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5 minutes

We gratefully acknowledge Karla Murga, Daniela Requena, and Megan Maloney at the Tufts Biomedical Repository for tissue support. This research was supported by the Find The Cause Breast Cancer Foundation and the Tufts CTSI NIH Clinical and Translational Science Award (UM1TR0043, G.R.).

Primary tissues that would otherwise have been discarded as medical waste following surgery were obtained in compliance with all relevant laws using protocols approved by the institutional review boards at Maine Medical Center and Tufts Medical Center. All tissues were anonymized prior to transfer and could not be traced to specific patients. For this reason, this research was granted exemption status by the Committee on the Use of Humans as Experimental Subjects at the Massachusetts Institute of Technology and at Tufts University Health Sciences (IRB #13521). All patients enrolled in this study signed an informed consent form agreeing to participate in the study and to the publication of the results.
1. Tissue Processing and Preparation
NOTE: Perform all procedures involving human tissue in accordance with institutional biosafety and ethical guidelines. Refer to the workflow schematics shown in Figure 1A and 1B for a visual overview of the protocol steps and organoid preparation workflow prior to beginning the procedure.
<ol>
	<li>Prepare MEGM using mammary epithelial basal medium supplemented with 52 &#181;g/mL bovine pituitary extract, 10 ng/mL human epidermal growth factor, 5 &#181;g/mL insulin, 500 ng/mL hydrocortisone, 1% (v/v) GlutaMAX, and 1% (v/v)&#160;Antibiotic-Antimycotic (100X).</li>
	<li>Prepare the dissociation medium by adding 1.5 mg/mL collagenase A (stored at &#8722;20&#176;C) and 100 U/mL hyaluronidase (stored at 4&#176;C)&#160;to mammary epithelial growth medium (MEGM).</li>
	<li>Receive human breast tissue obtained from prophylactic mastectomies and reduction mammoplasties within 24 h post-excision, unfixed in phosphate-buffered saline (PBS), and maintained at 4&#176;C. Weigh the tissue on a precision balance to determine the total tissue weight.</li>
	<li>Place the tissue in a sterile biosafety cabinet. Mechanically mince the tissue into 3&#8211;5 mm3 fragments using sterile scalpels. Transfer approximately 2&#8211;3 g of minced tissue into a 15 mL conical tube. Add 10 mL of dissociation medium to each tube.&#160;</li>
	<li>Incubate the tubes at 37&#176;C on an orbital rotator at 8 rpm for 12&#8211;18 h to enzymatically digest the tissue. Consider digestion complete when no large tissue fragments remain and a homogeneous suspension of evenly fragmented material is visible throughout the medium.</li>
	<li>Allow epithelial fragments to settle by gravity for 5 min. Confirm that no visible fragments remain suspended in the medium and that a distinct pellet is present. Carefully decant and discard the supernatant without disturbing the pellet. 
	NOTE:&#160;The supernatant contains stromal cells and matrix components, and may be washed and used or preserved for other studies.&#160;</li>
	<li>Resuspend the pellet in 10 mL wash medium consisting of 5% fetal bovine serum (FBS) in PBS. Centrifuge at 250 &#215; g for 5 min in a swing-bucket centrifuge at room temperature.</li>
	<li>Repeat the wash step three additional times or until the supernatant is clear and&#160;free of visible debris.</li>
	<li>Resuspend the final pellet in freezing medium consisting of 10% dimethyl sulfoxide (DMSO) in MEGM at a volume of 1 mL per gram of initial tissue weight. 
	CAUTION: DMSO is harmful upon contact or inhalation. Handle using appropriate personal protective equipment and work in a chemical safety hood if required by institutional guidelines.</li>
	<li>Aliquot 1 mL of the suspension into each cryovial. Place the cryovials at &#8722;80&#176;C in a freezing container prior to long-term storage in liquid nitrogen. 
	NOTE: This step represents a pause point. Store samples at &#8722;80&#176;C before transferring to long-term storage conditions.</li>
</ol>
2. Recovery and Optional Cell Preparation
<ol>
	<li>Thawing and Initial Recovery
	<ol>
		<li>Remove cryopreserved tissue from &#8722;80&#176;C storage after freezing for at least 24 h, or from the liquid nitrogen long term storage. Immediately submerge the cryovial up to the cap in a 37&#176;C water bath and thaw rapidly for 1 min to minimize cell death. 
		NOTE: Gently agitate the cryovial during thawing to ensure uniform warming.</li>
		<li>Once fully thawed, immediately transfer the contents of the cryovial to a 15 mL conical tube containing 10 mL of MEGM. Centrifuge at 250 &#215; g for 5 min.</li>
	</ol>
	</li>
	<li>Optional Fibroblast Depletion 
	NOTE: Fibroblast depletion by pre-plating is an optional enrichment step used to reduce rapidly adherent stromal or fibroblast populations when a more epithelial-enriched starting population is desired. This step is not required for organoid formation and may be adjusted depending on the experimental objective.
	<ol>
		<li>Resuspend the pellet in 10 mL of MEGM.</li>
		<li>Transfer the resuspended tissue to a 10 cm cell culture dish. Incubate at 37&#176;C in a humidified incubator with 5% CO&#8322; for 90 min to allow fibroblast attachment. 
		NOTE: Do not disturb the culture dish during incubation to ensure efficient fibroblast adherence.</li>
		<li>Collect the supernatant containing non-adherent cells using a 10 mL serological pipette while gently tilting the culture dish to approximately 45&#176;. Gently rinse the plate with 5 mL PBS and combine the rinse with the collected supernatant.</li>
		<li>Centrifuge the combined suspension at 250 &#215; g for 5 min to recover epithelial-enriched material. 
		NOTE:&#160; Cells that adhered to the plate are enriched for mammary gland fibroblasts, and may be propagated separately by adding medium composed of DMEM + 10% FBS and antibiotics.&#160;</li>
	</ol>
	</li>
	<li>Optional Dissociation to Single Cells
	<ol>
		<li>Resuspend the pellet in 500 &#181;L of prewarmed (37&#176;C) dissociation enzyme solution consisting of 0.25% trypsin. Alternative dissociation reagents may require optimization. Transfer the suspension to a 1.5 mL microcentrifuge tube. Triturate the sample 20 times using a P1000 pipette to promote dissociation. 
		CAUTION: Enzymatic dissociation reagents may be harmful. Handle using appropriate personal protective equipment and avoid skin or eye contact.</li>
		<li>Incubate the tube at 37&#176;C for 3-5 min. Mechanically dissociate the sample immediately following incubation by triturating 20 times using a P1000 pipette.</li>
		<li>Add 1 mL of serum-containing wash medium to neutralize the enzymatic reaction. Centrifuge at 500 &#215; g for 5 min.</li>
		<li>Resuspend the pellet in 300 &#181;L dispase solution (5 U/mL) and 30&#160;&#181;L DNase solution (1 mg/mL). Incubate at 37&#176;C for 3&#8211;5 min.</li>
		<li>Mechanically dissociate the sample by pipetting 15&#8211;20 times. Add 700 &#181;L serum-containing wash medium to the suspension.</li>
		<li>Filter the cell suspension through a 40 &#181;m cell strainer into a clean tube. Either a standard 40 &#181;m cell strainer or a Flowmi pipette-tip strainer may be used. Flowmi strainers may reduce sample loss when working with limited cell numbers.</li>
		<li>Determine viable cell number using trypan blue exclusion. Mix 10 &#181;L of the cell suspension with trypan blue at a 1:1 ratio and load 10 &#181;L of the mixture into a cell counting chamber or counting slide. Determine cell viability using an automated cell counter or hemocytometer.</li>
		<li>Centrifuge the suspension at 500 &#215; g for 5 min at room temperature.</li>
		<li>Resuspend the cell pellet in MEGM at the desired concentration for hydrogel seeding. 
		NOTE: For single-cell seeding experiments, typical inputs range from approximately 1,000&#8211;5,000 cells per 200 &#181;L hydrogel, depending on the experimental objective and donor sample characteristics. Lower seeding densities are generally used for live imaging and morphogenesis studies to facilitate tracking of individual organoid development, whereas higher densities are commonly used for endpoint molecular analyses, including RNA and protein collection.</li>
	</ol>
	</li>
</ol>
3. Hydrogel Preparation and Organoid Seeding
<ol>
	<li>Stock Preparation and Volume Calculations
	<ol>
		<li>Place laminin solution (1.18 mg/mL), hyaluronic acid solution (1 mg/mL in sterile water), fibronectin solution (2 mg/mL in sterile water), and PBS 1&#215; on ice prior to use. Store laminin and fibronectin aliquots at &#8722;80&#176;C and store hyaluronic acid solution at 4&#176;C. Thaw frozen components slowly on ice at 0&#176;C&#8211;4&#176;C prior to use.</li>
		<li>Prepare a 25&#215; extracellular matrix supplement stock solution. To prepare 1 mL, combine 425 &#181;L laminin, 250 &#181;L hyaluronic acid, 250 &#181;L fibronectin, and 75 &#181;L PBS in a tube maintained on ice. Mix gently by inverting the tube 3&#8211;4 times. Store the solution at 4&#176;C for up to 1 month.&#160;</li>
		<li>Determine the desired final hydrogel volume and composition prior to preparation. Prepare at least 20% excess volume to account for material loss during pipetting and transfer. 
		NOTE: The hydrogel formulation consists of collagen I, a 1&#215; final concentration of extracellular matrix supplements (from a 25&#215; stock), and 12.5% (v/v) 0.1 N sodium hydroxide relative to the collagen I volume for pH neutralization and polymerization. 
		NOTE: The final hydrogel concentrations are 1.7 mg/mL collagen I, 20 &#181;g/mL laminin, 20 &#181;g/mL fibronectin, and 10 &#181;g/mL hyaluronic acid.</li>
		<li>Calculate the required volume of collagen I (Vcollagen) using the following formula: 
		<img alt="Equation 1" src="/files/ftp_upload/71831/71831eq1.jpg" style="margin:auto;" /> 
		Here, Cfinal&#160;= 1.7 mg/mL, Vtotal&#160;is the desired final hydrogel volume, and&#160;Cstock is the concentration of the collagen stock solution. Use collagen stock concentrations between 2&#8211;12 mg/mL. 
		NOTE: Collagen stock concentration may vary between batches. Higher concentrations increase viscosity and should be pipetted slowly.</li>
		<li>Calculate the volume of 0.1 N sodium hydroxide (VNaOH) using the following formula: 
		VNaOH = 0.125 x Vcollagen 
		Prepare a 0.1 N sodium hydroxide working solution by diluting 1 N sodium hydroxide in sterile water.</li>
		<li>Calculate the volume of extracellular matrix supplement (VES) using the following formula: 
		<img alt="Equation 2" src="/files/ftp_upload/71831/71831eq2.jpg" style="margin:auto;" /></li>
		<li>Calculate the remaining volume to be filled with growth medium using the following formula: 
		Vgrowth medium = Vtotal -&#160;(Vcollagen +&#160;VNaOH +&#160;VES +&#160;Vcells/tissue) 
		NOTE: Determine the volume of cells or tissue fragments for each experiment individually. Use a typical range of 10&#8211;100 &#181;L within the allowable volume. Precise fragment counting is not performed because fragment size and composition vary substantially between preparations. Standardize seeding by visual assessment of fragment abundance and distribution.</li>
	</ol>
	</li>
	<li>Preparation of Hydrogel Master Mix
	<ol>
		<li>Place collagen I, extracellular matrix supplement, sodium hydroxide (0.1 N), and growth medium on ice at 0&#176;C&#8211;4&#176;C. Maintain all components at low temperature to prevent premature polymerization.</li>
		<li>Add the calculated volume of collagen I to a chilled microcentrifuge tube.</li>
		<li>Add the calculated volume of pre-chilled growth medium to the collagen solution.</li>
		<li>Add the calculated volume of 0.1 N sodium hydroxide to neutralize the solution. 
		NOTE: Previous optimization established that these conditions produce the appropriate gel pH; therefore, routine pH testing of the hydrogel mixture is not required. 
		NOTE: Perform all subsequent steps rapidly to prevent premature collagen polymerization.</li>
		<li>Mix the solution by shaking the tube vigorously at a rate of approximately one shake per second for at least 6&#160;shakes.</li>
		<li>Add the calculated volume of extracellular matrix supplement to the mixture.</li>
		<li>Add the calculated volume of single cells or tissue fragments using a P1000 pipette or wide-bore pipette tip. Mix immediately by shaking as described in Step 3.2.5 and proceed immediately to the next step.</li>
	</ol>
	</li>
	<li>Hydrogel Deposition
	<ol>
		<li>Pipette the hydrogel mixture into culture vessels at the desired volume per well. Use 200 &#181;L hydrogel per well for 4-well chamber slides, 100 &#181;L per well for 8-well chamber slides, and 20 &#181;L per well for 96-well plates.</li>
		<li>Spread the hydrogel into a thin pad across the surface when using chamber slides. Position the pipette tip at the center upper edge of the chamber well and drag the tip across the surface while dispensing the gel to create an even pad. For 96-well plates, position the pipette tip at the center of the well while maintaining contact with the surface and dispense the gel centrally to maintain a dome structure.&#160;Avoid pipetting air into the gel as this will create bubbles.</li>
		<li>Incubate the culture vessels at 37&#176;C in a humidified incubator with 5% CO2&#160;for 60 min to allow complete polymerization. 
		NOTE: Polymerization conditions described here were optimized for the culture formats used in this study. Minor adjustments in polymerization time may be required depending on culture vessel geometry, hydrogel volume, and incubation conditions.</li>
	</ol>
	</li>
	<li>Culture and Maintenance
	<ol>
		<li>Add prewarmed MEGM to each well. Adjust the volume according to the culture format used.</li>
		<li>Gently detach the hydrogel from the culture surface using a pipette tip to allow the gel to float. Slide the pipette tip around the perimeter of the gel and gently lift beneath the polymerized hydrogel to release it from the surface.</li>
		<li>Replace the MEGM twice weekly with fresh prewarmed medium. Maintain cultures in a humidified incubator with 5% CO&#8322; for approximately 3&#8211;4 weeks and terminate cultures before complete hydrogel collapse occurs. Identify collapsed hydrogels by the appearance of dense, opaque, plug-like structures resulting from extensive cellular outgrowth within the gel (see representative examples in Supplementary Figure 1).</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Rauner G, Gupta PB, Kuperwasser C. From 2D to 3D and beyond: the evolution and impact of in vitro tumor models in cancer research. Nat Methods. 2025;22:1776-87.</li>
	<li>Traugh N, et al. Immune-epithelial interactions via TGF-&#946; orchestrates stem-cell niche formation and morphogenesis [Preprint]. Available from: https://doi.org/10.1101/2025.05.22.655596. 2025.</li>
	<li>Rauner G, et al. Single-cell organogenesis captures complex breast tissue formation in 3D. Development. 2025. Available from: https://doi.org/10.1242/dev.204813.</li>
	<li>Trepicchio C, et al. DDR1 regulates RUNX1-CBF&#946; to control breast stem cell differentiation [Preprint]. bioRxiv. 2024. Available from: https://doi.org/10.1101/2024.02.21.581255.</li>
	<li>Rauner G, et al. Breast tissue regeneration is driven by cell-matrix interactions coordinating multi-lineage stem cell differentiation through DDR1. Nat Commun. 2021;12:7116.</li>
	<li>Schedin P, Keely PJ. Mammary gland ECM remodeling, stiffness, and mechanosignaling in normal development and tumor progression. Cold Spring Harb Perspect Biol. 2011;3:a003228.</li>
	<li>Fata JE, Werb Z, Bissell MJ. Regulation of mammary gland branching morphogenesis by the extracellular matrix and its remodeling enzymes. Breast Cancer Res. 2004;6:1-11.</li>
	<li>Sokol ES, et al. Growth of human breast tissues from patient cells in 3D hydrogel scaffolds. Breast Cancer Res. 2016;18:19.</li>
	<li>Parrish M, et al. Environmental chemical mixtures reprogram mammary epithelial development to epigenetic states associated with breast cancer [Preprint]. Available from: https://doi.org/10.64898/2026.04.01.715968. 2026.</li>
	<li>Stingl J, Emerman JT, Eaves CJ. Enzymatic dissociation and culture of normal human mammary tissue to detect progenitor activity. Methods Mol Biol. 2005;290:249-63.</li>
	<li>Antoine EE, Vlachos PP, Rylander MN. Review of collagen I hydrogels for bioengineered tissue microenvironments: characterization of mechanics, structure, and transport. Tissue Eng Part B Rev. 2014;20:683-96.</li>
	<li>Ngo P, Ramalingam P, Phillips JA, Furuta GT. Collagen gel contraction assay. Methods Mol Biol. 2006;341:103-9.</li>
	<li>Lancaster MA, Knoblich JA. Organogenesis in a dish: modeling development and disease using organoid technologies. Science. 2014;345:1247125.</li>
	<li>Linnemann JR, Meixner LK, Miura H, Scheel CH. An organotypic 3D assay for primary human mammary epithelial cells that recapitulates branching morphogenesis. Methods Mol Biol. 2017;1612:125-37.</li>
	<li>Bonnans C, Chou J, Werb Z. Remodelling the extracellular matrix in development and disease. Nat Rev Mol Cell Biol. 2014;15:786-801.</li>
	<li>Hynes RO. The extracellular matrix: not just pretty fibrils. Science. 2009;326:1216-9.</li>
	<li>Hughes CS, Postovit LM, Lajoie GA. Matrigel: a complex protein mixture required for optimal growth of cell culture. Proteomics. 2010;10:1886-90.</li>
	<li>Sokol ES, et al. Perturbation-expression analysis identifies RUNX1 as a regulator of human mammary stem cell differentiation. PLoS Comput Biol. 2015;11:e1004161.</li>
</ol>

C.K. is co-founder and consultant of Naveris.

The protocol described here enables the reproducible generation of three-dimensional human breast organoids within a defined hydrogel microenvironment that supports key features of mammary morphogenesis3. Several steps are critical for the success of this method. First, tissue processing and enzymatic dissociation must be carefully controlled to preserve epithelial viability while minimizing overdigestion, which can reduce cell yield and impair subsequent morphogenesis10. I...

Understanding human mammary gland development and the early events that predispose tissue to malignant transformation requires experimental systems that faithfully recapitulate tissue architecture, cellular hierarchy, and microenvironmental signaling. While two-dimensional epithelial cultures have provided important mechanistic insights, they lack the structural context necessary to model epithelial organization and morphogenesis1. Existing three-dimensional culture systems, including those based on basement membrane extracts, have advanced the field but remain limited by variable composition, incomplete control over extracellular matrix components, and inconsistent support of higher-order tissue architecture1. In addition, many organoid systems based on basement membrane extracts are constrained by their inability to consistently support the emergence of tissue-like organization1. In particular, many systems rely on exogenous stromal components rather than enabling the endogenous development of supportive microenvironments, thereby limiting their ability to model epithelial&#8211;mesenchymal interactions that are central to tissue development and disease. These limitations restrict the reproducible study of developmental processes, epithelial plasticity, and the effects of environmental or molecular perturbations on tissue organization.
To address these limitations, we developed a defined hydrogel-based three-dimensional human breast organoid model that enables primary epithelial cells to generate organized structures within a defined extracellular matrix2,3,4,5,8. The hydrogel consists of type I collagen, laminin, fibronectin, and hyaluronic acid, components selected based on their established roles in mammary gland development and epithelial morphogenesis. These extracellular matrix molecules engage distinct cellular receptors, including integrins, discoidin domain receptors, CD44, and RHAMM, and are known to regulate epithelial polarity, branching morphogenesis, stem cell maintenance, mechanotransduction, and tissue organization in the mammary gland6,7. Previous studies describing this hydrogel system demonstrated that incorporation of these extracellular matrix components markedly improved ductal-lobular morphogenesis and epithelial maturation compared to collagen-only or Matrigel-based conditions3,8. In addition, the physical properties of this hydrogel formulation were previously characterized by atomic force microscopy, demonstrating that the composite extracellular matrix hydrogel exhibited lower stiffness and increased swelling relative to collagen-only gels (Young&#8217;s modulus: 256.7 &#177; 20.0 Pa versus 559.2 &#177; 204.0 Pa, respectively), consistent with a softer and more hydrated matrix environment8.
Importantly, this model supports the emergence of a mesenchyme-like compartment that arises alongside epithelial structures, providing endogenous structural and signaling support that more closely reflects native tissue organization3. The physiological relevance of this hydrogel system has been evaluated through direct comparisons with conventional Matrigel-based organoid cultures using both morphological and transcriptomic analyses. Earlier studies demonstrated that hydrogel cultures supported more organized ductal-lobular tissue formation and multilineage differentiation than Matrigel-based cultures while also preserving hormone responsiveness8. More recently, integrated single-cell RNA sequencing analyses comparing hydrogel-derived organoids, Matrigel-grown organoids, and primary human breast tissue demonstrated that hydrogel-grown organoids more faithfully recapitulate the epithelial hierarchy, cellular diversity, and epithelial&#8211;mesenchymal interactions present in native human breast tissue3. In contrast, Matrigel-grown organoids were enriched for proliferative hybrid basal states and lacked stromal-like populations, consistent with epithelial self-assembly rather than directed organogenesis.
The protocol described here provides a reproducible and scalable method for generating organoids from primary human tissue, with optional steps for fibroblast depletion and dissociation to single cells. Because this platform is compatible with live imaging, high-content imaging, quantitative morphometric analysis, cell tracking, and genetic perturbation studies, it can be integrated with downstream approaches assessing organoid number, size, architecture, and growth dynamics. This platform therefore provides a biologically relevant system for studying human breast development, epithelial plasticity, epithelial&#8211;microenvironment interactions, and tissue-level responses to developmental, molecular, or environmental perturbations.
A schematic overview of the workflow, including tissue processing, hydrogel preparation, organoid culture, developmental progression, and downstream analyses, is provided in Figure 1, while representative organoid morphologies generated using this platform are shown in Figure 2.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/71831/71831fig1.jpg" /> 
Figure 1. Overview of the hydrogel-based organoid generation workflow. (A) Flowchart summarizing the protocol workflow, including tissue collection, tissue processing, cryopreservation, recovery and optional cell preparation, hydrogel preparation, organoid culture, organoid development, and downstream analytical applications. (B) Visual schematic depicting the major stages of the hydrogel-based organoid generation protocol, including tissue processing and preparation, recovery and optional cell preparation, and hydrogel preparation and organoid seeding. <a href="https://www.jove.com/files/ftp_upload/71831/71831fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/71831/71831fig2.jpg" /> 
Figure 2. Representative organoid morphologies generated in the defined hydrogel system. Representative brightfield images of organoids derived from different human tissues cultured in the defined hydrogel matrix. Breast organoids generated from reduction mammoplasty-derived single epithelial cells were imaged at day 21 of culture. Patient-derived xenograft organoids generated from tumor fragments were imaged at day 16 of culture. Salivary gland organoids generated from epithelial fragments were imaged at day 3 of culture. Kidney organoids generated from epithelial fragments were imaged at day 17 of culture. Scale bars = 50 &#181;m. <a href="https://www.jove.com/files/ftp_upload/71831/71831fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table><tbody><tr><td>15 mL conical tubes</td><td>VWR</td><td>89039-664</td><td>Sterile tubes for tissue processing and centrifugation</td></tr><tr><td>40 &amp;mu;m cell strainer</td><td>VWR</td><td>732-2757</td><td>Filtration device for single-cell suspension preparation</td></tr><tr><td>40 &amp;mu;m cell strainer for low volumes</td><td>Bel-Art</td><td>136800040</td><td>Filtration device for single-cell suspension preparation</td></tr><tr><td>Automated cell counter</td><td>Bio-Rad</td><td>1450102</td><td>Device used to count cells and determine viability</td></tr><tr><td>Bovine pituitary extract</td><td>Thermo Scientific</td><td>13028014</td><td>Supplement for epithelial cell media</td></tr><tr><td>Cell counting slides</td><td>Bio-Rad</td><td>145-0011</td><td>Dual-chamber slides used for cell counting</td></tr><tr><td>Centrifuge</td><td>N/A</td><td>N/A</td><td>Benchtop centrifuge/s need to have at least 500 x g speed capability and accommodation of 15 mL and 1.5 mL tubes.&amp;nbsp;</td></tr><tr><td>Collagen I</td><td>Millipore Sigma</td><td>08-115</td><td>Extracellular matrix protein used for hydrogel formation</td></tr><tr><td>Collagenase A</td><td>Sigma-Aldrich</td><td>11088793001</td><td>Enzyme used for tissue dissociation</td></tr><tr><td>Cryovials</td><td>Corning</td><td>976171</td><td>Sterile vials for cryogenic storage of samples</td></tr><tr><td>Culture vessels (e.g., chamber slides, multiwell plates)</td><td>Corning</td><td>354104&lt;br/&gt; 354108&lt;br/&gt; 3603</td><td>Platforms for hydrogel deposition and organoid culture.</td></tr><tr><td>Dimethyl sulfoxide</td><td>Millipore Sigma</td><td>317275</td><td>Cryoprotectant used in freezing medium</td></tr><tr><td>Dispase II</td><td>Roche</td><td>4942078001</td><td>Enzyme used for secondary tissue dissociation</td></tr><tr><td>DNase I</td><td>Roche</td><td>10104159001</td><td>Enzyme used during cell dissociation.</td></tr><tr><td>Fetal bovine serum</td><td>Gibco</td><td>10437</td><td>Serum supplement used in wash and neutralization media</td></tr><tr><td>Fibronectin</td><td>Sigma-Aldrich</td><td>F2006</td><td>Extracellular matrix protein component</td></tr><tr><td>GlutaMAX</td><td>Thermo Scientific</td><td>35050061</td><td>Supplement for epithelial cell media</td></tr><tr><td>Human epidermal growth factor</td><td>Sigma-Aldrich</td><td>E9644</td><td>Supplement for epithelial cell media</td></tr><tr><td>Hydrocortisone</td><td>Sigma-Aldrich</td><td>H0888</td><td>Supplement for epithelial cell media</td></tr><tr><td>Hyaluronic acid</td><td>Millipore Sigma</td><td>385908</td><td>Extracellular matrix component for hydrogel formulation</td></tr><tr><td>Hyaluronidase</td><td>Sigma-Aldrich</td><td>H3506</td><td>Enzyme used for tissue dissociation</td></tr><tr><td>Incubator</td><td>Thermo Scientific</td><td>3598</td><td>Device used for tissue culture incubation</td></tr><tr><td>Insulin</td><td>Sigma-Aldrich</td><td>I9278</td><td>Supplement for epithelial cell media</td></tr><tr><td>Laminin</td><td>Gibco</td><td>23017-015</td><td>Extracellular matrix protein component</td></tr><tr><td>Mammary epithelial basal medium</td><td>Thermo Scientific</td><td>M171500</td><td>Growth medium for epithelial cells</td></tr><tr><td>Microcentrifuge tubes (1.5 mL)</td><td>Thermo Scientific</td><td>3451</td><td>Tubes used for small-volume reactions</td></tr><tr><td>Orbital rotator</td><td>Thermo Scientific</td><td>400110</td><td>Rotator used for tissue dispersion during enzymatic dissociation</td></tr><tr><td>P1000 pipette</td><td>Gilson</td><td>P1000</td><td>Device used to mix and transfer volumes up to 1,000 &amp;mu;L</td></tr><tr><td>Penicillin-streptomycin</td><td>Thermo Scientific</td><td>15140122</td><td>Antibiotic supplement for epithelial cell media</td></tr><tr><td>Phosphate-buffered saline</td><td>Gibco</td><td>20012-027</td><td>Buffer solution used for washing and rinsing</td></tr><tr><td>Precision balance</td><td>Mettler Toledo</td><td>ML303E</td><td>Balance used for tissue weighing</td></tr><tr><td>Serological pipette</td><td>Nunc</td><td>170356N</td><td>Pipette used for harvesting non-adherent epithelial cells</td></tr><tr><td>Sodium hydroxide (1 N)</td><td>Fisher Chemical</td><td>SS261</td><td>Reagent used for collagen neutralization</td></tr><tr><td>Sterile scalpels</td><td>Bard-Parker</td><td>372615</td><td>Tools used for mechanical tissue mincing</td></tr><tr><td>Trypan blue solution</td><td>Gibco</td><td>15250061</td><td>Dye used for cell viability assessment</td></tr><tr><td>Trypsin (0.25%)</td><td>Gibco</td><td>25200056</td><td>Enzymatic reagent used for cell dissociation</td></tr><tr><td>Water bath (37 &amp;deg;C)</td><td>VWR</td><td>10LA</td><td>Device used for controlled thawing of samples</td></tr></tbody></table>

a defined hydrogel-based method for generating three-dimensional human breast organoids that recapitulate mammary morphogenesis

A Defined Hydrogel-Based Method For Generating Three-Dimensional Human Breast Organoids That Recapitulate Mammary Morphogenesis

The development of physiologically relevant human model systems that recapitulate tissue architecture and cell-state dynamics remains a major challenge in studying breast development and early events in carcinogenesis. Conventional two-dimensional cultures and many three-dimensional systems fail to capture the structural organization and microenvironmental cues that define the human mammary gland. Here, we describe a reproducible method for generating three-dimensional human breast organoids from primary epithelial cells embedded within a defined hydrogel matrix composed of type I collagen, laminin, fibronectin, and hyaluronic acid. This system supports the progression of single cells through key stages of mammary morphogenesis, including progenitor expansion, epithelial patterning, and the formation of terminal ductal lobular unit-like structures, as well as the emergence of a mesenchyme-like compartment, over a 21-day culture period. We provide a step-by-step protocol for hydrogel preparation, cell seeding and&#160;culture conditions. The method is compatible with high-content imaging and quantitative analysis of organoid number, size distribution, and architectural complexity. This platform enables mechanistic studies of epithelial plasticity and environmental perturbations, providing a scalable and biologically relevant system for investigating early tissue-level changes associated with breast cancer risk.

Successful execution of this protocol results in the formation of three-dimensional organoid structures that exhibit organized epithelial morphology and tissue-specific architectural features. Organoids begin to form within 3&#8211;7 days following seeding and continue to develop throughout the culture period. Previous characterization of this hydrogel organoid system demonstrated reproducible organoid formation across multiple independent primary human donors3. In that study, primary epithelial c...

This protocol describes a defined hydrogel-based method to generate human breast organoids that recapitulate key features of mammary morphogenesis in a controlled three-dimensional culture system.

The development of physiologically relevant human model systems that recapitulate tissue architecture and cell-state dynamics remains a major challenge in ...

Our research focuses on human breast development and lactation, tissue organization, environmental exposures, and genetic factors leading to biological changes in cancer. Existing models inaccurately capture human breast tissue architecture, developmental timing, and microenvironment. This protocol bridges 2D and animal models in a human system.Obtain digested human breast tissue cryo-preserved for at least 24 hours. Immediately submerge the cryo vial up to the cap in a 37 degrees Celsius water bath, and thaw rapidly for one minute to minimize cell death. Once fully thawed, transfer the contents of the cryo vial to a 15-milliliter conical tube containing 10 milliliters of mammary epithelial growth medium, or MEGM, and centrifuge at 250g for five minutes.After discarding the supernatant, re-suspend the pellet in 500 microliters of dissociation enzyme solution pre-warmed to 37 degrees Celsius containing 0.25%trypsin. Transfer the suspension to a 1.5-milliliter microcentrifuge tube. Using a P1000 pipette, triturate the sample 20 times to promote dissociation.Incubate the tube at 37 degrees Celsius for three to five minutes. Using a P1000 pipette, mechanically dissociate the sample by

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Research

JoVE Journal

Developmental Biology

With the intent of characterizing changes in miRNAs on differentiated human neural stem cells (hNSCs) we describe hNSC differentiation on a three dimensional system,the evaluation of changes in microRNA expression by miRNA PCR array, and computational analysis for miRNA target prediction and its validation by dual luciferase assay.

Differentiation of a Human Neural Stem Cell Line on Three Dimensional Cultures, Analysis of MicroRNA and Putative Target Genes

Dendritic spines of pyramidal neurons are the sites of most excitatory synapses in mammalian brain cortex. This method describes a 3D quantitative analysis of spine morphologies in human cortical pyramidal glutamatergic neurons derived from induced pluripotent stem cells.

Three-dimensional Quantification of Dendritic Spines from Pyramidal Neurons Derived from Human Induced Pluripotent Stem Cells

The method presented here describes a scalable and good manufacturing practice (GMP)-ready differentiation system to generate human hepatocyte-like cells from pluripotent stem cells. It serves as a cost-effective and standardized system to generate human hepatocyte-like cells for basic and applied human liver research.

Defined and Scalable Generation of Hepatocyte-like Cells from Human Pluripotent Stem Cells

This study presents a protocol for the reversible tissue clearing, immunostaining, 3D-rendering and analysis of vascular networks in human placenta villi samples on the order of 1 - 2 mm3.

Three-dimensional Rendering and Analysis of Immunolabeled, Clarified Human Placental Villous Vascular Networks

The goal of the protocol is to build an inflammatory human gingiva model in vitro. This tissue model co-cultivates three types of human cells, HaCaT keratinocytes, gingival fibroblasts, and THP-1 macrophages, under three-dimensional conditions. This model can be applied to investigating periodontal diseases, such as gingivitis and periodontitis.

Three-dimensional Inflammatory Human Tissue Equivalents of Gingiva

Three-dimensional organotypic cultures of the murine utricle and cochlea in optically clear collagen I gels preserve innate tissue morphology, allow for mechanical stimulation through adjustment of matrix stiffness, and permit virus-mediated gene delivery.

Three-dimensional Organotypic Cultures of Vestibular and Auditory Sensory Organs

Here, we present a protocol to generate insulin expressing 3D murine pancreatoids from free-floating e10.5 dissociated pancreatic progenitors and the associated mesenchyme.

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

Here, we present a protocol to precisely form hemogenic endothelium from human pluripotent stem cells in a feeder- and xeno-free condition. This method will have broad applications in disease modeling and screening for therapeutic compounds.

Hemogenic Endothelium Differentiation from Human Pluripotent Stem Cells in A Feeder- and Xeno-free Defined Condition

This protocol describes an approach to produce hepatospheres from human pluripotent stem cells using a defined culture system and cell self-assembly. This protocol is reproducible in a number of cell lines, cost effective and allows the production of stable human hepatospheres for biomedical application.

Serum Free Production of Three-dimensional Human Hepatospheres from Pluripotent Stem Cells

This protocol was generated as a means to produce brain organoids in a simplified, low cost manner without exogenous growth factors or basement membrane matrix while still maintaining the diversity of brain cell types and many features of cellular organization.

A Static Self-Directed Method for Generating Brain Organoids from Human Embryonic Stem Cells

Tufts University School of Medicine.  This protocol describes a defined hydrogel-based method to generate human breast organoids that recapitulate key features of mammary morphogenesis in a controlled three-dimensional culture system.

A Defined Hydrogel-Based Method For Generating Three-Dimensional Human Breast Organoids That Recapitulate Mammary Morphogenesis

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