Human intestinal organoids were generated following previously established protocols (Spence et al, 2011 (link)) using multiple pluripotent stem cell (PSC) lines (Table EV2 ). Briefly, PSCs were differentiated through the definitive endoderm stage via the addition of Activin A (Cell Guidance Systems) between day‐0 (D0) and D2, BMP4 (R&D Systems) at D1 and CHIR99021 (Stemgent)/FGF4 (R&D Systems) from D3 to D6. The addition of CHIR99021/FGF4 prompted the formation of midgut tube spheroids from the definitive endoderm monolayer. At D7 midgut tube spheroids were collected and suspended in a Matrigel dome (Corning). Spheroids were then grown in gut media: DMEM/F12 (Gibco) supplemented with N‐2 (Gibco), B‐27 (Gibco), HEPES solution (Millipore‐Sigma), recombinant murine epidermal growth factor (Peprotech), L‐Glutamine (Gibco), Penicillin/Streptomycin (Gibco). At D21, sample density was reduced to ≈1–3 per 50 µl Matrigel bubble. Samples were grown for an additional 14 days in gut media, resulting in terminal HIO cultures (Spence et al, 2011 (link)). Individual HIOs were considered as biological replicates for all experiments. All experimentation using human tissues described herein was approved by an IRB at CCHMC (IRB#2014‐0427). Informed consent for tissue collection, storage, and use of the samples was obtained from the donors at CCHMC.
Matrigel dome
Manufactured by Corning
Sourced in United States
Matrigel dome is a laboratory product developed by Corning. It is a gelatinous protein mixture that mimics the extracellular matrix found in many tissues. The core function of the Matrigel dome is to provide a three-dimensional environment for the culture and growth of cells.
Automatically generated - may contain errors
Lab products found in correlation
Activin a, by Cell Guidance Systems (2 mentions)
L glutamine, by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Bone morphogenetic protein 4 (bmp4), by R&D Systems (2 mentions)
Dmem f12, by Thermo Fisher Scientific (2 mentions)
Hepes solution, by Merck Group (2 mentions)
293ft cell, by Thermo Fisher Scientific (2 mentions)
Antibiotic antimycotic, by Thermo Fisher Scientific (2 mentions)
Celltiter glo reagent, by Promega (2 mentions)
Cell recovery solution, by Corning (2 mentions)
Murine recombinant epidermal growth factor, by Thermo Fisher Scientific (1 mentions)
Mda mb 436, by American Type Culture Collection (1 mentions)
Mcf 7, by American Type Culture Collection (1 mentions)
Mycoalert mycoplasma detection kit, by Lonza (1 mentions)
Cabazitaxel, by Sanofi (1 mentions)
R1881, by Merck Group (1 mentions)
Celltiter glo 3d, by Promega (1 mentions)
Enzalutamide, by Axon Medchem (1 mentions)
Docetaxel, by Sanofi (1 mentions)
Symphony s6, by BD (1 mentions)
10 protocols using matrigel dome
1
Generation of Human Intestinal Organoids
2
Establishment and Maintenance of Cell Lines
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MCF-7, T47D and MDA-MB-436 cell lines were purchased from ATCC. 293FT cells were purchased from Invitrogen. H596, H1048 and H1155 cell lines were kindly provided by Dr. John Minna. Du-145 cells were kindly provided by Dr. Ganesh Raj. MCF-7, MDA-MB-436 and 293FT cells were maintained in DMEM containing 10% FBS. T47D and Du-145 cells were maintained in RPMI containing 10% FBS. H596, H1048 and H1155 cells were maintained in RPMI containing 5% FBS. All culture media was supplemented with 1x antibiotic-antimycotic (Invitrogen). The PDxOs were maintained in Matrigel dome (Corning) supplemented with DMEM/F12 containing 250 ng/ml R-Spondin 3, 5 nM Heregulin β1, 5 ng/ml FGF7, 20 ng/ml FGF10, 5 ng/ml EGF, 100 ng/ml Noggin, 500 nM A83–01, 5 μM Y-27632, 500 nM SB202190, 1X B27 supplement, 1.25 mM N-Acetylcysteine, 5 mM Nicotinamide, 1X GlutaMax, 10 mM HEPES, 50 μg/ml primocin and 100 U/ml penicillin/100 μg/ml streptomycin.
3
Intestinal Organoid Perfusion Protocol
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Pluripotent stem-cell-derived HIOs were generated following the procedure defined in McCracken et al. [3 (link)] and Hill et al. [19 ]. The complete protocol for generation and maintenance of HIOs can be found in Sections 4 and 5 of Hill et al. [19 ]. HIOs were ~50 days old on the day of perfusion for both experiments. Control and test HIOs used in the bacterial perfusion experiment were from the same initial batch. HIOs were embedded in a growth-factor-reduced Matrigel dome (Cat# 356231, Corning Inc., Corning, NY, USA) with a protein concentration of >8 mg/mL to replicate physiological extracellular matrix support and stiffness. Then, 500–750 µL of HIO maintenance media [3 (link)] was added to each well and replaced every 2–3 days.
4
Comprehensive Cell Line Cultivation and Authentication
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MCF-7 (Cat. No. HTB-22), T47D (Cat. No. HTB-133), HCC1428 (Cat. No. CRL-2327), ZR-75-1 (Cat. No. CRL-1500), and MDA-MB-436 (Cat. No. HTB-130) cells were purchased from ATCC. 293FT cells were purchased from Invitrogen (Cat. No. R70007). H596, H1048 and H1155 cell lines were kindly provided by Dr. John Minna. Du-145 cells were kindly provided by Dr. Ganesh Raj. CAMA1 and KPL1 cells were kindly provided by Dr. Benjamin Neel. Short tandem repeat (STR) profiling was used to verify authenticity of the cell lines. Cell lines were routinely tested for mycoplasma using MycoAlert mycoplasma detection kit (Lonza, Cat. No. LT07-710). MCF-7, MDA-MB-436 and 293FT cells were maintained in DMEM containing 10% FBS. T47D and Du-145 cells were maintained in RPMI containing 10% FBS. HCC1428, H596, H1048, H1155, and ZR-75-1 cells were maintained in RPMI containing 5% FBS. All culture media was supplemented with 1x antibiotic-antimycotic (Invitrogen). The PDxOs were maintained in Matrigel dome (Corning) supplemented with DMEM/F12 containing 250 ng/ml R-Spondin 3, 5 nM Heregulin β1, 5 ng/ml FGF7, 20 ng/ml FGF10, 5 ng/ml EGF, 100 ng/ml Noggin, 500 nM A83-01, 5 μM Y-27632, 500 nM SB202190, 1X B27 supplement, 1.25 mM N-Acetylcysteine, 5 mM Nicotinamide, 1X GlutaMax, 10 mM HEPES, 50 μg/ml primocin and 100 U/ml penicillin/100 μg/ml streptomycin.
5
Intestinal Organoid Generation from Pluripotent Stem Cells
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HIOs were generated following previously established protocols 2 using multiple pluripotent stem cell (PSC) lines, both induced and embryonic. Briefly, PSCs were differentiated through the definitive endoderm stage via the addition of Activin A (Cell Guidance Systems) between day-0 (D0) and D2, BMP4 (R&D Systems) at D1 and CHIR99021 (Stemgent) / FGF4 (R&D Systems) from D3 to D6. The addition of CHIR99021/FGF4 prompted the formation of midgut tube spheroids from the definitive endoderm monolayer. At D7 midgut tube spheroids were collected and suspended in a Matrigel dome (Corning). Spheroids were then grown in gut media: DMEM/F12 (Gibco) supplemented with N-2 (Gibco), B-27 (Gibco), HEPES solution (Millipore-Sigma), recombinant murine epidermal growth factor (Peprotech), L-Glutamine (Gibco), Penicillin/Streptomycin (Gibco). At D21 sample density was reduced to ≈1-3 per 50µL Matrigel bubble. Samples were grown for an additional 14-days in gut media, resulting in terminal HIO cultures 2 . Individual HIOs were considered as biological replicates for all experiments.
6
Organoid-Based Tumor Lineage Profiling
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Human and mouse organoids were trypsinized into single cell solution and counted. MSK-PCa2 (5000 cells), Trp53-KO (2000 cells), Rb1-KO (2000 cells) or PTENΔ/Δ -Rosa26-ERG (2000 cells) single organoids were seeded in 4×15ul Matrigel domes (Corning #356231) in a 48-well plate with 300 ul organoid culture media and media was replenished every 3 days. After 6 days, media was withdrawn and 100 μl cell recovery solution (Corning #354253) was added. The organoid plate was then incubated at 4°C on a rotator for 60 minutes. Equal volume (100μl) of CellTiter-Glo reagents (Promega #G7571) was added into the organoid suspension, mixed and incubated in room temperature on an orbital shaker for 15 minutes to stabilize the reaction. A total 200μl reaction volume was transferred to a 96-well plate for CellTiter-Glo assay.
7
Organoid-Based Tumor Lineage Profiling
8
Organoid-based Anticancer Drug Screening
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The AR-negative (AR-) MSK-PCa1 and AR-positive (AR+) MSK-PCa2 organoid lines were kindly provided by Dr. Wouter Karthaus and Prof. Dr. Jack Schalken from the Radboud University Medical Center. Organoids were cultured and passaged as described previously [9 (link),33 (link)], with the addition of synthetic androgen R1881 (1 nM; Sigma-Aldrich, Saint Louis, Missouri, USA, cat. no. R0908) for MSK-PCa2 organoids. Viability assays were conducted in pre-warmed 96-well plates (Costar, Corning, New York, NY, USA, cat. no. 3595) at a plating density of 2500 MSK-PCa1 or 5000 MSK-PCa2 organoid cells in 8 µL Matrigel domes (Corning, cat. no. 356231) per well. Single cells or organoids were incubated for 7 days with a dose range of docetaxel, cabazitaxel (0.01–10 nM; provided by Sanofi, Paris, France), abiraterone (0.03–30 µM; provided by Sanofi) or enzalutamide (0.03–30 µM; Axon Medchem, Groningen, the Netherlands, cat. no. 1613). Viability was measured with CellTiter-Glo 3D according to the manufacturer’s protocol (Promega, Madison, WI, USA, cat. no. G9681).
9
Isolation and Organoid Culture of Endometrial Epithelial Subsets
10
Patient-Derived Tumor Organoid Generation
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Organoids were derived from primary human tumor samples in accordance with IRB 70703 at Mayo Clinic, Rochester, MN, USA. Freshly isolated human tumors were dissociated using a human tumor dissociation kit and MACS Dissociator (Miltenyi Biotec, Bergisch Gladbach, Germany). Dissociated cells were washed, strained (70 um), and resuspended in melted Matrigel domes (Corning, NY, USA). Organoids were cultured in complete media (supplementary materials and methods), changed twice weekly and passaged by physical dissociation. Experiments were performed within 10 passages of generation.
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