipment' section in a sterile environment and store properly.
Note: Prepare aliquots of media, small molecules and enzymes as indicated in the 'material and equipment' section, e.g., to avoid repeated freeze-thaw cycles.
2. Prepare the following reagents and keep or thaw on ice until use: a. Tumor tissue collection medium.b.Healthy tissue collection medium.c.Wash buffer.d.Crypts isolation medium.e. Fetal Bovine Serum (FBS) (thaw).f.Red Blood Cell (RBC) Lysis buffer (13).g.DPBS.3. If needed, reconstitute the following reagents according to manufacturer's recommendations and thaw on ice: a. Liberase DH (reconstitute).b.DNase I (reconstitute).c.Geltrex.4. Warm up the following media and reagents to 37 C: a. Advanced DMEM/F-12.b.Tumor organoid medium.

STEP-BY-STEP METHOD DETAILS
Isolation of peripheral blood mononuclear cells (PBMCs) from whole blood Timing: 1.5 h This section describes how to isolate mononuclear cells (PBMCs) from whole blood using density gradient centrifugation.The blood was drawn in 10 mL Sodium Heparin vacutainer anti-coagulation tubes.The blood sample should be processed as soon as possible after collection, ideally within 2-3 h (troubleshooting 1).Expect reduced cell recovery when sample processing is delayed and/or slow.The isolated PBMCs can be used directly or cryopreserved for future usage.Note: Keep the blood on ice from the time of blood draw and during transportation to the lab, as this drastically improves cell viability.Make sure not to exceed a maximum of one hour on ice to avoid blood coagulation.
Note: Perform the entire protocol under aseptic conditions in a cell-culture hood and adhering to local regulations for biological hazards.When handling blood products, extensive precautions should be implemented to safeguard the health and safety of personnel.
2. Prepare two 50 mL centrifuge tubes with each 15 mL of Lymphocyte separation medium.CRITICAL: Hold the pipette against the tube wall, tilting the tube while filling it up and use the lowest speed setting on the pipette gun to not disturb the interface, ensuring a separate layer of medium and blood (Figure 2A).Immediately proceed with the centrifugation.The blood and separation medium will slowly blend if not subjected to centrifugation promptly.
6. Centrifuge at 500 3 g for 30 min at 20 C using a swinging rotor with acceleration set at 3 and deceleration at 0 on the centrifuge.
CRITICAL: Keeping the break on, will interfere with the intactness of separate layers.
CRITICAL: Carefully move tubes following centrifugation not to disrupt the gradient.
Note: After centrifugation, a whitish cloudy layer consisting of mononuclear cells will be found on top of the separation medium (Figure 2B).
7. Aspirate supernatant containing plasma and platelets up to 1 cm above PBMC layer.8. Carefully collect PBMCs with a 10 mL serological pipet and place in a new 50 mL centrifuge tube.
Note: Refrain from sucking up the separation medium and the layer containing erythrocytes.9. Combine PBMCs from two 50 mL centrifuge tubes and fill up to 50 mL with DPBS.10.Centrifuge at 450 3 g for 10 min at 4 C.
CRITICAL: All following steps should be carried out with the cells placed on ice.
Note: Use a wide-bore pipette to not harm the cells.12. Fill up the centrifuge tube to 50 mL with cold DPBS.13.Centrifuge at 450 3 g for 10 min at 4 C. 14. Resuspend in 1 mL DPBS for cell counting and viability determination.
Note: Work fast and keep the cells on ice during the counting process.
Note: Anticipate noticeable contamination from red blood cells, a concern mitigated following cryopreservation and subsequent assay procedures.
Optional: Perform erythrolysis by incubating with 13 RBC lysis buffer for 10 min at 20 C G 2 C and washing three times with DPBS to remove residual buffer.
15. Mix 10 mL of cell suspension with 90 mL Trypan blue (1:10 dilution) rapidly and inject 10 mL of the mix into a hemocytometer for cell counting using an inverted microscope.Trypan blue negative cells are considered viable cells.16.After this step, continue by either using the cells directly for assays, for cryopreservation (see steps 17-23) or snap-freeze cell pellets for nucleic acid isolation (see steps 34-43).

Cryopreservation and subsequent thawing of PBMCs
This section describes how to cryopreserve PBMCs for long-term storage and how to successfully thaw cells for culturing and performing downstream experiments.

Cryopreservation of PBMCs
Timing: 30 min Note: Pre-label cryovials and ensure availability of freezing containers.
CRITICAL: Prolonged exposure of cells to the freezing medium at 20 C G 2 C may compromise cell viability and consequently, integrity upon thawing (troubleshooting 3).
Note: Frozen PBMCs, intended for storage less than two weeks, can be maintained at À80 C.However, for extended storage beyond two weeks, it is advisable to store cryovials in the vapor phase of a liquid nitrogen tank.
CRITICAL: Work on ice.
19. Centrifuge at 405 3 g for 5 min at 4 C. 20.Gently resuspend in 500 mL PBMC cryopreservation medium per aliquot.21.Transfer 500 mL of the cell suspension to a cryovial.22. Freeze at À80 C for 16-24 h in a Mr. Frosty freezing container.23.Transfer in the vapor phase of a liquid nitrogen tank the next day.
Pause point: PBMCs can be cryopreserved in liquid nitrogen for several years.

Thawing of PBMCs
Timing: 30 min Note: Ensure optimal recovery and viability of cells by strictly using pre-warmed (37 C) medium during thawing procedure.
Protocol 24.Warm up PBMC culture medium to 37 C in a water bath before beginning with the thawing procedure.25.Thaw PBMC cryovial at 37 C in a water bath by gently shaking the vial.
Note: Do not submerge the entire cryovial completely under water.
CRITICAL: Avoid leaving the cryovial unattended while thawing, as the process typically completes within a minute or two.Remove when a small amount of approximately 20% ice crystal remains.Before opening, dry the outside of the cryovial and wipe with 70% alcohol to prevent contamination.
26. Add 500 mL PBMC culture medium to the cryovial.27.Transfer PBMCs from the cryovial to a 50 mL centrifuge tube containing 1 mL of medium.28.Drop-wise, add pre-warmed (37 C) PBMC culture medium into the 50 mL centrifuge tube containing PBMCs at a rate of 1 drop per second while swirling the tube until it is filled up to 32 mL.
Note: Perform a serial dilution by first adding 2 mL, then 4 mL, 8 mL and finally 16 mL.
CRITICAL: Achieve optimal cell viability by gently rinsing cells with medium in a slow dropwise manner, preventing any potential osmotic shock.
29. Centrifuge sample at 300 3 g for 5 min at 20 C. 30.Aspirate the supernatant and resuspend the cells in 1 mL of pre-warmed (37 C) medium.31.Perform a cell count by removing 10 mL for a Trypan Blue exclusion using a hemocytometer or automated cell counter.
Note: Adhering to this protocol will result in a post-thaw cell viability exceeding 90%.
32. Culture in a T-25 (10 mL total volume) or T-75 flask (20 mL total volume) depending on the cell number and desired cell concentration (troubleshooting 4).
Note: Within our laboratory we seed the cells at a density of 1-2 3 10 6 cells/mL.
Note: Prior to performing cell-based assays, it is advised that you rest the cells for 18-20 h.

DNA extraction from PBMC cell pellets for exome sequencing analysis
Timing: $30 min This section describes how to isolate DNA from snap frozen PBMC pellets, containing $2 3 10 6 cells by using the PureLink Genomic DNA Mini Kit (Thermo Fisher Scientific) according to manufacturer's protocol https://tools.thermofisher.com/content/sfs/manuals/purelink_genomic_man.pdf with some adjustments.Note: E.g., for each 30 mL droplet: 70% Geltrex + 30% Advanced DMEM/F-12, add 21 mL of Geltrex to 9 mL of cell suspension.Note: Do not press the pipette beyond the first stop before dispensing, to avoid bubble formation.
96. Seed 4 drops of 30 mL per well in a 6-well plate.97.Let drops solidify for 30 min at 20 C G 2 C or place in the incubator (37 C) for 15 min.98. Add 2 mL of pre-warmed (37 C) Tumor organoid culture medium per well.
Note: Ensure that the culture medium is not cold when you add it to the wells.Pre-warmed (37 C) medium or medium at 20 C G 2 C better conserves Geltrex integrity.
Note: Carefully add the medium by slowly pipetting on the side of the well and not directly on the drops to avoid disruption (troubleshooting 7).
Note: Prior to reaching the second passage organoid cultures may contain residual debris from the isolation procedure.

Timing: 30-60 min
This section describes how to maintain organoid culture for expansion by weekly passaging and how to freeze and thaw samples for long-term storage in the vapor phase of a liquid nitrogen tank.
CRITICAL: Coat pipette tips and tubes with sterile 1% BSA in DPBS when splitting to avoid cells from sticking to plastic surfaces and minimize material loss.

Passaging of tumor organoids
Timing: $30 min Note: the splitting ratio depends on the individual proliferation rate of patient-derived organoids and will vary but as a rule of thumb split tumor organoids approximately every 3-4 days at a 1:6 to 1:8 ratio.Regular microscopic examination of the cultures is helpful to monitor organoid growth, morphological changes and accumulation of debris in the lumen and to ultimately ensure that passaging is carried out at optimal times.
102.Aspirate the medium and add 1 mL of pre-warmed (37 C) DPBS per well.103.Transfer organoids to a 15 mL tube using a cell lifter.
104.Centrifuge at 300 3 g for 5 min at 20 C. 105.Aspirate supernatant and incubate with 1 mL 13 Trypsin per 4 drops for 5-10 min in a water bath set at 37 C or incubator (37 C). 106.Add the same amount of medium to stop the reaction.107.Use a 1 mL tip and pipet 103 up and down.108.Centrifuge at 300 3 g for 5 min at 20 C. 109.Aspirate supernatant completely and resuspend with desired amount of medium (30% of the total volume).
CRITICAL: When removing the medium in step 109 try to leave as little as possible residual liquid as it dilutes the Geltrex concentration and could increase the fragility of drops.
110 Note: The number of organoids per drop is very important as too few or too many will affect the growth of the organoids.Before you plate all of your material, try with a small 10 mL drop first and check the density under the microscope.Start resuspending with a low volume and if the confluency is too high you can always adjust at this point and add more medium and Geltrex.

Timing: 30-45 min
This section describes how to generate single cells from tumor organoids with a high cell viability in order to perform downstream analysis e.g., single cell RNA sequencing.
CRITICAL: Organoids used for this protocol should not be too dense or contain dead cells.The morphology of the organoids needs to be considered as well, since an organoid with a cystic morphology contains cells only in the outer layer compared to an organoid with a budding morphology.Dissociation of 4 organoid culture drops leads to a cell yield which varies between 0.9-2.9 3 10 6 single cells with a viability between 90%-97%.

Protocol
This section describes how to isolate tumor-infiltrating lymphocytes from human tissue fragments after surgical tumor resection.Keep tissue on ice during the transport from the pathologist to the lab.

Tumor infiltrating lymphocytes (TILs) isolation
Timing: 30 min to 1 h 155.Wash the tumor tissue 53 according to the washing steps in step 74 (see section 'tissue sampling, processing and tumor organoid generation').156.After last washing step, remove fat and connective tissue by using a sterile scalpel and forceps.157.Mince tumor tissue into small fragments ($1 mm 3 pieces).158.Add 2 mL of pre-warmed (37 C) TILs isolation medium to the wells of a 24-well plate.159.Put one tumor fragment ($1 mm 3 ) into each well of a 24-well plate using sterile forceps and scalpel (troubleshooting 9).
Note: Tumor pieces settle onto the bottom of the well.Note: Do not mince the tumor piece further when pipetting up and down.

Culturing of TILs
Timing: $2 weeks culture time 163.Change medium every second day by tilting the plate.164.Carefully remove 1 mL of medium without touching the bottom of the plate.

Note:
The tissue with the extravasated cells is settled down to the bottom of the wells.
165.Add 1 mL of fresh pre-warmed (37 C) TILs isolation medium.166.Add 1000 U/mL IL-2 into each well (use a new tip for each well).167.Pipette up and down (use a new tip for every well).168.Keep tumor fragments in culture for $ 14 days (Figure 4, troubleshooting 10).

Cryopreservation of TILs after $2 weeks of isolation
Timing: $30 min CRITICAL: Before freezing down, make sure that the last medium change and the addition of IL-2 was $ 2 days ago, so that the cells are not in a stimulated state, since that would compromise the cell viability following freezing and thawing events.
169.Remove tissue fragments from the culture wells by using forceps.170.Collect the cells by straining them through a 100 mm strainer to remove remaining tissue fragments.171.Check the wells under a microscope and collect remaining cells by washing the wells with additional culture medium.
Note: Pre-wet strainer with 1 mL of TILs isolation medium to avoid cell loss.
172.Centrifuge at 405 3 g for 5 min at 20 C. 173.Remove supernatant and resuspend the cell pellet in 1 mL of pre-warmed (37 C) TILs isolation medium.
Note: Keep cells on ice during counting.
174.Count the cells manually with a hemocytometer and assess viability by Trypan blue exclusion method.175.Freeze 3-5 3 10 6 cells in 1 mL of TILs cryopreservation medium.176.Put cryotube(s) into Mr. Frosty freezing container at À80 C for 16-24 h and transfer into the N2 tank the next day for long-term storage.
Pause point: TILs can be cryopreserved in liquid nitrogen for several years.
Note: This protocol describes TILs isolation from primary tumor tissue by culturing tumor fragments.Addition of IL-2 leads to extravasation of immune cells from the tissue.Amount of fragments cultured depends on the size of the tumor piece obtained from the pathologist.The difference in the range of TILs obtained after $2 weeks of culturing tumor fragments elucidates the need of an additional expansion protocol (see section 'expansion of tumor infiltrating lymphocytes (TILs))' in order to have enough starting material for downstream experiments.
Optional: Subsequently a Fluorescence-Activated Cell Sorting (FACS) step of the cells of interest can be performed.E.g., CD45 + cells can be labeled with an anti-human CD45 antibody and sorted by using a FACS Aria Sorter (BD) and then used for further expansion.Especially when FACS is performed for more than 1 h, the cell viability can decrease.Use a FACS sorting medium with FBS, e.g., RPMI 1640 with the addition of 2 mM L-Glutamine, 1% Penicillin-Streptomycin and 1% FBS.Additionally a Live/Dead marker, e.g., 7-AAD can be included, to remove dead cells with the sorting process.

Expansion of tumor-infiltrating lymphocytes (TILs)
Timing: 1.5 h preparation, $2 weeks expansion time This section describes the expansion of TILs by using gamma irradiated (ɣ-irradiated) feeder cells.After $ 2 weeks of expansion, a high number of viable T cells are obtained, which can be used for downstream experiments.

Generation of feeder cells
Timing: $45 min preparation, $15 min irradiation This section describes the mitotically inactivation of PBMCs from healthy donors (Blood Bank, Tirol Kliniken, Innsbruck, Austria) by ɣ-irradiation in order to use them as feeder cells for the expansion of the TILs.
177.One day before expansion, thaw PBMCs from healthy donors according to the thawing steps 24-33 (section 'thawing of PBMCs').178.Culture them in PBMC culture medium in a T-75 flask.179.Incubate for 18-20 h at 37 C with 5% CO 2 .180.Next day, collect and transfer the PBMCs in their culture medium on ice to the clinics (Radiotherapy Medical University Innsbruck, Austria) for irradiation.181.Irradiate PBMCs with 30 Gy. 182.After irradiation, transfer them on ice back to the lab and continue with the expansion protocol.
Alternatives: If there is no access to a Radiotherapy unit and therefore ɣ-irradiation is not available, other common irradiation treatments can be used as well. 5
Note: By starting the expansion with 0.4 3 10 6 T cells, 40 3 10 6 feeder cells are added to the T cell suspension culture.
185.Pipette with a wide-bore serological pipette gently up and down.186.Incubate the flask(s) at 37 C with 5% CO 2 .187.Monitor the cells daily by observing the color of the expansion medium.
Note: When the pH of the medium drops below pH 6.8, the pH indicator phenol red turns yellow, which means that the culture needs to be splitted because the nutrients within the culture medium are used up.Additionally when cell density increases, T cell clusters can be observed in the TILs expansion medium.
223.Remove the supernatant with a P1000 tip and any remaining DPBS with a P100 and P10 tip.224.Snap freeze the pellet by placing the 1.5 mL microcentrifuge tube in liquid nitrogen and store at À80 C until further use.
Pause point: Snap-frozen organoid pellets can be cryopreserved at -80 C for several years.
Note: For proteomics/phosphoproteomics and transcriptomics analyses, directly use the frozen pellets.For protein extraction follow the appropriate protocol in accordance to the chosen downstream approach.
CRITICAL: Perform each step at 4 C. Ideally, perform every step in the cold room.
Alternatives: In case a properly equipped cold room is not available, fill an icebox with ice and place a metal plate directly on the ice.Place the dish with organoids on the metal plate and perform steps 199-209.This will ensure to perform every step at 4 C while minimizing the risk of ice pieces falling into the wells.

Preparation of organoid samples for exome sequencing analysis
Timing: $30 min This section describes how to prepare frozen pellets of organoids for exome sequencing analyses.Pause point: Extracted DNA can be cryopreserved at À20 C for at least 24 months.
Note: Make sure to add the DNA solution, the Wash Buffer and the ultrapure water onto the center of the column directly on the column membrane without touching or disrupting the membrane with the pipet tip.

Passaging of healthy organoids
Timing: $30 min Note: The splitting ratio depends on the individual proliferation rate of patient-derived organoids and will vary but as a rule of thumb split healthy organoids approximately once a week at a 1:2 to 1:4 ratio (troubleshooting 13).
313.Aspirate the medium and add 1 mL of pre-warmed (37 C) DPBS per well.314.Transfer organoids to a 15 mL centrifuge tube using the cell lifter.
315.Centrifuge at 300 3 g for 3 min at 20 C. 316.Aspirate supernatant and incubate with 1 mL Accutase per 4 drops for 3-5 min in water bath set to 37 C or incubator (37 C). 317.Inactivate Accutase by adding the same amount of medium.318.Put a 200 mL tip over a 1000 mL tip and pipette up and down for appr.20 times.
CRITICAL: Monitor fragmentation of organoids under the microscope and if needed continue with mechanical splitting until there are no remaining intact organoids visible under the microscope.This step is crucial to ensure successful expansion.If organoids remain intact you will not gain any new material.Ideally, cell clumps need to be obtained before seeding but strictly avoid dissociating so much that you only have single cells left (troubleshooting 14).
319.Centrifuge at 300 3 g for 3 min at 20 C. 320.Aspirate supernatant completely and resuspend with desired amount of medium.321.Put the vial on ice and add Geltrex.322.Seed 4 drops of 30 mL per well in a 6-well plate.
Note: Alternatively, we recommend seeding 10 mL Geltrex domes (in total 120 mL per well of a 6-well plate) for improved growth rate.323.Let drops solidify for 30 min at 20 C G 2 C or place in the incubator (37 C) for 15 min.324.Add 2 mL of Healthy organoid culture medium per well.325.Culture at 37 C and 5% CO 2 .
Pause point: Healthy organoids can be cryopreserved in liquid nitrogen for several years.This section describes in detail how to dissociate healthy and tumor colorectal tissue into viable single cells, which downstream can be used for molecular analysis, e.g., single cell RNA sequencing, flow cytometry measurements, etc.
Note: Prepare media and reagents needed (see section 'preparation of sample collection').
Note: Work under sterile conditions and keep tissue samples on ice at all times.
Note: Do not add FBS to the medium, because downstream it interferes with RNA sequencing. 66.Process and wash tissue according to the washing steps 74-78 (see section 'tissue sampling, processing and tumor organoid generation').327.Prepare 10 mL of Tissue digestion mix (23) and transfer it into a gentleMACS C tube.
Note: For tissue % 2 g use 10 mL of Tissue digestion medium.Adjust volume of the digestion mix according to the tumor weight.
CRITICAL: Working fast and on ice is essential for a viable single cell dissociation.Use different scalpels and forceps for healthy and tumor tissue otherwise tissues can be contaminated.

328.
Transfer tissue pieces into gentleMACS C tubes containing the digestion medium.329.Tightly close the C tubes and attach them upside down onto the sleeve of the gentleMACS dissociator.330.Put the heaters on top of the gentleMACS C tubes.

Note:
The gentleMACS heaters are needed for the combined enzymatic and mechanical dissociation at 37 C. CRITICAL: Make sure, no tissue piece is attached to the wall of the falcon.For an optimal dissociation, tissue pieces should be in the digestion medium.CRITICAL: If cells can be observed in the supernatant after centrifugation, centrifuge again at 300 3 g for max. 3 min at 20 C by switching on the deceleration (set to 7) of the centrifuge in order to gently stop the centrifuge, to collect all the cells without loss of material.

Select
Note: Use a P1000 to remove the supernatant instead of a vacuum pump, since the cell pellet might not be very firm.
336.Add 10 mL Tissue digestion medium and gently resuspend the cell pellet.337.Filter the cell suspension through a pre-wetted 100 mm strainer placed on a 50 mL centrifuge tube.
Note: After being pre-wetted, aqueous solutions can more easily pass through a hydrophilic membrane with reduced loss of material.
338.Wash the 100 mm strainer by passing again 10 mL Tissue digestion medium through and collecting the wash in the 50 mL centrifuge tube with the cell suspension.339.Centrifuge cell suspension at 300 3 g for 7 min at 20 C. 340.Aspirate supernatant completely.341.Perform Red blood cell lysis by using Red Blood Cell Removal Solution.
CRITICAL: Prolonged exposure to Red Blood Cell Removal Solution could lead to an unintended tumor cell lysis.

EXPECTED OUTCOMES
This protocol describes the processing of different specimens derived from human colorectal cancer tissue.We generated tumor and healthy organoid cultures and prepared tumor organoid samples for RNA-, DNA extraction, (Phospho-) proteomics and scRNA-seq analysis.The expected RNA yield after purification ranges from 320 to 1000 ng/mL for 4 drops of confluent tumor organoids.The estimated ratio for nucleic acid purity, measured at 260 nm and 280 nm wavelength (A 260 /A Dissociation of 4 confluent drops of tumor organoids into single cells yields in 1-3.5 3 10 6 single cells with a viability between 90%-97%.In order to perform phosphoproteomics analysis around 48 confluent drops of patient-derived tumor organoids consisting of 30 mL of 70% Geltrex are needed to generate one sample for phosphoproteomics analysis.This corresponds to a $1 mg protein sample.For proteomics analysis, six drops of confluent tumor organoid drops are required. TILs were isolated by using tumor fragments.From a tumor piece % 2 g , $12-17 tumor fragments (1 mm 3 ) can be cultured.After two weeks of TILs isolation, the cell yields from $2-30 3 10 6 cells with a viability > 85%.The viability will increase after subsequent expansion to > 95%.
We isolated PBMCs from autologous blood samples.Expected PBMC yield from adult whole blood ranges from 1.2-3.5 3 10 6 cells/mL.The expected DNA yield after purification ranges from 30-60 ng/mL for pellets consisting of $2 3 10 6 cells.The estimated A 260 /A 280 ratio is 1.78-1.90.A A 260 /A 280 ratio of $1.8 is accepted as pure DNA.The percentage of RNA contamination is < 4 ng/mL, which indicates almost no contamination.
Dissociation of healthy and tumor colorectal tissues (% 2 g) yields in 6-9 3 10 6 single cells with a viability > 95%.We did not observe any correlation between tissue weight and the number of single cells obtained after dissociation.
By following this protocol, generated organoids with their autologous PBMCs and TILs can further be used to perform co-culture experiments. 7,8Additionally, you can generate and sustain a culture of tumor and healthy colon organoids derived from surgical samples over an extended time period and expand them for biobanking.Accurate execution of the protocol is crucial for maintaining a high viability and purity of cells for various down-stream experimental applications.

LIMITATIONS
The main limitation of this protocol lies in the accessibility and quality of the tissues.In our experience, a minimum weight of 0.3 g of tissue is sufficient to generate organoids.Preparing samples especially for phosphoproteomics analysis is limited by the excessive amount of input material needed for preparing samples, in order to meet the need for replicates required to perform statistical analysis.Using organoids as a model system has certain limitations which need to be considered as well.Organoids derived from adult stem cells only consist of epithelial cells and lack stromal and immune cells.Besides, unlike in the human intestine, the apical surface of the epithelial cells is facing the inside of the lumen and the basal side is facing the exterior side instead.If experiments require contact to the apical side, the polarity of the organoids has to be switched 9 or microinjection has to be performed.Moreover, growing them in a 2D-monolayer or breaking them apart prior to incubation with cells or drugs provides alternative strategies to circumvent this spatial entity.

Figure 1 .
Figure 1.Transport box for tissue collection from the pathologist Items needed for pickup of the resected specimen from the pathologist.Keep transportation media, patient tissues and blood samples on ice and process immediately.

Protocol 3 .
Transfer the blood from the anti-coagulation tubes into one 50 mL centrifuge tube.4. Dilute the blood with DPBS by filling up the blood centrifuge tube to 50 mL and invert to mix thoroughly. 5. Gently layer 25 mL of the diluted blood on top of the separation medium of one of the two 50 mL centrifuge tubes (from Step 2) to form the density gradient.Repeat this procedure with the leftover 25 mL of the diluted blood and the second 50 mL centrifuge tube containing the separation medium (Figure2A; troubleshooting 2).

Figure 2 .
Figure 2. Workflow for isolation of PBMCs using density gradient centrifugation (A) Two distinct layers: blood diluted 1:2 with DPBS above Lymphocyte Separation medium before spin and density gradient after centrifugation (B).The marked circle indicates the layer of mononuclear cells.
Preparing snap-frozen PBMC cell pellets for RNA and DNA extraction Timing: 20 min This section describes how to freeze down PBMC pellets, e.g., for isolation of RNA and/or DNA.CRITICAL: Use RNase-and DNase-free microcentrifuge tubes.34.After the cell count, pool the desired amount of cells (e.g., 2 3 10 6 cells per aliquot) in a 15 mL centrifuge tube.35.Fill it up with ice-cold DPBS.36.Centrifuge at 720 3 g for 5 min at 4 C. 37. Aspirate and discard the supernatant.38.Fill up the tube with ice-cold DPBS.39.Distribute 2 3 10 6 cells in individual 1.5 mL microcentrifuge tubes.40.Centrifuge at 720 3 g for 5 min at 4 C. 41.Carefully remove the supernatant completely.CRITICAL: Residual DPBS can affect RNA/DNA extraction and therefore diminish RNA/ DNA quality.42.Instantly snap-freeze the tube with the cell pellets by dipping in liquid nitrogen.43.Store cell pellets at À80 C until further usage.Pause point: PBMC pellets can be cryopreserved at À80 C for several years.

Note:
Perform all centrifugation steps at 20 C. 58.Discard the collection tube and place the spin column into a clean PureLink collection tube.59.Add 500 mL Wash Buffer 1 prepared with EtOH to the column.60.Centrifuge column at 10.000 3 g for 1 min at 20 C. 61.Discard the collection tube and place the spin column into a clean PureLink collection tube.62. Add 500 mL Wash Buffer 2 prepared with EtOH to the column.63.Centrifuge the column at 21.130 3 g (full speed) for 3 min at 20 C. Discard the collection tube.64.Place the spin column in a sterile 1.5 mL microcentrifuge tube.STAR Protocols 5, 102887, March 15, 2024 Protocol Note: Work fast and on ice to sustain cell viability.75.Transfer tissue fragments to a petri dish.76.Remove surrounding fat and connective tissue using a scalpel and forceps.77.Weigh the specimens for documentation.78.Mince the tissue with sterile scalpel and forceps < 5 mm 3 .79. Transfer tumor pieces to a 50 mL centrifuge tube filled with 9 mL of Organoid digestion mix.80. Rinse Petri dish with 1 mL of Organoid digestion mix and add to the 50 mL centrifuge tube.81.Incubate for 1 h in a pre-warmed (37 C) Thermostable shaker at 180 rpm.82.Stop digestion by adding 8 mL of StemPro medium + 2 mL FBS.Note: Fetal Bovine Serum (FBS) contains protease inhibitors which inhibit the enzymatic activity of the protein and therefore stops the digestion process.83.Collect dissociated cells in a 50 mL centrifuge tube by filtering through a layer of 400 mM strainer on top and 100 mM strainer below attached to a vacuum pump with a connector ring (Figure3).84.Centrifuge at 200 3 g for 4 min at 20 C. 85. Wash once with 20 mL of DPBS.86.Centrifuge at 200 3 g for 4 min at 20 C and carefully aspirate the supernatant.87.Resuspend pellet with 3 mL 13 RBC-Lysis buffer.88.Incubate for 10 min in the dark at 20 C G 2 C .89. Centrifuge 350 3 g for 5 min at 20 C and carefully aspirate supernatant.90.Wash 33 with 20 mL of DPBS.91.Centrifuge at 200 3 g for 3 min at 20 C and carefully aspirate supernatant.92.Count cells manually in a Neubauer chamber (Hemocytometer).93.Centrifuge at 200 3 g for 5 min at 20 C and carefully aspirate supernatant.94.Resuspend cells in appropriate amount of Advanced DMEM/F-12 to have 1.5 3 10 5 cells in each 30 mL drop.95.Add required volume of Geltrex to the cells (troubleshooting 6).

Figure 3 .
Figure 3. Setup for filtering cells through strainer for obtaining a single cell suspension after dissociation A 400 mM strainer (neon green) is placed on top of a 100 mM strainer (yellow) in the middle and a connector ring (dark green) on the bottom, attached to a 50 mL centrifuge tube connected to a vacuum pump.

Figure 4 .
Figure 4. Isolation of tumor-infiltrating lymphocytes (TILs) by culturing tumor fragments (A) After 4 h (Day 0) of culturing tumor fragments (asterisks), TILs extravasate into the medium (arrow).After 11 days of culture, extravasated T cells form small clusters (arrows), which is a sign of T cell proliferation and activation.Images are acquired on an inverted light microscope (Zeiss).The black scale bars represent 100 mm.(B) Cell type distribution after two weeks of TILs isolation.Staining of isolated TILs with CD45 + , CD8 + , CD4 + and EpCAM + antibodies.Samples are measured via Flow cytometry on a FACS Fortessa (BD) and analyzed by using FlowJo Software.Bar diagrams are generated using Graph Pad Prism software.

Figure 5 .
Figure 5. Tumor organoid appearance after Geltrex removal Images represent a tumor organoid culture derived from human colorectal cancer.(A) Image shows the organoids cultured in Geltrex droplets before Geltrex removal.(B) Image represents the same organoid culture after removal of Geltrex.Images are acquired on an inverted light microscope (Zeiss).The black scale bars represent 50 mm.

Figure 6 .
Figure 6.Representative images of different stages of healthy Organoid generation and culture over time Day 0 shows crypts seeded in a Geltrex drop directly after isolation followed by day 2, 4 and 8 in culture.Images are acquired on an inverted light microscope (Zeiss).The black scale bars represent 100 mm.
342.Add 3 mL chilled 13 Red Blood Cell Removal Solution to the cell pellet.343.Gently resuspend the cells.Note: Do not vortex.344.Incubate for 10 min at 20 C G 2 C. 345.Add 10 mL chilled DPBS with 0.04% BSA.346.Centrifuge at 300 3 g for 10 min at 4 C. 347.Remove supernatant without disturbing the cell pellet.348.Wash again, by adding 5 mL of DPBS with 0.04% BSA.349.Centrifuge at 300 3 g for 10 min at 4 C. 350.Remove supernatant and count cells manually with a hemocytometer and assess viability by using Trypan Blue solution (Figure7).

Figure 7 .
Figure 7. Healthy and tumor colorectal tissue dissociation into single cells Healthy and tumor CRC tissue after enzymatic and mechanical dissociation into single cells.Images are acquired on an inverted light microscope (Zeiss).The black scale bars represent 20 mm.
Prepare in a sterile environment, store at 4 C for up to 1 month.Prepare aliquots of 18 mL and keep on ice until further use.Prepare in a sterile environment, store at 4 C for up to 1 month.Keep aliquots of 20 mL on ice until further usage.Prepare in a sterile environment, store at 4 C for up to 1 month.Prepare aliquots of 20 mL and keep on ice until further usage.Prepare in a sterile environment, store at 4 C for up to 1 month.Prepare aliquots of 20 mL and keep on ice until further usage.
Prepare in a sterile environment, store at 4 C for up to 4 weeks.Prepare aliquots of 50 mL for warming up to 37 C. Prepare fresh in a sterile environment, store at 4 C and use up within 10 days.Small molecule preparation for tumor and healthy organoid culture mediumReagentPreparation Storage / aliquots N-Acetylcysteine 500 mM stock in H2O À20 C for up to one year / 500 mL aliquots Nicotinamide 1 M stock in DPBS À20 C for up to one year / 2 mL aliquots (Continued on next page)Prepare in a sterile environment, store at À20 C for up to 3 months.Alternative for PBMC cryopreservation medium: this freezing medium can be used for cryopreservation of PBMCs as well.Prepare in a sterile environment, store at 4 C for up to 4 weeks, without addition of IL-2.Prepare aliquots of 50 mL for warming up to 37 C.
44.Before you start with the DNA extraction protocol, reconstitute all the components supplied with the Kit according to the manufacturer's recommendations.Incubate at 55 C for 10 min to promote protein digestion.53.Add 200 mL 96%-100% EtOH to the lysate.54.Mix well by vortexing to yield a homogenous solution.55.Remove a PureLink Spin Column in a Collection Tube (supplied with the Kit).56.Add the lysate ($640 mL) prepared with PureLink Genomic Lysis/Binding Buffer and EtOH to the Spin column.57.Centrifuge the column at 10.000 3 g for 1 min at 20 C.
Note: Make sure there is no precipitate visible in the PureLink Genomic Digestion Buffer of PureLink Genomic Lysis/Binding Buffer (troubleshooting 5).45.Set a water bath or a heat block to 55 C. 46.Add 20 mL Proteinase K to a sterile DNase free microcentrifuge tube.47.Resuspend your pellet in 200 mL DPBS.48.Transfer 200 mL cells in DPBS to the tube containing Proteinase K. 49.Add 20 mL RNase A to the sample.50.Mix well by brief vortexing and incubate for 2 min at 20 C G 2 C. CRITICAL: To avoid shearing of DNA at each vortexing step, vortex your samples not more than 5-10 s. 51.Add 200 mL PureLink Genomic Lysis/Binding Buffer and mix well by vortexing to obtain a homogenous solution.52.
Mechanically dissociate the organoids by putting a 200 mL tip on top of a 1000 mL tip and pipette 103 slowly up and down.149.Put cell suspension through a pre-wetted 40 mm cell strainer.150.Collect the suspension in a 15 mL centrifuge tube.151.Centrifuge at 405 3 g for 5 min at 4 C. 152.Remove the supernatant carefully without disturbing the cell pellet.
e. Two centrifuges, one at 20 C, one at 4 C. Note: By coating tips with 13 DPBS with 1% BSA cell loss can be avoided.137.Remove the medium in the wells.138.Add 2 mL of pre-warmed (37 C) DPBS into the wells.139.Scrape drops with a cell scraper.140.Collect organoids in a 15 mL centrifuge tube.141.Fill up to 10 mL with pre-warmed (37 C) DPBS.142.Centrifuge at 405 3 g for 5 min at 20 C. 143.Carefully discard supernatant.144.Resuspend in pre-warmed (37 C) 13 Trypsin: use 500 mL for each drop used in the beginning.145.Pipette 53 up and down with a P1000.146.Incubate 5 min in a water bath set to 37 C. 147.Stop Trypsin-reaction by adding the same amount of Advanced DMEM/F-12 medium (22 C G 2 C).148.Timing: 30 min to 1 h isolation, $2 weeks culture time, $30 min cryopreservation STAR Protocols 5, 102887, March 15, 2024 160.Add 1000 U/mL IL-2 into each well (use a new tip for each well).161.Mix the medium containing IL-2 of each well by pipetting carefully 23 up and down.162.Incubate at 37 C and 5% CO 2 .
Typically, for patient-derived CRC organoids, around 4 drops consisting of 30 mL of 70% Geltrex with confluent organoids are needed to generate 1 sample for exome sequencing analysis.Detach the droplets from the well using a cell lifter.233.Collect the droplets in DPBS with P1000 and transfer to a 15 mL centrifuge tube.234.Add 350 mL DPBS well.
the appropriate dissociation program for your species and organ of choice (tumor type:soft, program: 37C_h_TDK_1).
Note: Check if tissue pieces are completely dissociated.If not, proceed with the program.333.Detach C tube from the dissociator.334.Centrifuge at 300 3 g for 30 s at 20 C. 335.Remove the supernatant without disturbing the cell pellet.
280), is 2.07-2.10.A A 260 /A 280 ratio of $2.0 is accepted as pure RNA.The estimated RNA integrity has a RIN value of > 9. High-quality RNA contains a RIN value of R 8 (max.RIN value is 10).The percentage of contamination with genomic DNA is $ 3%, which indicates almost no contamination.The expected DNA yield after purification ranges from 30-120 ng/mL for 4 drops of confluent tumor organoid.The A 260 /A 280 ratio for nucleic acid purity is 1.8-2.0.The percentage of RNA contamination is < 4 ng/mL, which indicates almost no contamination.