Patient-Derived Three-Dimensional Tumor Organoid Model for In Vitro Chimeric Antigen Receptor T Cell Screening

Summary

This protocol establishes an in vitro co-culture model using patient-derived tumor organoids and chimeric antigen receptor T (CAR-T) cells to evaluate the specific cytotoxic activity of CAR-T cells against solid tumor cells.

Abstract

Chimeric antigen receptor T (CAR-T) cell therapy has achieved exciting clinical efficacy in hematological malignancies, but CAR-T cell therapy for solid tumors still requires further development. Patient-derived tumor organoids are in vitro disease models that retain patient heterogeneity and have been used to test the efficacy and safety of chemotherapy and targeted drugs. This method describes an in vitro efficacy testing model of co-culturing tumor organoids with CAR-T cells. Colorectal cancer samples from patients are constructed into tumor organoids with a three-dimensional (3D) structure in a basement membrane matrix. The tumor organoids can grow stably and be passaged continuously. Mature tumor organoids are separated from the matrix by washing and centrifugation, and CAR-T cells are added at different effector-to-target (E:T) ratios to form an immune-organoid co-culture system. After 6-24 h, the morphology and intercellular structures of the organoids are observed by bright-field imaging. This experiment further performs dead cell staining in the co-culture system, which can reflect the viability of the organoids and evaluate the cytotoxic effect of CAR-T cells on tumor organoids. This experiment can effectively observe the interaction between CAR-T cells and 3D tumor organoids, and the results can be used to assess the tumor-killing activity of CAR-T cells.

Introduction

Chimeric antigen receptor T (CAR-T) cell therapy has shown remarkable clinical success in treating hematological malignancies¹. However, its application in solid tumors remains limited due to several major obstacles, including tumor heterogeneity and antigen loss, physical and chemical barriers within the tumor mass, and the immunosuppressive tumor microenvironment^2,3. These challenges contribute to the limited efficacy of CAR-T cell therapy in solid tumor settings.

In vitro evaluation of CAR-T cell efficacy is a critical step during preclinical development. The most commonly used models are tumor cell lines, which may either express specific tumor-associated antigens or be engineered to carry reporter genes such as luciferase^4,5. Consequently, results from conventional cell-line-based assays often fail to predict clinical outcomes. Patient-derived tumor models offer a promising alternative for assessing CAR-T function more accurately in the context of solid tumors.

While several studies have begun to utilize PDTOs for CAR-T cell evaluation, challenges remain in establishing standardized, robust, and accessible co-culture protocols that effectively balance the preservation of 3D tumor architecture with efficient immune cell-tumor cell interaction. Patient-derived tumor organoids (PDTOs) are three-dimensional (3D) ex vivo disease models generated from patient tumor tissues or cells. PDTOs have been successfully established for a wide range of tumor types, including colorectal, breast, pancreatic, and lung cancers. These structures retain the histological architecture, molecular profiles, and functional heterogeneity of the original tumor⁶. Alongside primary tumor cells and patient-derived xenografts (PDX), PDTOs represent a valuable platform for modeling patient-specific disease features⁷. Compared to conventional tumor cell lines or primary cultures, PDTOs more closely recapitulate the in vivo tumor environment, displaying complex cellular compositions and structural organization⁸. They also maintain genomic stability and preserve gene expression patterns over extended passages, allowing more accurate assessment of therapeutic responses^9,10. PDTOs have been successfully employed in studies of tumor biology, drug screening, and precision medicine, and are increasingly recognized for their utility in immunotherapy testing, including identification of novel CAR targets, CAR design optimization, and cytotoxicity assessment^6,11,12,13. A common limitation of organoid culture is its dependence on extracellular matrix (ECM) components like Matrigel (henceforth referred to as basement membrane matrix [BMM]) for 3D structure maintenance, which can hinder immune cell infiltration in co-culture assays. To ensure consistency and optimal cell interaction in co-culture, organoids within a defined size range (e.g., 100-200 µm in diameter) are typically selected.

This protocol describes a co-culture platform for evaluating CAR-T cell cytotoxicity using colorectal cancer-derived PDTOs. Patient tumor samples are enzymatically dissociated and cultured in BMM to generate 3D organoids. Once matured and expanded, organoids of uniform size are isolated and fluorescently labeled. They are seeded in multi-well plates and co-cultured with CAR-T cells at varying effector-to-target (E:T) ratios. A key feature of this approach is the deliberate removal of PDTOs from BMM prior to co-culture, enabling direct and unhindered access of CAR-T cells to the tumor organoids while maintaining their 3D integrity in suspension. After 24 h, bright-field microscopy reveals progressive PDTO disintegration, characterized by disrupted 3D morphology, tumor cell shrinkage, and loss of viability. Live-cell imaging and fluorescence microscopy further visualize interactions between fluorescently labeled CAR-T cells and tumor targets. Quantitative cell death assays are used to assess cytotoxicity. This organoid-based immune co-culture provides a robust and patient-specific platform for assessing the antitumor efficacy of CAR-T cells ex vivo, enabling therapeutic optimization and supporting clinical translation.

Protocol

Colorectal cancer patient-derived organoids were used. Tumor tissue fragments were obtained from treatment-naïve patients with histologically confirmed primary colorectal adenocarcinoma (CRC). All tissues were procured from endoscopic biopsy specimens collected during standard diagnostic procedures at Shenzhen Qianhai Shekou Free Trade Zone Hospital. All donor tissues were confirmed as CRC through histopathological examination by institutional pathologists. The protocol follows institutional guidelines. Written informed consents were obtained from all patients. The study was approved by the Shenzhen Qianhai Shekou Free Trade Zone Hospital Ethical Committee (2024KY-009-01K).

1. PDTO culture

1. Tissue preparation
   1. Place tumor tissue fragments obtained via colonoscopy biopsy from patients with histologically confirmed primary colorectal adenocarcinoma (CRC) into a sterile 50-mL tube.
   2. Add 30 mL of cold tissue wash solution composed of Hanks' Balanced Salt Solution (HBSS), 1% Bovine Serum Albumin (BSA), 100 U/mL penicillin, 0.1 mg/mL streptomycin, 50 µg/mL gentamicin, 5 µg/mL amphotericin B, and 10 µM Rho-associated kinase inhibitor (Y-27632).
   3. Place the tube on ice and rock gently for 5 min to wash the tissue.
   4. Repeat step 1.1.3. two additional times with new tissue wash solution.
2. Tissue digestion
   1. Trim away necrotic or adipose tissue and cut the tumor into tissue fragments.
   2. Warm 8 mL of digestion medium (DMEM medium, 500 U/mL collagenase IV, 1.5 mg/mL collagenase II, 20 µg/mL hyaluronidase, 0.1 mg/mL dispase type II, 10 µM Y-27632, and 1% fetal bovine serum) to 37 °C.
   3. Transfer fragments into the digestion buffer and incubate at 37 °C in a water bath. Swirl the tube gently by hand for ~10 s every 5 min to facilitate enzyme penetration.
      NOTE: Adjust digestion buffer volume according to tissue block size. Generally, 8-10 mL is sufficient for endoscopic biopsy sizes.
   4. Observe under a microscope after 15 min to check the dissociation status. If tissue fragments remain, continue digestion up to a maximum of 30 min.
   5. When clusters are visible, pipette gently (5-10 times) using a 10-mL pipette to mechanically dissociate. Centrifuge at 350 × g for 5 min.
   6. Discard supernatant; resuspend pellet in 10 mL of wash solution and centrifuge at 350 × g for 5 min. Repeat the wash for twice.
      NOTE: PDTOs typically began forming compact spherical structures within 3-5 days of culture. Organoids exhibited robust proliferation and reached a suitable size for experimentation (approximately 100-200 µm in diameter) by day 10-14. Passaging was performed every 7-10 days to maintain healthy growth and prevent over-confluence or central necrosis. Generally, 8-10 mL digestion buffer is used per biopsy (2 mm³ fragments), ensuring TrypLE Express at a 1× final working concentration.

2. Construction of PDTOs

1. Matrix embedding
   1. Place 200 µL of BMM (pre-chilled at 4 °C) into a clean tube and pipette the cell pellet up and down at least 10-15 times using a pre-cooled pipette tip until the suspension appears homogeneous, without visible clumps.
   2. Dispense approximately 30-µL droplets into each well of a 24-well plate. Incubate at 37 °C for 10 min to solidify.
   3. Add 500 µL of organoid culture medium (Advanced DMEM/F12 medium, containing 50 µg/mL R-spondin 1, 10 µg/mL Noggin, 500 µg/mL EGF, 100x HEPES, 100x Glutamax, 500x Normocin, 500x Gentamicin/amphotericin B, 50x N2, 100x B27, 500 mM n-Acetylcysteine, 1 M Niacinamide, 5 mM Alk 4/5/7 inhibitor, 30 M p38 inhibitor, 100 µM Gastrin, and 100 µM Prostaglandin E2) per well.
      NOTE: Use approximately 30 µL of BMM per droplet; adjust based on cell density.
2. Culture maintenance
   1. Culture organoids at 37 °C with 5% CO₂; change medium every 3 days.
   2. Monitor organoid formation daily. Expect small spheroids by day 3.
   3. Evaluate successful organoid establishment during days 6-10. Formation time (ranges between 3 to 14 days) depends on tissue viability and stemness.

3. Passaging PDTOs

1. Disaggregation
   1. Pre-cool the centrifuge to 4°C. Remove medium and add 1 mL of ice-cold HBSS and pipette gently 3-5 times to disrupt BMM.
      NOTE: Well-grown organoids can be observed as small white dots with the naked eye.
   2. Transfer the suspension into a sterile 15-mL tube and centrifuge at 350 × g for 5 min.
2. Enzyme dissociation
   1. Discard the supernatant and add 1 mL of TrypLE. Place in a 37 °C-water bath for 2 min.
   2. Add 2 mL of HBSS to neutralize TrypLE; centrifuge at 350 × g for 5 min.
   3. Remove the supernatant and resuspend in 2 mL of HBSS. Centrifuge again at 350 × g for 5 min.
3. Re-embedding
   1. Add fresh BMM to the pellet in a ratio of 1:3-1:6 (organoid pellet: BMM, v/v); resuspend gently. Each droplet contains approximately 30 µL final volume. Place droplets into a new 24-well plate; incubate at 37 °C for 10 min.
   2. Add 500 µL of culture medium per well and incubate at 37 °C with 5% CO₂.
   3. Examine wells daily; observe 3D re-assembly within approximately 48 h.
   4. After 3-5 passages, select healthy organoids for downstream assays or cryopreservation.

4. Co-culture of CAR-T cells and PDTOs

1. Co-culture medium preparation
   1. Supplement base organoid culture medium with 10 ng/L IL-2 and 0.05 mM β-mercaptoethanol to support CAR-T viability.
      NOTE: Base medium supports 3D structure and viability of PDTOs.
2. PDTO preparation
   1. Remove the BMM droplets containing mature PDTOs (e.g., Day 4-6) from the culture plate using a sterile cell scraper or pipette tip.
   2. Transfer the BMM/PDTO mixture to a 15-mL conical tube.
   3. Add 5-10 mL of ice-cold HBBS or tissue wash solution (as described in step 1.1.2) to the tube. Invert gently 5-10 times to dislodge organoids from BMM.
   4. Allow the mixture to stand on ice for 5 min. The BMM will become liquid, while dissociated PDTOs remain in suspension.
   5. Carefully aspirate the supernatant containing BMM, leaving the PDTO pellet behind. Avoid disturbing the pellet.
   6. Repeat steps 4.2.3 to 4.2.5 two additional times to ensure complete removal of BMM.
   7. After the final wash, collect the PDTO-containing supernatant and centrifuge at 300 × g for 5 min at 4 °C.
   8. Aspirate the supernatant and resuspend the PDTO pellet in 1-2 mL of organoid culture medium or co-culture medium.
   9. (Optional for counting) Prepare a single-cell suspension.
      1. If precise counting is required, transfer the resuspended organoids to a new tube and incubate with pre-warmed TrypLE Express at 37 °C for 3-5 min, followed by gentle pipetting to achieve a single-cell suspension. Proceed with cell counting (e.g., using a hemocytometer or automated cell counter).
         NOTE: This step dissociates the organoids and is only for counting purposes before re-plating.
   10. Size selection and standardization: To ensure consistent organoid size for co-culture, pass the resuspended PDTOs (from step 4.2.8) through a 70-µm cell strainer placed on top of a 50-mL conical tube. This step removes large organoid aggregates and debris, representing a standardized size fraction suitable for uniform co-culture.
   11. Centrifuge the standardized organoid suspension at 300 × g for 5 min at 4 °C.
   12. Aspirate the supernatant and resuspend the standardized PDTOs in an appropriate volume of pre-warmed co-culture medium.
   13. Count the standardized PDTOs (e.g., using a hemocytometer under a microscope, counting the number of organoids per field).
       NOTE: Counting is based on organoid number, not cell number.
   14. Seed the standardized PDTOs into the desired multi-well plate (e.g., 96-well U-bottom plate) at the target number per well.
   15. Add CAR-T cells to the wells at the desired effector-to-target (E:T) ratios.
3. Labeling PDTOs
   1. Pre-cool the centrifuge to 4 °C. Aspirate the medium; add 1 mL of HBSS to dislodge droplets.
   2. Transfer to a 15-mL centrifuge tube, add 4 mL of HBSS, and pipette gently. Centrifuge at 350 × g for 5 min.
   3. Discard the supernatant, add 5 mL of HBSS, and pipette gently. Centrifuge again at 350 × g for 5 min.
   4. Prepare Hoechst 33342 working solution.
   5. Resuspend pellet in 1 mL Hoechst 33342 solution (final concentration 5 µg/mL). Mix gently by inversion 3-5 times to ensure uniform dye penetration. Protect from light by wrapping the tube in aluminum foil. Incubate at 37 °C for 10 min. After staining, wash twice with HBSS to remove excess dye.
4. Plating PDTOs
   1. Estimate organoid density at a gel droplet-to-well ratio of 1:5; resuspend in co-culture medium at approximately 1 × 10³ organoids/mL.
   2. Add 100 µL per well into a 96-well plate; include at least triplicates per condition.
      NOTE: Do not perform cell counting at this stage; organoids remain intact.
5. CAR-T co-culture
   NOTE: This protocol focuses on the co-culture and efficacy assessment of CAR-T cells with PDTOs. The generation and characterization of the CAR-T cells used in this study are described in Section 8.
   1. Harvest CAR-T cells in log phase. Stain with Trypan Blue to confirm viability >95%.
   2. Centrifuge cells at 350 × g for 5 min; resuspend in co-culture medium.
   3. Adjust concentrations to 4 × 10⁶/mL, 2 × 10⁶/mL, and 1 × 10⁶/mL.
   4. Add 100 µL of CAR-T suspension per well according to the experimental design. Add 100 µL of co-culture medium to control wells.
   5. Incubate plate at 37 °C, 5% CO₂ for 6-24 h.

5. Dead cell staining

1. SYTOX green staining
   1. For each 1 × 10⁵ cells, prepare 100 µL of Nuclear Green staining solution by diluting 1 µL of SYTOX Green (1000x) into 1 mL of assay buffer.
   2. At the 6- and 24-h co-culture timepoints, add 100 µL of SYTOX Green staining solution per well. Incubate at 37 °C for 20 min in the dark.

6. Image acquisition

1. Aspirate the supernatant and add 100 µL of DMEM/F12.
2. Capture bright-field and fluorescence images using a microscope (e.g., confocal):
   Hoechst 33342 (PDTOs): excitation (Ex)/emission (Em) = 350 nm/461 nm
   mCherry (CAR-T): Ex/Em = 587 nm/610 nm
   SYTOX Green (dead cells): Ex/Em = 504 nm/528 nm
3. Capture at least triplicate images per well, focusing on the central field.

7. Data analysis

1. Launch ImageJ (version 1.54, NIH, https://imagej.nih.gov/ij/); import SYTOX Green channel images. Set the threshold manually to distinguish fluorescence signals from background (Image > Adjust > Threshold).
2. Use the Analyze > Measure function to calculate integrated density. Export data as .csv for subsequent statistical analysis in an appropriate statistical analysis software (e.g., GraphPad Prism).
3. Set threshold to separate fluorescence signals from background.
4. Select the entire field; run Analyze > Measure to calculate the summed fluorescence signal intensity.
5. Export data and perform statistical analysis to compare treated versus control at 24 h.

8. Generation and characterization of CAR-T Cells

1. CAR construct design
   NOTE: The CAR-T cells used in this study target B7-H3 (encoded by the CD276 gene), an antigen frequently overexpressed in colorectal cancer¹⁴. The CAR construct consists of:
   Extracellular domain: A single-chain variable fragment (scFv) derived from a monoclonal antibody against B7-H3 clone 376.96.
   Hinge and transmembrane domain: CD8α hinge and transmembrane domain.
   Co-stimulatory domain: human CD28.
   ​Signaling domain: human CD3ζ.
2. CAR-T cell generation
   1. Isolation: Isolate peripheral blood mononuclear cells (PBMCs) from healthy donor buffy coats using Ficoll-Paque density gradient centrifugation¹⁴.
   2. Activation: Activate human PBMCs with plate-bond anti-CD3/CD28 antibodies for 48 h in T cell media supplemented with 10 ng/mL IL-7 and 5 ng/mL IL-15. Prepare the T cell media by mixing equal volumes (250 mL each) of RPMI 1640 and Click's Media, and supplemented with 10% fetal bovine serum (FBS), 1x GlutaMAX, and 1% penicillin-streptomycin (Pen/Strep).
   3. Transduction: Transduce the activated T cells with retrovirus supernatant using a retronectin-coated plate as described previously¹⁵.
   4. Expansion: Harvest and culture T cells 3 days post transduction in complete human T-cell medium containing IL-7 and IL-15 for an additional 7-9 days before characterization and functional assays.
3. CAR-T cell characterization
   1. CAR expression: Assess the CAR expression 5-7 days post-transduction by flow cytometry using a recombinant B7-H3-Fc protein, followed by a fluorescently labeled secondary antibody against human Fc¹⁴. Typical transduction efficiency (CAR+ cells) ranges from 80% to 90%.
   2. Phenotype: Characterize the T cell subsets (e.g., CD4+, CD8+, etc) by flow cytometry using antibodies against CD3, CD4, CD8, CD45RA, CCR7, etc¹⁴.
   3. Functional validation (Optional pre-co-culture): Prior to co-culture experiments, validate the CAR-T cells functionally by co-culturing with B7-H3-positive and B7-H3-negative target cell lines and measuring cytokine release (e.g., IFN-γ by ELISA) or cytotoxicity (e.g., using flow cytometry¹⁵).
4. Preparation for co-culture (PDTO Assay)
   1. Use CAR-T cells for co-culture during their log-phase expansion (typically between days 10-14 post-activation). Before use, wash the cells twice with HBSS to remove IL-7/IL-15 and resuspend them in the co-culture medium described in step 4.1.
   2. Confirm viability to be >95% using Trypan Blue staining.
   3. Visual checkpoints: Check for small spherical clusters (50-100 µm) at days 3-5 and mature spheroids (100-200 µm) at days 10-14.

Results

Colorectal cancer organoids were successfully derived from endoscopic biopsy samples (Figure 1), with tumor identity confirmed by pathological diagnosis (Figure 2). The tumor fragments cultured in BMM began forming 3D structures within 24 h. First-generation organoids (P0) were passaged between days 7-14. Organoids exhibited diverse morphologies, including spherical, cystic, and circular structures (Figure 2A).

Discussion

This article presents a method for evaluating the cytotoxic effect of CAR-T cells on PDTOs, combining bright-field imaging and fluorescent staining to assess organoid viability. This dual-modality approach enables robust visualization of tumor cell death in a 3D model system and provides a semi-quantitative assessment of CAR-T cytotoxicity.

A critical component of this protocol is the culture medium. Because CAR-T cells and tumor organoids have distinct nutrient requirements, the compatibility...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
15-mL tube	SAINING	3030100
24-well plate	CORNING	3524
50-mL tube	SAINING	3030000
96-well plate	CORNING	3599
Advanced DMEM/F12	Gibco	C11330500BT
Alk 4/5/7 inhibitor	MCE	HY-10432
B27	Gibco	17504044
Biosafety Cabinet (Class II)	Thermo Fisher	N/A
Bovine Serum Albumin	Sigma Aldrich	A9056
Cell culture dish	BIOFIL	TCD010060
Centrifuge	Eppendorf	5810R
Click's Medium	FUJI FILM	9195
CO2 Incubator	Yamata	IP610
Collagenase II	Solarbio	C8150
Collagenase IV	Sigma Aldrich	C9407
Confocal laser microscope	Zeiss	LSM900
Cryopreservative medium	ZENOAQ	CELLBANKERTM2
Digestion buffer	Reagents prepared in-house	N/A	Composition provided in the manuscript
Digital Shaker	MIULAB	HS-25
Dispase type II	Sigma-aldrich	D4693
DMEM	Gibco	C11995500BT
Dynabeads Human T-Activator CD3/CD28	Thermo Fisher	11131D
EGF	PeproTech	AF-100-15-500UG
Fetal bovine serum	Gibco	1631389
Gastrin	GlpBio	GA20228
Gentamicin/amphoteritin B	Gibco	R0151
Glutamax	Gibco	35050061
Hanks' Balanced Salt Solution	Gibco	C14175500BT
HEPES	Gibco	15630080
Hoechst 33342	Beyotime	C1027
Hyaluronidase	Solarbio	h8030
IL-15	Peprotech	200-15-10UG
IL-2	Novoprotein	CK24-10
IL-7	Peprotech	200-07-10UG
ImageJ	NIH (open-source)	https://imagej.nih.gov/ij/
Inverted  fluorescent microscope	Mshot	MF52-N
Matrigel	Corning	356231
N2	Gibco	17502048
n-Acetylcysteine	Sigma Aldrich	A7250-5G
Niacinamide	Sigma Aldrich	N0636-100G
Noggin	PeproTech	120-10C-20
Normocin	InvivoGen	ant-nr-1
Organoid culture medium	Reagents prepared in-house	N/A	Composition provided in the manuscript
p38 inhibitor	MCE	HY-10295
Penicillin-Streptomy	Gibco	15140122
Prostaglandin E2	MCE	HY-101952
Rho-associated kinase inhibitor	Stem cell	72304
RPMI 1640	HyClone	SH30809.01B
R-spondin 1	PeproTech	120-38-20UG
S1 Pipet Fillers	Thermo Scientific	9501
Serological Pipette	BIOFIL	GSP-010-050
Single-channel pipette	Eppendorf	3123000063
Single-channel pipette	Eppendorf	3123000055
Single-channel pipette	Eppendorf	3123000098
Single-channel pipette	Eppendorf	3123000022
Sterile Disposable Scalpels	Tech-S	SSD10
SYTOX Green	Beyotime	C1181S
TexMACS Medium	Miltenyi Biotec	130-097-196
Thermostat water tank	Bluepard	BWS-10
Trypan Blue	Phygene	PH0519
TrypLE Express	Gibco	12605028
β-mercaptoethanol	Gibco	21985023