We thank Dr. Laura L. Bronsart for providing GFP and dTomato-labeled RAW 264.7 macrophages. This research was financially supported by NIH grant #R00CA201304.

Animal studies were performed in accordance with institutional guidelines and protocols approved by the Vanderbilt University Institutional Animal Care and Use Committee.
1. Preparation of mice and cell acquisition (adapted from Nguyen-Ngoc et al.11)
<ol>
	<li>Sacrifice athymic Nu/Nu mice (8-10 weeks old) using CO&#173;2 asphyxiation followed by cervical dislocation. Clean the skin using 70% ethanol.</li>
	<li>Resect abdominal and inguinal mammary glands from mice using pre-sterilized scissors and forceps. Remove lymph nodes before resection. Rinse in sterile 1x phosphate buffered saline (PBS) (Figure 1A).</li>
	<li>Place it in 15 mL tubes with 10 mL of Dulbecco&#8217;s Modified Eagle Media/Nutrient Mixture F12 (DMEM/F12) for transport. Samples can be kept overnight at 4 &#176;C or processed immediately. Keep on ice.</li>
	<li>Irradiate samples at 20 Gy using a cesium source (Figure 1B).</li>
	<li>45 min after irradiation, place mammary glands in a 35 mm sterile cell plate and mince with scalpels (Figure 1C,D). Mince with approximately 40 strokes until the tissue relaxes and pieces are obtained that are no larger than approximately 1 mm2 in area.</li>
	<li>Transfer to the collagenase solution in a 50 mL centrifuge tube. Collagenase solution consists of 2 mg/mL collagenase (see Table of Materials), 2 mg/mL trypsin, 5% v/v fetal bovine serum (FBS), 5 &#956;g/mL insulin, and 50 &#956;g/mL gentamicin in DMEM/F12 media. Use 10 mL collagenase solution per mouse.</li>
	<li>Place in a water bath at 37 &#176;C, vortexing every 10 min for 30-60 min. Digestion is complete when the collagenase solution is cloudy (Figure 1E,F).</li>
	<li>Spin down the digested solution at 450 x g for 10 min at room temperature (RT). Three layers will be observed. The supernatant is composed of fat, the middle layer is an aqueous solution, and the bottom is a pellet. The pellet will appear red as it is a mixture of epithelial cells, individual stromal cells, and red blood cells (Figure 1G).</li>
	<li>Precoat all pipettes, pipette tips, and centrifuge tubes with bovine serum albumin (BSA) solution prior to contact. BSA solution consists of 2.5 w/v % BSA in Dulbecco&#8217;s Phosphate Buffered Saline (DPBS). For pre-coating, simply add then remove BSA solution to the inside of the pipette tip and tubes. BSA solution can be reused, although it should be sterile filtered before each experiment.</li>
	<li>For additional recovery, transfer the supernatant to a fresh BSA coated 15 mL tube. Pipette up and down vigorously to disperse fat layer. Centrifuge at 450 x g for 10 min at RT. Aspirate the supernatant, leaving a small amount of media in the tube to avoid aspirating the cell pellet.</li>
	<li>Aspirate the aqueous layer from the tube with the original pellet.</li>
	<li>Add 10 mL of DMEM/F12 to the tube with the original pellet and transfer to the second tube. Pipette vigorously to combine and resuspend the two pellets.</li>
	<li>Centrifuge at 450 x g for 10 min at RT. Aspirate the supernatant and add 4 mL of DMEM/F12 to the tube.</li>
	<li>Add 40 &#956;L of deoxyribonuclease (DNase) to the suspension and gently shake by hand for 2-5 min at RT. DNase solution consists of 4 U/mL DNase in DMEM/F12.</li>
	<li>Add 6 mL of DMEM/F12 and pipette thoroughly. Centrifuge the tube at 450 x g for 10 min at RT.</li>
	<li>Aspirate supernatant to the 0.5 mL mark. Resuspend in 10 mL of DMEM/F12 and pipette thoroughly.</li>
	<li>Pulse to 450 x g and stop 4 s after reaching that speed.</li>
	<li>Repeat steps 1.16-1.17 three more times to purify organoids via centrifugal differentiation. The pellet should now be an off-white color consisting of only epithelial organoids (Figure 1H). 
	NOTE: Organoids can also be filtered using sterile mesh 40 &#956;m filters. After step 1.16, pipette media containing organoids through a filter into a centrifuge tube, and then rinse with 5 to 10 mL of DMEM/F12 media. Flip the filter over a new 50 mL centrifuge tube. Pass 10 mL of DMEM/F12 media through, going the opposite way to rinse off any retentate. The retentate should consist of organoids, and the filtrate should consist mainly of stromal cells, which can be discarded or kept if desired.</li>
</ol>
2. Determining density and plating organoids
<ol>
	<li>Resuspend pellet in 10 mL of DMEM/F12. Pipette thoroughly to create a homogenous solution.</li>
	<li>Transfer 50 &#181;L to a 30 mm Petri dish, and view under a phase contrast microscope at 20x. Count the number of organoids with a tally counter. 
	NOTE: Here pipette tips have been consistently used with a minimal diameter of 457 &#956;m, which is 5-10 times the diameter of the organoids that are seeded. For transferring volumes of 2 mL or larger (e.g., steps 1.16 and 2.1), use serological pipettes with tip diameters excess of 1,500 &#956;m.</li>
	<li>Calculate the organoid density using the following equation: 
	<img alt="Equation" src="/files/ftp_upload/59293/59293eq1.jpg" /> 
	The desired density is 1,000 organoids/mL to simplify further dilution. If the density is too low, centrifuge at 450 x g for 5 min and aspirate media. Add media necessary to reach 1,000 organoids/mL, and pipette thoroughly to create a homogenous mixture.
	<ol>
		<li>To grow organoids in a protein matrix, seed organoids at a concentration of 1 organoid/&#956;L in collagen type 1 diluted to 87% or in basement membrane extracted from Engelbreth-Holm-Swarm mouse sarcoma. While working with samples, keep on ice.</li>
		<li>To freeze organoids, transfer the desired volume to a separate centrifuge tube. Spin down at 450 x g for 5 min. Aspirate media, and then add the same volume of 90% FBS/10% DMSO. Resuspend the organoids, and then aliquot into cryotubes. Transfer to -80 &#730;C, and then to liquid nitrogen within one week.</li>
		<li>To thaw, warm in a 37 &#730;C water bath for one min. Centrifuge at 450 x g for 5 min, and then aspirate freezing media. Rinse with sterile DPBS, and then centrifuge again. Aspirate DPBS and add organoid media.</li>
	</ol>
	</li>
	<li>Pipette 50 &#181;L (50 organoids) into each well of the low adhesion plate (Figure 1I).</li>
	<li>Add 150 &#181;L of organoid media to bring the total working volume to 200 &#181;L. Organoid media consists of 1% penicillin-streptomycin and 1% insulin-transferrin-selenium (ITS) in DMEM/F12 media.</li>
	<li>Every 2 days replace media carefully. 
	NOTE: Low adhesion plates are not tissue culture treated; therefore, the cells can be easily detached. Aspirate media slowly by tilting the plate and inserting the pipette tip at the edge of each well. Leave a small amount of media in the bottom of the well. Add new media slowly to avoid applying unnecessary shear forces to organoids.</li>
</ol>
3. Co-culturing with macrophages
<ol>
	<li>Maintain GFP or dTomato-labelled RAW 264.7 macrophages in DMEM media supplemented with 10% FBS and 1% penicillin-streptomycin. Seed 1 x 104, 5 x 104, or 1 x 105 cells/mL into organoid media.</li>
	<li>Use live cell phase contrast and fluorescent imaging to monitor macrophage infiltration over time.</li>
</ol>
4. Immunofluorescence staining of organoids
NOTE: Organoids can be stained in low adhesion wells or can be transferred to chamber slides. To transfer, gently pipette up and down until organoids have detached from plates. Transfer to chamber slides and incubate for 4-8 h to allow organoids to adhere to the plate surface.
<ol>
	<li>Remove organoid medium from the wells by carefully aspirating. Fix samples with 10% neutral buffered formalin for 15 min at RT.</li>
	<li>Wash 3x 5 min in 1x PBS. If desired, fixed samples can be stored at 4 &#176;C for one week for further staining.</li>
	<li>Permeabilize with 0.1% 4-(1,1,3,3-Tetramethylbutyl) phenyl-polyethylene glycol for 5 min. 
	NOTE: To stain for F-actin, incubate samples with phalloidin diluted 1:1,000 and 1.67 nM bisbenzimide nuclear dye in 1% PBS/BSA for 1 h at RT. Then, proceed to step 4.8.</li>
	<li>Bermeabilize with 0.5PBS. Samples can be stored at 4opy images and immunofluorescent images. mechanisms that contribute to CTC rlock with 5% normal goat serum in 0.1% PBS/Polyethylene glycol sorbitan monolaurate (PBST) for 1 h at RT. Wash 3x 5 min with PBS.</li>
	<li>Incubate with Anti-Cytokeratin 14 diluted 1:1,000, E-Cadherin diluted 1:200, or Tight Junction Protein One diluted 1:100 in 1% NGS in PBST for 1 h at RT. Wash 3x 5 min in PBST.</li>
	<li>Incubate with Goat Anti-Rabbit secondary diluted 1:200 with 1% NGS/PBST for 1 h at RT. Cover with foil to avoid light exposure.</li>
	<li>Wash 3x 5 min in PBS. Use the nuclear dye (see Table of Materials) to stain nuclei.</li>
	<li>Wash 3x 5 min in PBS. If using chamber slide, mount with a coverslip. Store wrapped in foil at 4 &#176;C for up to 2 weeks.</li>
</ol>

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The authors have nothing to disclose.

In this protocol, we have developed a method for reproducible growth and characterization of irradiated mammary organoids (Figure 1). An irradiation dose of 20 Gy was applied to mirror previous in vivo models of tumor cell recruitment5. Irradiation of mammary glands ex vivo prior to organoid formation allowed for isolation of radiation damage effects without a corresponding infiltration of immune cells. The development of an in vitro irradiated normal tissue model ena...

Approximately 60% of the triple negative breast cancer (TNBC) patients choose breast-conserving therapy (BCT) as a form of treatment1. In this treatment modality, the tumor containing part of the breast tissue is removed, and the surrounding normal tissue is exposed to ionizing radiation to kill any residual tumor cells. Treatment reduces recurrence in much of the breast cancer population; however, approximately 13.5% of treated patients with TNBC experience locoregional recurrences2. Therefore, studying how radiation may recruit circulating tumor cells (CTCs) will lead to important insights into local recurrence3,4.
Previous work has shown that radiation of the normal tissue increases recruitment of various cell types5. In pre-clinical models of TNBC, irradiation of normal tissue increased macrophage and subsequently tumor cell recruitment to normal tissues5. Immune status influenced tumor cell recruitment to irradiated sites, with tumor cell migration observed in immunocompromised subjects. Recapitulating these interactions using organoids derived from mammary glands will allow the observation of cell migration and cell-stromal interactions in real time with microscopy and live cell imaging to determine the role of radiation damage in altering tumor cell behavior.
Mouse mammary organoids have helped elucidate key steps in the development of the mammary gland. A mammary organoid is a multicellular, three dimensional construct of isolated mammary epithelium that is larger than 50 &#956;m6,7,8,9,10. Using primary epithelial organoids, Simian et al. evaluated necessary factors for branching in the mammary gland7. Shamir et al. discovered that dissemination can occur without an epithelial to mesenchymal transition, providing insight into the metastatic cascade8. Methods for generating and characterizing organoids from mammary gland tissue are well established6,11,12,13. However, to our knowledge, methods for growing irradiated organoids from mammary glands have not been reported. A protocol for growing and characterizing irradiated organoids would be a critical step in recapitulating radiation-induced immune and tumor cell recruitment.
In this paper, we report a method for growing and characterizing irradiated mammary epithelial organoids in low adhesion microplates coated with a hydrophilic polymer that supports the formation of spheroids. These organoids were co-cultured with macrophages to examine immune cell infiltration kinetics. This work can be extended to include co-culturing organoids with adipose cells to recapitulate mammary characteristics, breast cancer cells to visualize tumor cell recruitment, and CD8+ T cells to study tumor-immune cell interactions. Previously established protocols may be used to evaluate irradiated organoids. Earlier models co-culturing mammary organoids and immune cells have shed light on mechanisms of metastasis and dissemination. DeNardo et al. found that CD4+ T cell regulation of tumor associated macrophages enhanced a metastatic phenotype of mammary adenocarcinomas14. Co-culture models have also been used to elucidate mechanisms of biological development. Plaks et al. clarified the role of CD4+ T cells as down-regulators of mammary organogenesis15. However, our group is the first to establish a procedure of visualizing how normal tissue irradiation influences immune cell behavior. Because normal tissue irradiation has been shown to enhance tumor cell recruitment5, this protocol can be further developed to analyze how tumor cell behavior is altered by irradiation of normal tissue and cells, leading to a greater understanding of cancer recurrence.

<table><tbody><tr><td>10% Neutral Buffered Formalin</td><td>VWR</td><td>16004-128</td><td></td></tr><tr><td>Anti-cytokeratin 14</td><td>abcam</td><td>ab181595</td><td>Lot: GR3200524-3</td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma</td><td>A1933-25G</td><td></td></tr><tr><td>Collagen Type I</td><td>Corning</td><td>354236</td><td></td></tr><tr><td>Collagenase from Clostridium Histolyticum, Type VIII</td><td>Sigma</td><td>C2139</td><td></td></tr><tr><td>Collagenase I</td><td>Gibco</td><td>17018029</td><td></td></tr><tr><td>DMEM/F12</td><td>Thermofisher</td><td>11320-033</td><td></td></tr><tr><td>DNAse</td><td>Roche</td><td>10104159001</td><td></td></tr><tr><td>DPBS</td><td>Fisher</td><td>14190250</td><td></td></tr><tr><td>E-Cadherin</td><td>Cell Signaling</td><td>24E10</td><td>Lot: 13</td></tr><tr><td>FBS</td><td>Sigma</td><td>F0926</td><td></td></tr><tr><td>Gentamicin</td><td>Gibco</td><td>15750</td><td></td></tr><tr><td>Goat anti-rabbit secondary</td><td>abcam</td><td>ab150077</td><td>green&lt;br/&gt; Lot: GR3203000-1</td></tr><tr><td>Goat anti-rabbit secondary</td><td>abcam</td><td>ab150080</td><td>red&lt;br/&gt; Lot: GR3192711-1</td></tr><tr><td>Hoechst 33342</td><td>Fisher</td><td>62249</td><td>Lot: TG2611041</td></tr><tr><td>Insulin (10 mg/mL)</td><td>Sigma</td><td>I9278</td><td></td></tr><tr><td>Insulin-Transferrin-Selenium, 100x</td><td>Gibco</td><td>51500-056</td><td></td></tr><tr><td>Matrigel Basement Membrane (basement membrane extracted from Engelbreth-Holm-Swarm mouse sarcoma)</td><td>Corning</td><td>356237</td><td></td></tr><tr><td>Normal Goat Serum</td><td>Vector Laboratories</td><td>S-1000</td><td></td></tr><tr><td>Nuclon Sphera 96 well plates</td><td>Thermo</td><td>174927</td><td></td></tr><tr><td>PBS</td><td>VWR</td><td>10128-856</td><td></td></tr><tr><td>Pen/strep</td><td>Fisher</td><td>15140122</td><td></td></tr><tr><td>Phalloidin</td><td>abcam</td><td>ab176757</td><td>Lot: GR3214582-16</td></tr><tr><td>Tight Junction Protein 1</td><td>Novus</td><td>NBP1-85047</td><td>Lot: C115428</td></tr><tr><td>Triton X-100 (4-(1,1,3,3-Tetramethylbutyl)phenyl-polyethylene glycol)</td><td>Sigma</td><td>X100-100ML</td><td></td></tr><tr><td>Trypsin</td><td>Gibco</td><td>27250-018</td><td></td></tr><tr><td>Tween-20 (Polyethylene glycol sorbitan monolaurate)</td><td>Sigma</td><td>P1379-100ML</td><td></td></tr></tbody></table>

growth and characterization of irradiated organoids from mammary glands

Organoids derived from the digested tissue are multicellular three-dimensional (3D) constructs that better recapitulate in vivo conditions than cell monolayers. Although they cannot completely model in vivo complexity, they retain some functionality of the original organ. In cancer models, organoids are commonly used to study tumor cell invasion. This protocol aims to develop and characterize organoids from the normal and irradiated mouse mammary gland tissue to evaluate the radiation response in normal tissues. These organoids can be applied to future in vitro cancer studies to evaluate tumor cell interactions with irradiated organoids. Mammary glands were resected, irradiated to 20 Gy and digested in a collagenase VIII solution. Epithelial organoids were separated via centrifugal differentiation, and 3D organoids were developed in 96-well low-adhesion microplates. Organoids expressed the characteristic epithelial marker cytokeratin 14. Macrophage interaction with the organoids was observed in co-culture experiments. This model may be useful for studying tumor-stromal interactions, infiltration of immune cells, and macrophage polarization within an irradiated microenvironment.

Irradiated epithelial mammary organoids were successfully obtained from mouse mammary glands, processed, and cultured on low-adhesion plates (Figure 1). Organoid yield was tested by seeding in different growth environments (Figure 2A-G). Seeding cells directly onto tissue culture treated 10 cm cell plates yielded an overgrowth of fibroblast cells. Fibroblasts were identified under phase contrast microscopy in or near the same plane of focus as organoids, and they quickly gre...

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Growth and Characterization of Irradiated Organoids from Mammary Glands

Organoids developed from mouse mammary glands were irradiated and characterized to assess epithelial traits and interactions with immune cells. Irradiated organoids can be used to better evaluate cell-cell interactions that may lead to tumor cell recruitment in irradiated normal tissue.

Organoids derived from the digested tissue are multicellular three-dimensional (3D) constructs that better recapitulate in vivo conditions than cell ...

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Research

JoVE Journal

Bioengineering

8.0K Views.  Vanderbilt University. Dit protocol kan worden gebruikt en verder ontwikkeld om te analyseren hoe ioniserende straling de rekrutering van tumorcellen beïnvloedt, wat leidt tot een beter begrip van recidief van kanker. Borstorganoïden zijn krachtige modellen. Ze bootsen fundamentele in vivo kenmerken na, maar ze zorgen voor een eenvoudige isolatie van biologische variabelen.Het gebruik van lage-hechtingsplaten ontkent werk-flow uitdagingen in verband met eiwit matrices. Drievoudig negatieve borstkankerpati?...

Video: Growth and Characterization of Irradiated Organoids from Mammary Glands

Establishment and Analysis of Three-Dimensional (3D) Organoids Derived from Patient Prostate Cancer Bone Metastasis Specimens and their Xenografts

Three-dimensional (3D) culture of organoids from tumor specimens of human patients and patient-derived xenograft (PDX) models of prostate cancer, referred to as patient-derived organoids (PDO), are an invaluable resource for studying the mechanism of tumorigenesis and metastasis of prostate cancer. Their main advantage is that they maintain the distinctive genomic and functional heterogeneity of the original tissue compared to conventional cell lines that do not. Furthermore, 3D cultures of PDO can be used to predict the effects of drug treatment on individual patients and are a step towards personalized medicine. Despite these advantages, few groups routinely use this method in part because of the extensive optimization of PDO culture conditions that may be required for different patient samples. We previously demonstrated that our prostate cancer bone metastasis PDX model, PCSD1, recapitulated the resistance of the donor patient&rsquo;s bone metastasis to anti-androgen therapy. We used PCSD1 3D organoids to characterize further the mechanisms of anti-androgen resistance. Following an overview of currently published studies of PDX and PDO models, we describe a step-by-step protocol for 3D culture of PDO using domed or floating basement membrane (e.g., Matrigel) spheres in optimized culture conditions. In vivo stitch imaging and cell processing for histology are also described. This protocol can be further optimized for other applications including western blot, co-culture, etc. and can be used to explore characteristics of 3D cultured PDO pertaining to drug resistance, tumorigenesis, metastasis and therapeutics.

Establishment and Analysis of Three-Dimensional 3D Organoids Derived from Patient Prostate Cancer Bone Metastasis Specimens and their Xenografts

Three-dimensional cultures of patient BMPC specimens and xenografts of bone metastatic prostate cancer maintain the functional heterogeneity of their original tumors resulting in cysts, spheroids and complex, tumor-like organoids. This manuscript provides an optimization strategy and protocol for 3D culture of heterogeneous patient derived samples and their analysis using IFC.

Three-dimensional (3D) culture of organoids from tumor specimens of human patients and patient-derived xenograft (PDX) models of prostate cancer, referred ...

Cancer Research

Rat Mammary Epithelial Cell Transplantation into the Interscapular White Fat Pad

As early as the 1970s, researchers have successfully transplanted mammary epithelial cells into the interscapular white fat pad of rats. Grafting mammary epithelium using transplantation techniques takes advantage of the hormonal environment provided by the adolescent rodent host. These studies are ideally suited to explore the impact of various biological manipulations on mammary gland development and dissect many aspects of mammary gland biology. A common, but limiting, feature is that transplanted epithelial cells are strongly influenced by the surrounding stroma and outcompeted by endogenous epithelium; to utilize native mammary tissue, the abdominal-inguinal white fat pad must be cleared to remove host mammary epithelium prior to the transplantation. A major obstacle when using the rat model organism is that clearing the developing mammary tree in post-weaned rats is not efficient. When transplanted into gland-free fat pads, donor epithelial cells can repopulate the cleared host fat pad and form a functional mammary gland. The interscapular fat pad is an alternative location for these grafts. A major advantage is that it lacks ductal structures yet provides the normal stroma that is necessary to promote epithelial outgrowth and is easily accessible in the rat. Another major advantage of this technique is that it is minimally invasive, because it eliminates the need to cauterize and remove the growing endogenous mammary tree. Additionally, the interscapular fat pad contains a medial blood vessel that can be used to separate sites for grafting. Because the endogenous glands remain intact, this technique can also be used for studies comparing the endogenous mammary gland to the transplanted gland. This paper describes the method of mammary epithelial cell transplantation into the interscapular white fat pad of rats.

This article describes a transplantation method to graft donor rat mammary epithelial cells into the interscapular white fat pad of recipient animals. This method can be used to examine host and/or donor effects on mammary epithelium development and eliminates the need for pre-clearing, thereby extending the usefulness of this technique.

As early as the 1970s, researchers have successfully transplanted mammary epithelial cells into the interscapular white fat pad of rats. Grafting mammary ...

Three-Dimensional Imaging of Organoids to Study Primary Ciliogenesis During ex vivo Organogenesis

Organoids are stem cell-derived three-dimensional structures that reproduce ex vivo the complex architecture and physiology of organs. Thus, organoids represent useful models to study the mechanisms that control stem cell self-renewal and differentiation in mammals, including primary ciliogenesis and ciliary signaling. Primary ciliogenesis is the dynamic process of assembling the primary cilium, a key cell signaling center that controls stem cell self-renewal and/or differentiation in various tissues. Here we present a comprehensive protocol for the immunofluorescence staining of cell lineage and primary cilia markers, in whole-mount mouse mammary organoids, for light sheet microscopy. We describe the microscopy imaging method and an image processing technique for the quantitative analysis of primary cilium assembly and length in organoids. This protocol enables a precise analysis of primary cilia in complex three-dimensional structures at the single cell level. This method is applicable for immunofluorescence staining and imaging of primary cilia and ciliary signaling in mammary organoids derived from normal and genetically modified stem cells, from healthy and pathological tissues, to study the biology of the primary cilium in health and disease.

Three-Dimensional Imaging of Organoids to Study Primary Ciliogenesis During ex vivo Organogenesis

Stem cell-derived organoids facilitate the analysis of molecular and cellular processes that regulate stem cell self-renewal and differentiation during organogenesis in mammalian tissues. Here we present a protocol for the analysis of the biology of the primary cilium in mouse mammary organoids.

Organoids are stem cell-derived three-dimensional structures that reproduce ex vivo the complex architecture and physiology of organs. Thus, organoids ...

Genetics

Generating and Imaging Mouse and Human Epithelial Organoids from Normal and Tumor Mammary Tissue Without Passaging

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.

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 ...

Establishment and Culture of Patient-Derived Breast Organoids

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.

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.

Breast cancer is a complex disease that has been classified into several different histological and molecular subtypes. Patient-derived breast tumor ...

Generation of Multicellular Human Primary Endometrial Organoids

The human endometrium is one of the most hormonally responsive tissues in the body and is essential for the establishment of pregnancy. This tissue can also become diseased and cause morbidity and even death. Model systems to study human endometrial biology have been limited to in vitro culture systems of single cell types. In addition, the epithelial cells, one of the major cell types of the endometrium, do not propagate well or retain their physiological traits in culture, and thus our understanding of endometrial biology remains limited. We have generated, for the first time, endometrial organoids that consist of both epithelial and stromal cells of the human endometrium. These organoids do not require any exogenous scaffold materials and specifically organize so that epithelial cells encompass the spheroid-like structure and become polarized with stromal cells in the center that produce and secrete collagen. Estrogen, progesterone and androgen receptors are expressed in the epithelial and stromal cells and treatment with physiological levels of estrogen and testosterone promote the organization of the organoids. This new model system can be used to study normal endometrial biology and disease in ways that were not possible before.

A protocol to generate human primary endometrial organoids that consist of epithelial and stromal cells and retain characteristics of the native endometrial tissue is presented. This protocol describes methods from uterine tissue acquisition to the histologic processing of endometrial organoids.

The human endometrium is one of the most hormonally responsive tissues in the body and is essential for the establishment of pregnancy. This tissue can ...

Developmental Biology

Generation of Mosaic Mammary Organoids by Differential Trypsinization

Organoids offer self-organizing, three-dimensional tissue structures that recapitulate physiological processes in the convenience of a dish. The murine mammary gland is composed of two distinct epithelial cell compartments, serving different functions: the outer, contractile myoepithelial compartment and the inner, secretory luminal compartment. Here, we describe a method by which the cells comprising these compartments are isolated and then combined to investigate their individual lineage contributions to mammary gland morphogenesis and differentiation. The method is simple and efficient and does not require sophisticated separation technologies such as fluorescence activated cell sorting. Instead, we harvest and enzymatically digest the tissue, seed the epithelium on adherent tissue culture dishes, and then use differential trypsinization to separate myoepithelial from luminal cells with ~90% purity. The cells are then plated in an extracellular matrix where they organize into bilayered, three-dimensional (3D) organoids that can be differentiated to produce milk after 10 days in culture. To test the effects of genetic mutations, cells can be harvested from wild type or genetically engineered mouse models, or they can be genetically manipulated prior to 3D culture. This technique can be used to generate mosaic organoids that allow investigation of gene function specifically in the luminal or myoepithelial compartment.

The mammary gland is a bilayered structure, comprising outer myoepithelial and inner luminal epithelial cells. Presented is a protocol to prepare organoids using differential trypsinization. This efficient method allows researchers to separately manipulate these two cell types to explore questions concerning their roles in mammary gland form and function.

Organoids offer self-organizing, three-dimensional tissue structures that recapitulate physiological processes in the convenience of a dish. The murine ...

The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse Intestinal Organoids

Primary intestinal organoids are a valuable model system that has the potential to significantly impact the field of mucosal immunology. However, the complexities of the organoid growth characteristics carry significant caveats for the investigator. Specifically, the growth patterns of each individual organoid are highly variable and create a heterogeneous population of epithelial cells in culture. With such caveats, common tissue culture practices cannot be simply applied to the organoid system due to the complexity of the cellular structure. Counting and plating based solely on cell number, which is common for individually separated cells, such as cell lines, is not a reliable method for organoids unless some normalization technique is applied. Normalizing to total protein content is made complex due to the resident protein matrix. These characteristics in terms of cell number, shape and cell type should be taken into consideration when evaluating secreted contents from the organoid mass. This protocol has been generated to outline a simple procedure to culture and treat small intestinal organoids with microbial pathogens and pathogen associated molecular patterns (PAMPs). It also emphasizes the normalization techniques that should be applied when protein analysis are conducted after such a challenge.

The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse Intestinal Organoids

Here, a protocol to harvest, maintain, and treat mouse small intestinal organoids with pathogen associated molecular patterns (PAMPs) and Listeria monocytogenes is described, as well as emphasis on gene expression and proper normalization techniques for protein.

Primary intestinal organoids are a valuable model system that has the potential to significantly impact the field of mucosal immunology. However, the ...

Immunology and Infection

Non-enzymatic, Serum-free Tissue Culture of Pre-invasive Breast Lesions for Spontaneous Generation of Mammospheres

Breast ductal carcinoma in situ (DCIS), by definition, is proliferation of neoplastic epithelial cells within the confines of the breast duct, without breaching the collagenous basement membrane. While DCIS is a non-obligate precursor to invasive breast cancers, the molecular mechanisms and cell populations that permit progression to invasive cancer are not fully known. To determine if progenitor cells capable of invasion existed within the DCIS cell population, we developed a methodology for collecting and culturing sterile human breast tissue at the time of surgery, without enzymatic disruption of tissue.
Sterile breast tissue containing ductal segments is harvested from surgically excised breast tissue following routine pathological examination. Tissue containing DCIS is placed in nutrient rich, antibiotic-containing, serum free medium, and transported to the tissue culture laboratory. The breast tissue is further dissected to isolate the calcified areas. Multiple breast tissue pieces (organoids) are placed in a minimal volume of serum free medium in a flask with a removable lid and cultured in a humidified CO2 incubator. Epithelial and fibroblast cell populations emerge from the organoid after 10 - 14 days. Mammospheres spontaneously form on and around the epithelial cell monolayer. Specific cell populations can be harvested directly from the flask without disrupting neighboring cells. Our non-enzymatic tissue culture system reliably reveals cytogenetically abnormal, invasive progenitor cells from fresh human DCIS lesions.

Primary cell culture using intact tissue organoids provides a model system that mimics the multi-cellular in vivo microenvironment. We developed a serum-free primary breast epithelium tissue culture model that perpetuates mixed cell culture lineages and exhibits differentiated morphology, without enzymatic tissue disruption. Breast organoids remain viable for &#62;6 months.

Breast ductal carcinoma in situ (DCIS), by definition, is proliferation of neoplastic epithelial cells within the confines of the breast duct, without ...

Biology

Generation and Characterization of Murine Oral Mucosal Organoid Cultures

The mucous lining covering the inside of our mouth, the oral mucosa, is a highly compartmentalized tissue and can be subdivided into the buccal mucosa, gingiva, lips, palate, and tongue. Its uppermost layer, the oral epithelium, is maintained by adult stem cells throughout life. Proliferation and differentiation of adult epithelial stem cells have been intensively studied using in vivo mouse models as well as two-dimensional (2D) feeder-cell based in vitro models. Complementary to these methods is organoid technology, where adult stem cells are embedded into an extracellular matrix (ECM)-rich hydrogel and provided with a culture medium containing a defined cocktail of growth factors. Under these conditions, adult stem cells proliferate and spontaneously form three-dimensional (3D) cell clusters, the so-called organoids. Organoid cultures were initially established from murine small intestinal epithelial stem cells. However, the method has since been adapted for other epithelial stem cell types. Here, we describe a protocol for the generation and characterization of murine oral mucosal organoid cultures. Primary epithelial cells are isolated from murine tongue tissue, embedded into an ECM hydrogel, and cultured in a medium containing: epidermal growth factor (EGF), R-spondin, and fibroblast growth factor (FGF) 10. Within 7 to 14 days of initial seeding, the resulting organoids can be passaged for further expansion and cryopreservation. We additionally present strategies for the characterization of established organoid cultures via 3D whole-mount imaging and gene-expression analysis. This protocol may serve as a tool to investigate oral epithelial stem cell behavior ex vivo in a reductionist manner.

We present a method for the generation and characterization of oral mucosal organoid cultures derived from the tongue epithelium of adult mice.

The mucous lining covering the inside of our mouth, the oral mucosa, is a highly compartmentalized tissue and can be subdivided into the buccal mucosa, ...

Growth and Characterization of Irradiated Organoids from Mammary Glands

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