This protocol discusses an approach for generating epithelial organoids from primary normal and tumor mammary tissue through differential centrifugation. Furthermore, instructions are included for three-dimensional culturing as well as immunofluorescent imaging of embedded organoids.
Organoids are a reliable method for modeling organ tissue due to their self-organizing properties and retention of function and architecture after propagation from primary tissue or stem cells. This method of organoid generation forgoes single-cell differentiation through multiple passages and instead uses differential centrifugation to isolate mammary epithelial organoids from mechanically and enzymatically dissociated tissues. This protocol provides a streamlined technique for rapidly producing small and large epithelial organoids from both mouse and human mammary tissue in addition to techniques for organoid embedding in collagen and basement extracellular matrix. Furthermore, instructions for in-gel fixation and immunofluorescent staining are provided for the purpose of visualizing organoid morphology and density. These methodologies are suitable for myriad downstream analyses, such as co-culturing with immune cells and ex vivo metastasis modeling via collagen invasion assay. These analyses serve to better elucidate cell-cell behavior and create a more complete understanding of interactions within the tumor microenvironment.
The ability to model epithelial cells in vitro has been the foundation of modern biomedical research because it captures cellular features that are not accessible in vivo. For instance, growing epithelial cell lines in a two-dimensional plane can provide an assessment of the molecular changes that occur in an epithelial cell during proliferation1. Furthermore, measuring the dynamic regulation between signaling and gene expression is limited in in vivo systems2. In cancer research, cancer epithelial cell line modeling has enabled the identification of molecular drivers of disease progression and potential drug targets3. However, growing cancer epithelial cell lines on a two-dimensional plane has limitations, as most are genetically immortalized and modified, often clonal in nature, selected for their ability to grow in non-physiologic conditions, limited in their assessment of three-dimensional (3D) tumor tissue architecture, and do not adequately model microenvironment interactions within a realistic tissue environment4. These constraints are particularly evident in modeling metastasis, which in vivo includes several distinct biological stages, including invasion, dissemination, circulation, and colonization at the distant organ site5.
Cancer epithelial organoids have been developed to better recapitulate the 3D environment and behavior of tumors6,7,8. Organoids were first developed from single LRG5+ intestinal crypt cells and differentiated to represent the 3D structure of crypt-villus units that maintained the hierarchical structure of the small intestine in vitro9. This approach permitted real-time visualization and characterization of self-organizing tissue architecture under homeostatic and stress conditions. As a natural extension, cancer epithelial organoids were developed to model many different cancer types, including colorectal10, pancreatic11, breast12, liver13, lung14, brain15, and gastric cancers16. Cancer epithelial organoids have been exploited to characterize cancer evolution17,18 and metastatic spatiotemporal behaviors19,20 and interrogate tumor heterogeneity21, and test chemotherapies22. Cancer epithelial organoids have also been isolated and collected during ongoing clinical trials to predict patient response to anticancer agents and radiation therapy ex vivo8,23,24,25. Furthermore, systems incorporating cancer epithelial organoids can be combined with other non-cancer cells, such as immune cells, to form a more comprehensive model of the tumor microenvironment to visualize interactions in real-time, uncover how cancer epithelial cells change the fundamental nature of cytotoxic effector immune cells such as natural killer cells, and test potential immunotherapies and antibody-drug dependent cytotoxic activity26,27,28. This article demonstrates a method of generating epithelial organoids without passaging and embedding in collagen and basement extracellular matrix (ECM). Additionally, techniques for downstream imaging of isolated organoids are also shared.
All mouse tissue utilized in this manuscript has been ethically collected in accordance with the Institutional Animal Care and Use Committee (IACUC) regulations and guidelines of the University of Texas Southwestern Medical Center. Likewise, all the patients consented prior to tissue donation under the oversight of an Institutional Review Board (IRB), and the samples were deidentified.
NOTE: This protocol describes the generation of organoids from primary tissue.
1. Overnight preparation of materials
2. Preparing collagenase and bovine serum albumin (BSA) coating solution
3. Preparing media
4. Collecting and digesting tissue
5. Differential centrifugation
6. Collecting small organoids
7. Embedding organoids in BECM
8. Embedding organoids in collagen
9. Fixing embedded organoids
10. Immunofluorescent staining of embedded organoids
The images featured in Figure 1 provide an example of wild-type and tumorous mammary epithelial organoids from human and mouse tissues. An at-a-glance illustration of the method for isolating epithelial organoids through differential centrifugation is provided in the cartoon workflow in Figure 1A, showing that primary tissues from different species can be processed in near-identical ways while yielding epithelial tissue as shown in the brightfield images (Figure 1B). Furthermore, these inter-species tissue composition similarities can be seen in the immunofluorescent images of embedded organoids in either basement extracellular matrix or collagen displayed in Figure 2A–D. The organoid structure can be visualized through either membrane Tomato (mTomato)-labeling or phalloidin staining of actin. These figures also demonstrate the expected organoid composition and size using this method. Organoids embedded in collagen matrix can be used for an invasion assay and analyzed by tracking the expansion of tendrils branching out from the organoid itself, as seen in Figure 2C. Lastly, hematoxylin and eosin (H&E) staining of paraffin-embedded organoids shows that organoids maintain the same histology of breast cancer (Figure 2E).
Figure 1: Workflow for the epithelial organoid generation with example organoids. (A) Schema of workflow for organoid generation from mouse or human tissue without passaging. (B) Representative images of isolated epithelial organoids from mouse WT mammary glands, mouse mammary tumors, and human breast tumors in media following isolation. Each image was adjusted individually for brightness and contrast for enhanced visualization. Images were taken in brightfield on an inverted epi-fluorescent microscope at 10x magnification. Scale bar represents 20 µm. Mammary tumors were isolated from MMTV-PyMT mice; normal mammary tissue was isolated from FVB mice. Mice ranged from 8-14 weeks of age29. Please click here to view a larger version of this figure.
Figure 2: Imaging organoids in the extracellular matrix. Each image was adjusted individually for brightness and contrast for enhanced visualization for this collection. Prior to imaging, samples were fixed with 4% paraformaldehyde. Wild-type samples were stained with phalloidin 568 to visualize the cell membrane and all samples were stained with Hoechst to visualize nuclei, showing organoid morphology and density. Images were taken at 10x magnification on a confocal microscope and further enlarged with a scanning zoom of 3.003. The laser wavelength used to detect DAPI was 405 nm with a power of 5 and the laser wavelength used to detect phalloidin was 561.0 nm for channel 3 with a power of 0.5. Confocal pinhole size was maintained at 19.16 for all images. (A) Representative brightfield image of mouse mammary WT organoid embedded in BECM at Day 0. (A') 1:250 Hoechst labeling nuclei and (A'') 1:250 phalloidin labeling actin immunofluorescent images of A. Scale bar represents 20 µm. Normal mammary tissue was isolated from FVB mice. Mice ranged from 8-12 weeks of age. (B) Representative brightfield image of mouse mammary tumor organoid embedded in BECM at Day 0. (B') 1:250 Hoechst labeling nuclei and (B'') mTomato-labeled immunofluorescent image of B. Scale bar represents 20 µm. Mammary tumors were isolated from MMTV-PyMT mice. Mice ranged from 12-14 weeks of age. (C) Representative brightfield image of mouse mammary tumor organoid embedded in Collagen I at Day 3. The image represents an organoid demonstrating "invasive" property. (C') 1:250 Hoechst labeling nuclei and (C') mTomato-labeled immunofluorescent image of C. Scale bar represents 20 µm. Mammary tumors were isolated from MMTV-PyMT mice. Mice ranged from 12-14 weeks of age. (D) Representative brightfield image of human breast tumor organoid embedded in BECM at Day 0. (D') Hoechst labeling nuclei and (D'') 1:250 Actin-targeting phalloidin-labeled immunofluorescent image of D. Scale bar represents 20 µm. (E) Sectioned image of an organoid embedded in BECM and stained with H&E taken at 20x magnification. Scale bar represents 20 µm. H&E staining was performed by the University of Texas Southwestern Tissue Management Shared Resource30. Please click here to view a larger version of this figure.
Culture plate | ECM dome Volume | Recommended number of organoids | Media volume |
6-well plate | 200 μL | 300 | 3 mL |
12-well plate | 150 μL | 225 | 2 mL |
24-well plate | 100 μL | 150 | 1 mL |
48-well plate | 40 μL | 60 | 300 μL |
96-well plate | 20 μL | 30 | 150 μL |
Table 1: Recommended volume of ECM components for domes, density of organoids, and volume of media needed per well on varying culture plates.
Problem | Potential cause | Solution | |||
ECM domes have collapsed. | The plate was shaken, the ECM volume was too large for the dome shape to be sustained, or the domes were touching the edge of the well, causing them to stick and fall. | Avoid moving the plate too vigorously before domes have set, reduce the volume of ECM used for the resuspension of organoids, or be very careful to pipette the domes in the center of the plate. | |||
There is tissue that does not look like organoids. | Muscle tissue or nerve may have been collected in the process of harvesting the mammary fat pad. | Only cut away what is clearly a mammary fat pad. Avoid removing the tissue that is adhered tightly to the skin. | |||
There are many dead organoids. | Necrotic tumor tissue was harvested. | Avoid collecting the tissue from tumors that are necrotic, cystic, excessively soft, or darker in color. Tumor tissue should be firm. | |||
There are more single cells than organoids. | Tissue was minced too finely, or collagenase digestion was run for too long. | Avoid over-mincing the tissue or reduce the collagenase incubation time. | |||
There are bubbles in ECM. | Resuspension by pipetting was too vigorous, and ejection while pipetting domes was too fast. | Slowly resuspend the organoids in the ECM and slowly pipette them into domes. If the issue persists, avoid going to the second stop while pipetting. | |||
There is no invasion of organoids within collagen. | Collagen was not properly polymerized or over-polymerized. | Do not resuspend the organoid in collagen until properly polymerized. Check every 30 min. If no polymerization is visible, resuspend at the 2 h mark and plate immediately. | |||
There are no collagen fibers visible while watching for polymerization. | Microscope settings were not favorable. | Adjust the phase contrast for maximum darkness, then bring the brightness of the microscope all the way up. Additionally, increasing the magnification may enhance viewing. | |||
ECM domes have dissolved. | Fixation time was too long, or PFA was not thoroughly removed from the ECM-embedded samples. | Keep fixation of ECM domes time below 5 min and follow up with five washes on a rotating shaker for 5 min each. |
Table 2: Table of potential problems, causes, and solutions.
Different methods have been described in the literature to generate tumor organoids. This protocol highlights a method for generating tumor organoids directly from the tumor without passaging. Using this method, tumor organoids are producible within hours of initiating the procedure and generate close to 100% viable organoids compared to 70% reported in the literature31. In comparison, other methods require serial passaging of cells into organoids over several weeks. Thus, the downstream applications, such as determining and visualizing immune cell interactions with matched organoid and immune samples from the same host without the impact of long-term culture, become more feasible. Further, as highlighted in Figure 2C, embedding tumor organoids in different extracellular matrices can permit the identification of key phenotypes throughout the metastatic cascade, such as invasion out of the primary tumor. Other downstream applications include phenotypic assays of branching morphogenesis32, invasion, dissemination, and colony formation33 to assess for various epithelial cell behaviors. Immune interactions can also be functionally and visually captured with these organoid-based assays. Further, gels can be dissolved to isolate embedded cells for downstream analysis of genetic and protein content using standard biochemical and flow-based assays. Finally, because large quantities of organoids can be quickly generated from tumor tissue, these assays can be scaled for drug screening applications and integration into clinical trial workflows.
There are several key steps that are critical to this protocol. First, the amount of time required for collagenase digestion is dependent on the tissue composition and amount of tissue being digested. For example, when working with smaller pieces of tissue, such as human breast tumor surgical samples (on average 100-250 mg), a shorter time of digestion is required. However, mammary tumors harvested from a mouse are much larger in size (500-800 mg) and may require 30-60 min of enzymatic digestion. Secondly, to ensure a maximum yield of epithelial organoids, it is also important to coat all pipette tips and serological pipets with BSA to avoid loss from cell adhesion to plastic. Third, a brief differential centrifugation time is crucial for eliminating non-epithelial tissue components. This approach permits heavier epithelial organoids to pellet while lighter stromal and immune compartments remain in the supernatant. For creating invasion assays by embedding organoids in collagen, it is critical to allow for proper polymerization of the collagen before embedding organoids. This step should be checked visually by confirming collagen fiber formation under a light microscope. Finally, for best imaging results, take care to avoid producing bubbles when resuspending organoids in ECM and plating. Bubbles will obscure organoids within the gel and distort images. Table 2 lists potential problems that have been encountered and solutions to overcome these challenges.
Certain steps in the protocol permit modifications to customize the size of organoids generated or reduce the time required to execute the protocol. For example, increasing the duration of mechanical digestion time can result in a shorter collagenase digestion time, smaller organoids, and more individual cells. Centrifugation following collagenase digestion can be shortened to 5 min if working with a large amount of tumor tissue. Media used for culturing and growing organoids can be prepared a day prior to save time during the organoid generating steps. Similarly, tumor tissue can be stored for up to 24 h in appropriate media before organoid preparation. If time is extremely limited, this protocol includes pausing steps by freezing tumor tissue on the day of collection. Then, these frozen tissues can be used to generate viable organoids at a later date. Approximately 90% of organoids derived from the frozen tissue were viable, confirmed visually under a light microscope and with a trypan blue solution.
There are some limitations to this protocol. While this approach generates viable organoids quickly, the quantity of organoids generated is limited by the amount of tumor tissue. This limitation becomes especially apparent when working with clinical samples in which the amount of tumor tissue is less or, at times, even restricted to a few cells. In those extreme cases where only a few cells can be recovered as starting material, passaging may be a better option. Another limitation is the reductionist approach of this method. Removal of stromal compartments such as fibroblasts or endothelial cells enrich epithelial organoid generation. However, these cell populations are critical to the function of the tumor. Therefore, their removal limits the interpretation of tumor biology that is derived solely from epithelial organoid models. In conclusion, this protocol provides an approach for the quick generation of epithelial organoids for immediate use in downstream imaging, functional (including immune interactions), and drug-screening applications.
The authors have nothing to disclose.
This study was supported by funding provided by METAvivor, the Peter Carlson Trust, Theresa's Research Foundation, and the NCI/UTSW Simmons Cancer Center P30 CA142543. We acknowledge the assistance of the University of Texas Southwestern Tissue Management Shared Resource, a shared resource at the Simmons Comprehensive Cancer Center, which is supported in part by the National Cancer Institute under award number P30 CA142543. Special thanks to all members of the Chan Lab.
10 mM HEPES Buffer | Gibco | 15630080 | |
100x Antibiotic-Antimycotic | Gibco | 15240-096 | |
100x Glutamax | Life Technologies | 35050-061 | Glutamine supplement |
100x Insulin-Transferrin-Selenium (ITS) | Life Technologies | 51500-056 | |
100x Penicillin/Streptomycin (Pen/Strep) | Sigma | P4333 | |
10x DMEM | Sigma | D2429 | |
50 mL/0.2 µm filter flask | Fisher | #564-0020 | |
Amphotericin B | Life Technologies | 15290-018 | |
bFGF | Sigma | F0291 | |
BSA Solution (32%) | Sigma | #A9576 | |
Cholera Toxin | Sigma | C8052 | |
CO2-Independent Medium | Gibco | 18045-088 | |
Collagenase A | Sigma | C2139 | |
Deoxyribonuclease I from bovine pancreas (DNase) | Sigma | D4263 | |
DMEM with 4500 mg/L glucose, sodium pyruvate, and sodium bicarbonate, without L-glutamine, liquid, sterile-filtered, suitable for cell culture | Sigma | D6546 | Common basal medium |
D-MEM/F12 | Life Technologies | #10565-018 | Basal cell medium |
Dulbecco's Phosphate Buffered Saline (D-PBS) | Sigma | #D8662 | PBS |
Fetal bovine serum (FBS) | Sigma | #F0926 | |
Gentamicin | Life Technologies | #15750-060 | |
Human epidermal growth factor (EGF) | Sigma | E9644 | |
Hydrocortisone | Sigma | H0396 | |
Insulin | Sigma | #I9278 | |
Matrigel | Corning | #354230 | Basement Extracellular Matrix (BECM) |
NaOH (1 N) | Sigma | S2770 | |
Rat Tail Collagen I | Corning | 354236 | |
RPMI-1640 media | Fisher | SH3002701 | |
Trypsin | Life Technologies | 27250-018 |