ECM-Embedded Organoid (Matrigel/Dome) Culture
Materials Required
Principle
ECM-embedded organoid dome culture embeds epithelial stem cells, crypts, organoid fragments, or tumor-derived epithelial cells in a basement-membrane-like hydrogel such as Matrigel, allowing 3D growth, self-organization, lumen formation, budding or cystic morphogenesis, and lineage maintenance under defined niche-factor-containing medium[1][2][3][4][5]. The primary readouts are organoid establishment efficiency, growth, morphology, passaging capacity, lineage-marker expression, and, when fluorescently labeled lines are used, microscopy- or flow-cytometry-based quantification of population behavior in 3D culture[3][4][5][6].
MCE has not independently verified the accuracy of these methods. They are for reference only.
Experimental Materials
• Matrigel or EHS-derived basement membrane matrix is used as the ECM scaffold because EHS matrix contains basement-membrane components including laminin, type IV collagen, and heparan sulfate proteoglycan, and Matrigel supports biologically active 3D epithelial culture[1][2][3][4][5][6].
• Organoid culture medium should be matched to the tissue model and may include EGF, Noggin, R-spondin, and Wnt-pathway support when reported for intestinal epithelial organoids[3][4][5][6].
• PBS or equivalent buffered saline is used for washing organoid pellets and wells during recovery, passaging, or preparation for downstream analysis[5][6].
• Cell Recovery Solution is used in published organoid protocols to dissolve Matrigel on ice before collecting organoids for passaging or analysis[6].
• TrypLE is used when single-cell dissociation is required for flow cytometry or sorting, with published incubation at 37°C for 4-5 min in intestinal organoid workflows[6].
• DAPI is used as a dead-cell exclusion dye for flow-cytometry analysis of dissociated organoid cells, with one reported final concentration of 10 µM[6].
• Lineage-marker immunostaining or immunofluorescence can be used to assess organoid differentiation and tissue identity after culture[5].
• Required equipment includes a Class II biosafety cabinet, centrifuge or microcentrifuge, inverted light microscope, humidified 37°C incubator with 5% CO2, pre-warmed multiwell tissue-culture plates, chilled tubes and pipette tips for Matrigel handling, and flow cytometer or fluorescence microscope when fluorescent organoid analysis is performed[5][6].
• Organoid culture medium should be matched to the tissue model and may include EGF, Noggin, R-spondin, and Wnt-pathway support when reported for intestinal epithelial organoids[3][4][5][6].
• PBS or equivalent buffered saline is used for washing organoid pellets and wells during recovery, passaging, or preparation for downstream analysis[5][6].
• Cell Recovery Solution is used in published organoid protocols to dissolve Matrigel on ice before collecting organoids for passaging or analysis[6].
• TrypLE is used when single-cell dissociation is required for flow cytometry or sorting, with published incubation at 37°C for 4-5 min in intestinal organoid workflows[6].
• DAPI is used as a dead-cell exclusion dye for flow-cytometry analysis of dissociated organoid cells, with one reported final concentration of 10 µM[6].
• Lineage-marker immunostaining or immunofluorescence can be used to assess organoid differentiation and tissue identity after culture[5].
• Required equipment includes a Class II biosafety cabinet, centrifuge or microcentrifuge, inverted light microscope, humidified 37°C incubator with 5% CO2, pre-warmed multiwell tissue-culture plates, chilled tubes and pipette tips for Matrigel handling, and flow cytometer or fluorescence microscope when fluorescent organoid analysis is performed[5][6].
Experimental Procedure
• Keep Matrigel and Matrigel-containing suspensions cold during handling, because published dome workflows emphasize ice-cold handling before plating and then solidification after transfer to 37°C[6].
• Prepare organoid fragments, crypts, or dissociated cells according to the tissue-specific published protocol; intestinal organoids have been established from single Lgr5-positive stem cells, mouse or human intestinal crypts, human colon, adenoma, adenocarcinoma, Barrett’s epithelium, and fresh human intestinal biopsies[3][4][5].
• Pre-warm the culture plate and complete organoid medium before dome plating when using the published 24-well Matrigel dome workflow[6].
• Collect organoids from existing Matrigel domes by adding Cell Recovery Solution at 250 µL per 50 µL Matrigel dome, mechanically disrupting the dome with a P1000 tip, transferring the suspension to a sterile tube, incubating on ice for 30 min, and centrifuging at 480 × g for 4 min to pellet organoids[6].
• For passaging as organoid fragments, remove the supernatant after centrifugation and resuspend the pellet in cold Matrigel; published intestinal organoid protocols used 100 µL Matrigel to seed two 50 µL domes in a pre-warmed 24-well plate[6].
• For a 3D dome setup, plate 50 µL Matrigel-organoid suspension per well of a pre-warmed 24-well plate, mix between wells to maintain a uniform suspension, transfer carefully to a 37°C, 5% CO2 incubator, and allow the dome to solidify for approximately 15 min before adding medium[6].
• After dome solidification, add 500 µL pre-warmed organoid medium per well and maintain cultures in a humidified 37°C, 5% CO2 incubator[6].
• For single-cell analysis, dissociate organoids with TrypLE at 37°C for 4 min, neutralize with FACS buffer, centrifuge at 480 × g for 4 min, wash once more, and add DAPI to 10 µM immediately before flow-cytometry analysis[6].
• Acquire brightfield or fluorescence microscopy images to document organoid morphology, dome integrity, organoid number, and growth pattern; for fluorescent co-culture experiments, published workflows analyze 3D cultures using microscopy and flow cytometry[5][6].
• Use positive controls consisting of previously expandable organoid lines cultured under the same matrix and medium conditions, and use negative or comparator controls defined by the experimental question, such as genetically distinct fluorescent organoid populations in co-culture studies[6].
• Use technical replicate wells when comparing conditions; one published 3D co-culture workflow illustrates four technical replicates for control and experimental organoid co-cultures[6].
• Prepare organoid fragments, crypts, or dissociated cells according to the tissue-specific published protocol; intestinal organoids have been established from single Lgr5-positive stem cells, mouse or human intestinal crypts, human colon, adenoma, adenocarcinoma, Barrett’s epithelium, and fresh human intestinal biopsies[3][4][5].
• Pre-warm the culture plate and complete organoid medium before dome plating when using the published 24-well Matrigel dome workflow[6].
• Collect organoids from existing Matrigel domes by adding Cell Recovery Solution at 250 µL per 50 µL Matrigel dome, mechanically disrupting the dome with a P1000 tip, transferring the suspension to a sterile tube, incubating on ice for 30 min, and centrifuging at 480 × g for 4 min to pellet organoids[6].
• For passaging as organoid fragments, remove the supernatant after centrifugation and resuspend the pellet in cold Matrigel; published intestinal organoid protocols used 100 µL Matrigel to seed two 50 µL domes in a pre-warmed 24-well plate[6].
• For a 3D dome setup, plate 50 µL Matrigel-organoid suspension per well of a pre-warmed 24-well plate, mix between wells to maintain a uniform suspension, transfer carefully to a 37°C, 5% CO2 incubator, and allow the dome to solidify for approximately 15 min before adding medium[6].
• After dome solidification, add 500 µL pre-warmed organoid medium per well and maintain cultures in a humidified 37°C, 5% CO2 incubator[6].
• For single-cell analysis, dissociate organoids with TrypLE at 37°C for 4 min, neutralize with FACS buffer, centrifuge at 480 × g for 4 min, wash once more, and add DAPI to 10 µM immediately before flow-cytometry analysis[6].
• Acquire brightfield or fluorescence microscopy images to document organoid morphology, dome integrity, organoid number, and growth pattern; for fluorescent co-culture experiments, published workflows analyze 3D cultures using microscopy and flow cytometry[5][6].
• Use positive controls consisting of previously expandable organoid lines cultured under the same matrix and medium conditions, and use negative or comparator controls defined by the experimental question, such as genetically distinct fluorescent organoid populations in co-culture studies[6].
• Use technical replicate wells when comparing conditions; one published 3D co-culture workflow illustrates four technical replicates for control and experimental organoid co-cultures[6].
Troubleshooting
Problem: Matrigel remains in the recovered organoid preparation.
• Possible Cause: Incomplete Matrigel dissolution during recovery.• Literature-supported Solution: Incubate the disrupted Matrigel-organoid suspension on ice for 30 min, centrifuge at 480 × g for 4 min, and, if residual Matrigel is visible, wash with ice-cold PBS and repeat centrifugation[6].
Problem: Organoid fragments do not grow efficiently after passaging.
• Possible Cause: Over-dissociation into single cells when fragment culture is intended.• Literature-supported Solution: Generate small organoid clumps by vigorous pipetting rather than a full single-cell suspension for fragment-based outgrowth, because the cited protocol notes that single-cell suspensions do not grow out as efficiently in that context[6].
Problem: Flow cytometer clogging or doublets during cell sorting.
• Possible Cause: Incomplete single-cell dissociation before flow cytometry.• Literature-supported Solution: Use TrypLE dissociation at 37°C for 4 min and prepare a single-cell suspension before analysis[6].
References:
- [1]. Kleinman HK, et al. Isolation and characterization of type IV procollagen, laminin, and heparan sulfate proteoglycan from the EHS sarcoma. Biochemistry. 1982;21(24):6188-6193. [Content Brief]
- [2]. Kleinman HK, et al. Matrigel: basement membrane matrix with biological activity. Semin Cancer Biol. 2005;15(5):378-386. [Content Brief]
- [3]. Sato T, et al. Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature. 2009;459(7244):262-265. [Content Brief]
- [4]. Sato T, et al. Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett’s epithelium. Gastroenterology. 2011;141(5):1762-1772. [Content Brief]
- [5]. Pleguezuelos-Manzano C, et al. Establishment and Culture of Human Intestinal Organoids Derived from Adult Stem Cells. Curr Protoc Immunol. 2020;130(1):e106. [Content Brief]
- [6]. van Neerven SM, et al. Intestinal organoid co-culture protocol to study cell competition in vitro. STAR Protoc. 2022;3(1):101050. [Content Brief]
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