Establishment and Culture of Patient-Derived Breast Organoids

Summary

A detailed protocol is provided here for establishing human breast organoids from patient-derived breast tumor resections or normal breast tissue. The protocol provides comprehensive step-by-step instructions for culturing, freezing, and thawing human patient-derived breast organoids.

Abstract

Breast cancer is a complex disease that has been classified into several different histological and molecular subtypes. Patient-derived breast tumor organoids developed in our laboratory consist of a mix of multiple tumor-derived cell populations, and thus represent a better approximation of tumor cell diversity and milieu than the established 2D cancer cell lines. Organoids serve as an ideal in vitro model, allowing for cell-extracellular matrix interactions, known to play an important role in cell-cell interactions and cancer progression. Patient-derived organoids also have advantages over mouse models as they are of human origin. Furthermore, they have been shown to recapitulate the genomic, transcriptomic as well as metabolic heterogeneity of patient tumors; thus, they are capable of representing tumor complexity as well as patient diversity. As a result, they are poised to provide more accurate insights into target discovery and validation and drug sensitivity assays. In this protocol, we provide a detailed demonstration of how patient-derived breast organoids are established from resected breast tumors (cancer organoids) or reductive mammoplasty-derived breast tissue (normal organoids). This is followed by a comprehensive account of 3D organoid culture, expansion, passaging, freezing, as well as thawing of patient-derived breast organoid cultures.

Introduction

Breast cancer (BC) is the most commonly occurring malignancy in females, with 287,850 new cases estimated to be diagnosed in the United States in 2022¹. Despite the recent advances in early detection with annual screenings, targeted therapies, and a better understanding of genetic predisposition, it prevails to be the second leading cause of cancer deaths in females in the United States, with >40,000 deaths attributed to breast cancer annually¹. Breast cancer is currently classified into multiple subtypes based on histopathological and molecular evaluation of the primary tumor. Better subtype stratification has improved patient outcomes with subtype-specific treatment options². For instance, the identification of HER2 as a proto-oncogene³ has led to the development of Trastuzumab, which has made this highly aggressive subtype manageable in most patients⁴. Further research into the genetics and transcriptomics of this complex disease in a patient-specific manner will aid in developing and predicting better patient-specific personalized treatment regimens^2,5. Patient-derived organoids (PDOs) are a promising new model to gain insights into cancer at the molecular level, identifying novel targets or biomarkers and designing new treatment strategies^6,7,8.

PDOs are multicellular, three-dimensional (3D) structures derived from freshly resected primary tissue samples^8,9. They are grown three-dimensionally by being embedded in a hydrogel matrix, typically composed of a combination of extracellular matrix (ECM) proteins, and hence can be used to study tumor cell-ECM interactions. PDOs represent patient diversity and recapitulate cellular heterogeneity and genetic features of the tumor^10,11,12. Being in vitro models, they allow for genetic manipulation and high-throughput drug screens^13,14,15. Further, PDOs can be plausibly utilized to evaluate patient drug sensitivity and treatment strategies in parallel to the clinic and help predict patient outcomes^16,17,18. Besides chemotherapy, certain organoid models have also been used to examine individual patient responses to chemoradiation^19,20. Given the promising applicability of PDOs for research and clinical use, the National Cancer Institute has initiated an international consortium, The Human Cancer Models Initiative (HCMI)²¹, to generate and provide these tumor-derived novel cancer models. Many of the organoid models of various cancer types developed through the HCMI are available via the American Type Culture Collection (ATCC)²².

Normal breast organoids have been shown to be comprised of different epithelial cell populations present in the mammary gland^11,23 and thus serve as great models to study basic biological processes, to analyze driver mutations causing tumorigenesis, and for cancer cell-of-origin lineage studies^6,15. Breast tumor organoid models have been used to identify novel targets that are encouraging prospects for developing new therapies, particularly for resistant tumors^24,25,26. Using patient-derived xenograft (PDX) and matched PDX-derived organoid (PDxO) models of treatment-resistant breast tumors, Guillen et al. showed that organoids are powerful models for precision medicine, which can be leveraged to evaluate drug responses and direct therapy decisions parallelly²⁸. Furthermore, the development of new co-culture methods for culturing PDOs with various immune cells^27,28,29, fibroblasts^30,31 and microbes^32,33 presents an opportunity to study the impact of the tumor microenvironment on cancer progression. While many such co-culture methods are actively being established for PDOs derived from pancreatic or colorectal tumors, similar established co-culture methods for breast PDOs have only been reported for natural killer cells³⁴ and fibroblasts³⁵.

The first biobank of >100 patient-derived organoids representing different breast cancer subtypes was developed by the Hans Clevers group^36,37. As part of this effort, the Clevers group also developed the first complex culture medium for breast organoid growth, which is currently widely used³⁶. A follow-up study provided a comprehensive account of the establishment and culture of breast PDOs and patient-derived organoid xenografts (PDOXs)³⁸. The Welm lab developed a large collection of BC PDX models and PDxOs that are cultured in a comparatively simpler growth medium containing fetal bovine serum (FBS) and fewer growth factors^39,40. We have independently developed and characterized a large array of naïve patient-derived breast cancer organoid models¹¹, and participated in developing BC PDO models as part of the HCMI initiative²¹. Here, we aim to provide a practical guide detailing the methodology employed by us in generating patient-derived breast organoid model systems.

Protocol

Tumor resections from breast cancer patients, along with the distal and adjacent normal tissue, were obtained from Northwell Health in accordance with Institutional Review Board protocols IRB-03-012 and IRB 20-0150, and with written informed consent from the patients.

NOTE: All procedures mentioned below were performed in a mammalian tissue culture BSL2 room designated for patient samples upon approval of the biosafety committee. All procedures should be performed following safety protocols maintaining aseptic conditions in biosafety cabinets. Each centrifugation step is performed at room temperature (RT) unless stated otherwise. Tissue/organoids and basement membrane matrix stocks are always placed on ice unless stated otherwise. New plates are incubated overnight for pre-warming. Plating domes on pre-warmed plates ensures the best results to obtain rounded domes that don't flatten out while plating or lifting off later from the plate surface.

1. Medium preparation and recipes

1. Prepare R-spondin1 conditioned media as described below (alternatively can be bought commercially).
   1. Prepare two separate growing media composed of Dulbecco's modified Eagle's medium (DMEM), 10% FBS, 5% penicillin-streptomycin, and with or without 300 µg/mL zeocin supplementation.
   2. Thaw a vial of HEK293T-HA-Rspondin1-Fc cells (obtained from Calvin Kuo's lab at Stanford University and also commercially available) in a 75 cm² tissue culture flask with 15 mL of medium.
   3. Split the cells into 2 x 175 cm² culture flasks, each with 35 mL of medium containing zeocin.
   4. Passage the cells again to obtain as many flasks as needed for generating the desired batch of R-spondin1 conditioned medium. Use growth medium without zeocin.
   5. When the flasks containing medium without zeocin are confluent, remove and replace the growing medium with Ad-DF+++ (step 1.2; Table 1). Place the cells in a 37 °C incubator with 5% CO₂ for 1 week.
   6. Spin down the medium at 400 x g for 5 min to remove any unattached cells. Filter the medium through a 0.22 µm filter. Make 25 mL aliquots and store them at -20 °C (can be stored for up to 6 months).
   7. Using HEK293T cells, perform a dual luciferase TOPFLASH^41,42 assay using a commercial DNA transfection reagent at a 4.8:1 reagent to DNA ratio with the diluent DMEM (high glucose, pyruvate). Add 100 µL of R-spondin1 conditioned medium (or basal medium for the negative control) to 100 µL of transfected cells. Then, perform the dual luciferase assay as per the manufacturer's protocol to verify Wnt activity of the R-spondin1 conditioned medium.
      NOTE: The assay uses a TOP plasmid with Firefly luciferase activity read-out, and the FOP plasmid is used as a negative control.
2. Prepare basal medium (Ad-DF+++ medium, as previously published by Sachs et al.³⁶).

   Reagent	Stock Concentration	Final Concentration
   Advanced DMEM/F12	1x	1x
   GlutaMax	100x	1x
   HEPES	1 M	10 mM
   Penicillin-Streptomycin	10,000 U/mL; 10,000 µg/mL	100 U/mL; 100 µg/mL

   
   Table 1: Basal Ad-DF+++ medium composition.
3. Prepare complete medium for patient-derived breast organoids as previously published by Sachs et al.³⁶.

   Reagent	Stock Concentration	Final Concentration
   Ad-DF+++ medium	1x	1x
   R-spondin in-house	100%	10%
   B-27 supplement	50x	1x
   Nicotinamide	1 M	5 mM
   NAC	500 mM	1.25 mM
   Primocin	50 mg/mL	50 µg/mL
   Noggin	100 µg/mL	100 ng/mL
   Human EGF	5 µg/mL	5 ng/mL
   Human Heregulin β1/Neuregulin1	75 µg/mL	37.5 ng/mL
   Y-27632 Dihydrochloride (Rho-Kinase)	100 mM	5 µM
   A83-01	5 mM	500 nM
   Human FGF-7	100 µg/mL	5 ng/mL
   Human FGF-10	1 mg/mL	20 ng/mL
   p38i	30 mM	498 nM

   
   Table 2: Complete medium composition for patient-derived breast organoids.
4. Prepare 2 mg/mL collagenase IV solution by weighing the required amount of collagenase IV powder and solubilizing it in Ad-DF+++ basal medium. Filter through a 0.22 µm filter before use.
5. Prepare 0.5% bovine serum albumin (BSA) solution from 7.5% stock solution using 1x Dulbecco's phosphate buffered saline (DPBS) as a diluent. Filter through a 0.22 µm filter before use.

2. Establishing breast tumor/normal organoids from resected tissue (Figure 1)

1. Transport a surgically resected breast tumor/normal tissue specimen to the lab in a 50 mL conical tube containing Ad-DF+++. Store the tube on ice until the start of isolation.
2. Thaw a bottle of basement membrane matrix on ice or overnight at 4 °C.
3. Transfer the resected tissue to a 10 cm sterile Petri dish. Examine the tissue macroscopically and make a note if it appears morphologically fatty, vascularized, or necrotic. Additionally, record the size and shape of the tissue. Ideally, take a picture of the tissue with a ruler in view (Figure 2).
4. Mince the tissue into very small pieces (~1 mm³) with a sterile no. 10 scalpel, and transfer it into a 50 mL conical tube.
5. Add 10 mL of 2 mg/mL collagenase IV solution and seal the tube with a transparent film. Place the tube on an orbital shaker at 37 °C at 140 rpm for 30-90 min in an angled position (~30° angle).
6. During the incubation, place an aliquot of complete medium to pre-warm in a 37 °C bead/water bath.
7. Every 15 min, resuspend the tissue by mixing up and down vigorously using a 5 mL sterile serological pipette. Pre-coat the pipette with 0.5% BSA solution to prevent tissue loss caused by the sample sticking to the pipette. Monitor dissociation over time by observing the conical tube under the microscope at a 5x or higher magnification.
8. Once the tissue is dissociated, centrifuge at 400 x g for 5 min. Aspirate the supernatant, add 10 mL of Ad-DF+++, and centrifuge at 400 x g for 5 min.
9. Discard the supernatant carefully. Tissue pellets can occasionally be loose, so be careful when aspirating. Repeat the step once more.
10. If the tissue pellet is partially red, add 2 mL of red blood cell lysis buffer and incubate for 5 min at room temperature. Do not incubate for longer, as this will significantly reduce cell viability.
11. Add 10 mL of Ad-DF+++ to the tube, centrifuge at 400 x g for 5 min, and discard the supernatant. Resuspend the pellet in 50-300 µL of undiluted, cold basement membrane matrix and mix by pipetting carefully to avoid forming bubbles.
    NOTE: The volume of the basement membrane matrix added depends on the pellet size. If unsure, plate a small dome of ~10 µL from the resuspended pellet and observe the confluency under the microscope. Adjust by adding more basement membrane matrix if needed. Refer to Figure 3 (day 1 image) for a reference seeding density.
12. Plate 300 µL of basement membrane matrix dome containing organoids in each well of a pre-warmed, labeled, 6-well tissue culture plate. Refer to Table 3 for recommended basement membrane matrix and medium volumes if using different plates.

    Number of wells per plate	Basement Membrane Matrix per dome (µL)	Medium per well (µL)
    6	300	3000
    12	100	1000
    24	50	500
    48	25	250
    96	10 (suspension instead of dome)	100

    
    Table 3: Amount of basement membrane matrix and growth medium recommended per well based on plate size.
13. Leave the plate undisturbed in the hood for 5 min and then carefully place in the incubator at 37 °C for 20-30 min for the basement membrane matrix dome to fully solidify.
14. Add 3 mL of pre-warmed complete medium dropwise to each well of a 6-well plate and place in the incubator at 37 °C and 5% CO₂. Take images of the organoids using a 5x objective on an inverted brightfield microscope.
15. Replenish the medium every 4-8 days based on the growth of organoids. Carefully aspirate the old medium without disturbing the dome, and add fresh, pre-warmed medium dropwise.
    NOTE: The protocol is the same for establishing breast cancer organoids derived from the resected tumor and for normal organoids derived from resected healthy breast tissue (either from adjacent and normal breast tissue of the same patient or healthy breast tissue obtained from a reductive mammoplasty surgery).

3. Passaging and expanding patient-derived breast organoids in culture

1. Thaw a bottle of basement membrane matrix on ice or overnight at 4 °C.
2. Lift the basement membrane matrix dome into the medium in the well using a cell scraper or a 1 mL pipette tip.
3. Pre-coat the pipette tip with 0.5% BSA solution and then transfer the floating dome of the organoids with medium to a 15 mL or 50 mL conical tube, depending on the number of wells being harvested. For more than two wells containing 300 µL of basement membrane matrix domes, use a 50 mL conical tube. Add 1x DPBS to increase the volume to at least 5 mL.
4. Spin the tubes at 400 x g for 5 min. The basement membrane matrix with organoids forms a layer at the bottom. Carefully aspirate to remove the supernatant.
5. Add 5-20 mL of 1x DPBS, depending on the number of wells pooled in a tube. Pre-coat a sterile disposable pipette with 0.5% BSA and mix the organoid-basement membrane matrix pellet in DPBS uniformly by pipetting up and down.
6. Spin the tubes at 400 x g for 5 min. Carefully aspirate and discard the supernatant.
7. Add cell dissociation reagent at three times the volume of the basement membrane matrix to the tube. Using a 0.5% BSA-coated pipette tip, resuspend the organoids in the cell dissociation reagent.
8. Place the tube on an orbital shaker at 37 °C at 140 rpm for 8-15 min in an angled position. Monitor the tube by observing it under the microscope every 5 min to ensure the organoids are broken down into smaller clusters.
9. Add basal medium (i.e., Ad-DF+++) at a volume equal to or greater than the cell dissociation reagent, and pipette to mix the organoids. Spin at 400 x g for 5 min at RT to obtain an organoid pellet.
10. If the pellet contains organoids still embedded within the undissolved basement membrane matrix, repeat steps 3.7-3.9 for an additional 5-8 min of trypsinization.
11. Once a white organoid pellet with no undissolved basement membrane matrix is obtained, discard the supernatant, and add 1 mL of Ad-DF+++ to resuspend the pellet by pipetting. Then add more Ad-DF+++ up to 10 mL.
12. Spin at 400 x g for 5 min at RT for the wash step. Aspirate and discard the supernatant.
13. Add the required amount of basement membrane matrix to the digested organoids based on the appropriate split ratio (this will vary widely for each organoid line based on growth rate). Refer to Figure 3 (day 1 image) for an example of seeding density. Mix by gently pipetting up and down to avoid creating bubbles. Immediately place on ice.
14. Open and label a pre-warmed 6-well plate. It is recommended to plate a 10-20 µL dome in the corner of a well to observe the confluency under the microscope. If the organoids are confluent, more basement membrane matrix can be added to increase the split ratio.
15. Plate 300 µL domes of organoids resuspended in basement membrane matrix. Make sure to keep the tube on ice while plating multiple wells so that the basement membrane matrix remains in solution.
16. Leave the plate undisturbed in the hood for 5 min and then carefully place in a 37 °C incubator with 5% CO₂ without disturbing the domes.
17. After 20-30 min, the domes solidify. Add 3 mL of pre-warmed complete medium to each well and place the plate back in the incubator. Add fresh complete medium every 5-7 days. Perform routine testing of the cultures for detecting mycoplasma contamination.
    NOTE: The protocol for the culture of breast tumors and normal organoids is the same. However, in our experience, normal organoids tend to grow well up to passage 6-8 before slowing down. It is advisable to make as many early passage stocks as possible for future research.

4. Freezing patient-derived breast organoids

1. Passage confluent wells of organoids to remove any basement membrane matrix, as stated in steps 3.1-3.12.
2. Resuspend the pellet of organoids in cell freezing medium (thawed overnight at 4 ºC) with 1 mL of medium per 200-300 µL of basement membrane matrix volume before passaging. Perform a cell count before freezing for reference. Aliquot a small volume (at least 100 µL) of cells to undergo further trypsinization, if needed, to get single cells, and then count them using a cell counter machine.
3. Transfer 1 mL of cells into 2 mL cryovials labeled with organoid line number, date, and passage number. Be sure to label the tubes with a permanent marker or use cryolabels.
4. Move the vials to a cell freezing container and transfer the container to a -80 °C freezer. After incubation overnight, the vials are ready to be moved to a liquid nitrogen storage freezer for long-term storage.

5. Thawing patient-derived breast organoids

1. Thaw a bottle of basement membrane matrix on ice or overnight at 4 °C. Pre-warm Ad-DF+++ medium at 37 °C. Aliquot 9 mL of pre-warmed medium into 15 mL conical tubes for each frozen vial.
2. Rapidly thaw the frozen vial of organoids by placing it in a 37 °C bead bath. Spray 70% ethanol and transfer the tube to the biosafety cabinet.
3. Rinse the pipette tip with 0.5% BSA solution to avoid organoid loss. Mix the thawed organoids gently by pipetting up and down to mix any settled organoids in the tube.
4. Gently transfer 1 mL of frozen organoids to 9 mL of pre-warmed Ad-DF+++ in a dropwise manner. Spin the cells at 400 x g for 5 min and then carefully discard the supernatant.
5. Wash the pellet by adding 10 mL of fresh pre-warmed Ad-DF+++ to the organoid pellet, and mix gently with a pipette pre-coated with 0.5% BSA to resuspend the pellet. Spin the cells at 400 x g for 5 min and carefully discard the supernatant.
6. Place the organoid pellet on ice and remove any remnant Ad-DF+++ carefully using a smaller volume pipette tip. Resuspend the pellet in 300 µL of basement membrane matrix and plate in a pre-warmed 6-well plate. Place the plate in a 37 °C incubator with 5% CO₂.
   NOTE: It is recommended to plate a small 10 µL dome from the resuspended pellet in a corner of the well to discern the confluency under the microscope. If the organoids look confluent, dilute by adding 300 µL of basement membrane matrix to plate into two wells of a 6-well plate.
7. After a 20-30 min incubation, add 3 mL of pre-warmed complete medium to the solidified dome.

Results

We have established a biobank of patient-derived breast tumor organoids comprising various subtypes¹¹. Additionally, we have established multiple normal breast organoid lines derived from reductive mammoplasty tissue samples or adjacent/distal normal breast from BC patients using the approach outlined in Figure 1.

The various patient-derived breast tumor organoid lines differ in their morphology (Figure 2) and ...

Discussion

Our lab has successfully employed the above protocols to establish organoids from naïve tumor resections or scrapings. We have also utilized this protocol to develop normal organoids from breast tissue obtained via reductive mammoplasties or from cancer patients' adjacent or distal normal breast tissue. About 30%-40% of the resected primary tumors resulted in successful long-term (>passage 8) tumor organoid cultures. The tumor organoid lines that tapered off after a few passages either had an outgro...

Materials

Name	Company	Catalog Number	Comments
15 mL conical tubes	VWR	525-1068
175 cm2 tissue culture flask	VWR (Corning)	29185-308
37 °C bead bath
37 °C CO2 incubator
50 mL conical tubes	VWR	525-1077
50 mL vacuum filtration system (0.22 µm Filter)	Millipore Sigma	SCGP00525	SCGP00525
500 mL Rapid-Flow Filter Unit, 0.2 µm aPES membrane, 75 mm diameter	Nalgene	566-0020
6-well culture plates	Greiner Cellstar	82050-842
75 cm2 tissue culture flask	VWR (Corning)	29185-304
96-well opaque plates	Corning	353296	For CTG assay
A83-01	Tocris	2939
Advanced DMEM/F12	Gibco	12634-010
B-27 supplement	Life Technologies	12587010
BioTek Synergy H4 Hybrid Microplate Reader	Fisher Scientific (Agilent)		For dual luciferase assay and CTG assay
BSA fraction V (7.5%)	Thermo Fisher	15260037
Cell Titer-Glo (CTG) Reagent	Promega	G9683	luminescent cell viability assay
Centrifuge	Eppendorf	5804
Collagenase from Clostridium histolyticum	Millipore Sigma	C5138	Type IV
Cryolabels	Amazon	DTCR-1000	Direct Thermal Cryo-Tags, White, 1.05 x 0.5"
Cryovials	Simport Scientific Inc.	T311-1
Countess 3 Automated Cell Counter	Thermo Fisher	AMQAX2000
DMEM, high glucose, pyruvate	Thermo Fisher (Gibco)	11995040
Dual Luciferase Reporter Assay System	Promega	E1910
Dulbecco’s Phosphate Buffered Saline (1X)	Gibco	14190-144	DPBS
Epidermal growth factor (hEGF)	Peprotech	AF-100-15
Fetal Bovine Serum (FBS)	Corning	35-010-CV
FGF-10 (human)	Peprotech	100-26
FGF-7/KGF (human)	Peprotech	100-19
GlutaMax	Life Technologies	35050061
HEK293T cells	ATCC	CRL-3216	For TOPFlash Assay
HEK293T-HA-Rspondin1-Fc cells	R&D Systems	3710-001-01	Cultrex HA-R-Spondin1-Fc 293T Cells
HEPES	Life Technologies	15630-080
Heregulinβ-1 (human)	Peprotech	100-03
Matrigel Growth Factor Reduced (GFR) Basement Membrane Matrix (~10 mg/mL protein concentration)	Corning	356231	Phenol-red free, LDEV-free; basement membrane matrix
Mr. Frosty Cell Freezing Container	Thermo Fisher	5100-0001
Mycoplasma detection kit	Lonza	LT07-418
N-acetyl-l-cysteine	Millipore Sigma	A9165
Nalgene Rapid-Flow Sterile Disposable Filter Units with PES Membranes	Thermo Fisher	166-0045
Nicotinamide	Millipore Sigma	N0636
Noggin (human)	Peprotech	120-10C
P1000, P200, P10 pipettes with tips
p38 MAPK inhibitor (p38i) SB 202190	Millipore Sigma	S7067
Parafilm			transparent film
Penicillin-Streptomycin	Life Technologies	15140122
Plasmid1: pRL-SV40P	Addgene	27163
Plasmid2: M51 Super 8x FOPFlash	Addgene	12457
Plasmid3: M50 Super 8x TOPFlash	Addgene	12456
pluriStrainer 200 µm	pluriSelect	43-50200-01
Primocin	Invivogen	ANT-PM-1
Recovery Cell Culture Freezing Medium	Thermo Fisher (Gibco)	12648-010	cell freezing medium
Red Blood Cell lysis buffer	Millipore Sigma	11814389001
R-spondin conditioned media	In-house or commercial from Peprotech	120-38
Scalpel (No.10)	Sklar Instruments	Jun-10
Shaker (Incu-shaker Mini)	Benchmark	H1001-M
TGF-β receptor inhibitor A 83-01	Tocris	2939
Trypan Blue Stain (0.4%)	Gibco	15250-061
TrypLE Express Enzyme (1X), phenol red	Life Technologies	12605028	cell dissociation reagent
X-tremeGENE 9 DNA transfection reagent	Millipore Sigma	6365779001
Y-27632 Dihydrochloride (RhoKi)	Abmole Bioscience	Y-27632
Zeocin	Thermo Fisher	R25001