All methods described here have been approved by the Institutional Animal Care and Use Committee (IACUC) of the University of California, Santa Cruz.
1. Day 1: Mammary gland digestion
2. Day 2: Isolation of mammary epithelial tissue fragments
3. Day 3: Differential trypsinization of myoepithelial and luminal epithelial cells
4. Day 3: Combining and embedding cell fractions in an extracellular matrix
NOTE: Once the MyoEC and LEC fractions have been collected and counted, they can be combined. The typical MyoEC/LEC ratio is 1:3 (Figure 3A)19. Different studies can be performed. For example, to perform mosaic studies, fractions can be generated from wild type (WT) and mutant (Mut) mice and combined (MyoEC/LEC: WT/WT; WT/Mut; Mut/WT; Mut/Mut)21, or fractions can be combined using different ratios of MyoECs/LECs19.
5. Day 5 or 10: Fixing and immunostaining organoids
6. Day 11: Complete immunofluorescence
The protocol presented here describes a method for investigating specific lineage contributions of mammary epithelial cells by making use of mosaic organoids. To obtain primary murine cells for organoids, the mammary gland epithelium must first be isolated from the surrounding adipocyte rich stroma (Figure 1). This process is described briefly here and is also described in a previously published study18. To obtain enough cells, it is recommended that #2, 3, 4, and 5 MGs be removed (Figure 1A). An important step key to isolating a pure population of epithelial cells is removal of the lymph nodes from the #4 MGs, which are rich in immune cells that will contaminate the preparation (Figure 1A,B). The MGs were minced to generate fragments ~0.1 mm in size (Figure 1B). The tissue fragments were then enzymatically digested, a process occurring in the presence of collagenase, to release epithelia from stroma, and in the absence of trypsin, to prevent the digestion of proteins such as cadherins that maintain cell-cell contacts. The digested tissue was then centrifuged to remove lipids and filtered through a cell strainer and washed (Figure 1C). Epithelial fragments, adhering to the strainer, were released by inverting the filter and washing the membrane, which transferred the epithelial fragments onto a polystyrene dish (Figure 1D). These fragments appeared as small, branched structures (Figure 1E).
The purified epithelial fragments were incubated for 24 h. They settled down onto the dish and adhered, forming flat, pancake-like structures with an outer layer of MyoECs encircling inner LECs (Figure 2A-B). Figure 2C shows the edge of such a pancake-like structure from a wild type animal. Trypsin treatment differentially detached the MyoECs, which detached first and appeared as bright, rounded cells that encircled the core of remaining cuboidal LECs (Figure 2C,F). The detachment of the MyoECs was carefully monitored using brightfield microscopy and occurred within 3-6 min. Once the MECs were collected, LECs were subsequently detached through a second, longer trypsin treatment of 7-15 min. The time required for cell detachment depends on the trypsin concentration and freshness. The overall purity of the two cell compartments was ~90%, as assayed by counting cells that were KRT14-positive and E-Cadherin (CDH1)-negative in the MyoEC fraction and cells that were KRT14-negative and E-Cadherin-positive in the LEC fraction (Figure 2D-E)19. We discovered that some of the MyoECs were removed from the top of the pancake-like structure as well as from the outer edges. This was observed by using tissue fragments collected from mice labeled with an inducible, fluorescent basal marker (Cytokeratin 14 (KRT14)-CreERT1; R26RYFP/+) and injected with 75 mg/kg tamoxifen 5 days prior to harvest. In Figure 2F,G the detachment of MyoECs from around the edges of the pancake structure is readily apparent. This occurred within the first 2 min of trypsin treatment (Figure 2F). In addition, YFP-KRT14-positive cells were observed on top of the structure, where they rounded up after trypsin treatment and were removed by the rinse/collection step (Figure 2G). The unlabeled core of LECs (Figure 2H), which contained few or no YFP-KRT14-positive cells, (Figure 2I) subsequently detached in the second round of trypsin treatment.
The MyoEC and LEC fractions were collected, combined, and embedded into 10% ECM plated onto a 50% ECM base. This allowed for better optical resolution of the organoids that grew primarily along the base layer (Figure 3A). After 24 h, the cells assembled into aggregated structures that largely lacked a lumen (Figure 3B). After 48 h, nascent organoids formed as the central lumens hollowed and appeared as a lighter internal space (Figure 3C). After 10 days, the organoids were large, branched structures with well-developed lumens. Mosaic organoids generated from MyoECs harvested from wild type mice and LECs harvested from ACTb-EGFP mice were fixed in situ, immunostained with an antibody against the basal marker alpha-smooth muscle actin (SMA), and stained with the Hoechst DNA stain to show the nuclei. In the figures, the top and section views show different sets of images collected as a Z-stack and reconstructed into a 3D view (Figure 3D). The top view reveals the branched morphology of the organoids (Figure 3E'). The section view shows the bilayered epithelial structure and open lumen of these organoids (Figure 3E''). These organoids can also be differentiated at Day 5 using Alveologenesis Medium and incubated for an additional 5 days (Figure 3F). The organoids grew larger, had more branches, and contained milk. Differentiated organoids were generated as described above and immunostained with an antibody directed against the milk marker, whey acidic protein (WAP, Figure 3F). WAP is a soluble protein secreted into milk. Much of this liquid was lost when the cells were fixed and immunostained in situ. Therefore, in the top and section views, WAP staining is visible intracellularly in secreting cells and extracellularly in milk that was trapped at the cell surface during fixation (Figure 3F), although in section view a small organoid appears to contain liquid milk (Figure 3F'' boxed overlay).
Figure 1: Mammary fragment isolation. (A) Labeled schematic of a mouse's 5 MGs with unlabeled, contralateral paired MGs. (B) Images of mouse MGs with the #4 MG boxed and magnified to show how to identify the lymph node for removal. (C) Image of chopped MGs in a 6 well low adhesion plate with a ruler showing the size of the tissue pieces (~0.1 mm each). (D-E) Schematic illustrating protocol steps 2.7-2.9. (D) MG fragments were filtered through a 70 µm strainer and rinsed 4X. (E) The strainer was then inverted over a 60 mm polystyrene tissue culture dish and fragments were released into the dish. (F) Image showing the filtered tissue fragments collected on a 60 mm dish that are free of stroma. The arrows point to the smallest fragments that are collected on the 70 µm strainer. Scale bar = 100 µm. Please click here to view a larger version of this figure.
Figure 2: Differential trypsinization. (A) Brightfield image showing a tissue fragment adhered on a polystyrene dish, forming a pancake-like structure. (B-C) The first differential trypsinization step detached MyoECs that are clearly visible as bright, rounded cells after 3 min. (D) Immunofluorescent images of MyoECs (bottom) and LECs (top) using the Cytokeratin 14 (KRT14) cell marker for MyoECs, and E-Cadherin (CDH1) cell marker for LECs. (E) The expression of KRT14 and CDH1 was used to quantify the yield and purity of the differentially trypsinized cell fractions. (F-I). Representative phase-contrast and fluorescence (YFP) images of tissue fragments from Cytokeratin 14 (KRT14)-CreERT1; R26RYFP/+ MGs. Mice were injected with 75 mg/kg tamoxifen 5 days prior to harvest. (F) Detaching MyoECs (arrows) during the initial trypsin-EDTA (0.05%) 2 min after incubation. (G) A sprinkling of KRT14-YFP-MyoECs (arrows) on top of a pancake of unlabeled LECs. (H) Brightfield image of LECs after initial trypsinization and MyoEC detachment. (I) After MyoEC detachment, KRT14-YFP-MyoECs are no longer visible as shown by the absence of YFP expression. Scale bars = 30 µm (A, brightfield) 100 µm (F, brightfield), 50 µm (G, fluorescence), 100 µm (H,I). Panels A-E of this figure are modified from Macias et al.19. Please click here to view a larger version of this figure.
Figure 3: Three-dimensional organoid culture. (A) Schematic representation of single cells embedded in 10% ECM/90% Growth Medium and grown on a 50% ECM/50% DMEM base layer (protocol step 4.5). (B-C) Illustrations and phase-contrast images showing the rapid self-organizing capacities of mammary organoids generated from differentially trypsinized and recombined MyoECs and LECs at 24 h (B) and 48 h (C). Images collected using a digital widefield microscope (D) Schematic representation illustrating the top (left) or section (right) views used in E-F to show immunostained organoids. (E) Schematic representation of a single well of an 8 well chamber slide containing mammary organoids grown for 5-10 days in Growth Medium. (E'-E") Top view (E') and section view (E") of immunostained organoids at day 10 of growth. MyoECs are marked with smooth actin muscle (SMA) in pseudocolor magenta. The LECs are from ACTb-EGFP mice and are shown in pseudocolor green. Nuclei were stained with Hoechst dye. (F) Schematic representation of a single well of an 8 well chamber slide containing mammary organoids grown for 5 days in Growth Medium and 5 days in Alveologenesis Medium. (F'-F"). Top view (F') and section view (F") of immunostained organoids at day 10 of growth. The MyoECs are unmarked. The LECs from ACTb-EGFP mice are shown in pseudocolor green. The milk protein, whey acidic protein (WAP), is shown in pseudocolor yellow in the LECs and coating the inside of the organoids' lumens. Nuclei were stained with Hoechst dye. Images collected using a spinning disk confocal microscope and reconstructed in 3D using Imaris (E', E') or bottom section ~30 slices (F', F"). Scale bars = 100 µm (C), 20 µm (E), 40 µm (F). Please click here to view a larger version of this figure.
10 mL | Digestion Medium | |
Amount | Reagent | Notes |
9.45 mL | DMEM/F12 | |
100 µL | Antibiotic-Antimycotic (100X) | |
0.04 g | Class 3 Collagenase | |
0.04 g | Class 2 Dispase | |
50 µL | Gentamicin | Final Concentration: 500 µg |
2.5 mL | Fetal Bovine Serum | Final concentration: 5% (v/v) |
Pass through 0.22µm filter | ||
50 mL | Maintenance Medium | |
Amount | Reagent | Notes |
49.47 mL | DMEM/F12 | |
0.5 mL | Antibiotic-Antimycotic (100X) | |
2.5 mL | Fetal Bovine Serum | Final concentration: 5% (v/v) |
25 µL | Insulin | Final concentration: 250 µg |
5 µL | EGF | Final concentration: 500 ng |
10 mL | Growth Medium | |
Amount | Reagent | Notes |
9.6455 mL | DMEM/F12, no phenol red | |
100 µL | N-2 Supplement (100x) | |
200 µL | B27 supplement without vitamin A (50x) | |
10 µL | Nrg1 | Stock: 100µg/mL |
42.5 µL | R-spondin | Stock: 10 µg/mL |
1 µL | Rho inhibitor Y-27632 | Stock: 10 µM |
1 µL | EGF | Stock: 0.1 µg/µL |
10 mL | Alveologenesis Medium | |
Amount | Reagent | Notes |
9.6355 mL | DMEM/F12, no phenol red | |
100 µL | N-2 Supplement (100x) | |
200 µL | B27 supplement without vitamin A (50x) | |
10 µL | Nrg1 | Stock: 100µg/mL |
42.5 µL | R-spondin | Stock: 10 µg/mL |
1 µL | Rho inhibitor Y-27632 | Stock: 10 µM |
5 µL | Ovine Pituitary Prolactin | Final concentration: 1 µg/mL |
1 µL | Dexamethasone | Final concentration: 5 µg/mL |
5 µL | Insulin | Final concentration: 5 µg/mL |
1 L | 10X DPBS | |
Amount | Reagent | Notes |
80 g | NaCl | |
2 g | KCl | |
14.4 g | NaH2PO4 | |
2.4 g | KH2PO4 | |
1 L | di H2O | |
Fill to 800 mL before adding dry reagents and dissolve. Fill volume to 1 L. Adjust pH to 7.4. Autoclave to sterilize. | ||
1 L | 1X DPBS | |
Amount | Reagent | Notes |
100 mL | 10X PBS | |
900 mL | di H2O | |
1 L | PBST | |
Amount | Reagent | Notes |
100 mL | 10X PBS | |
2.5 mL | Triton X-100 | |
250 mL | 4% Paraformaldehyde | |
Amount | Reagent | Notes |
10 g | Paraformaldehyde | |
200 mL | di H20 | water must be at 60 °C |
25 mL | 10X DPBS | |
50 µL | 10 N Sodium Hydroxide | |
Pass through a 0.45 µm filter to sterilize and assure pH is 7.4 | ||
10 mL | 1 % Donkey Serum | |
Amount | Reagent | Notes |
100 µL | Sterile Filtered Donkey Serum | |
9.9 mL | 1X DPBS | |
10 mL | 0.2% Glycine | |
Amount | Reagent | Notes |
0.02 g | Glycine | |
10 mL | 1X DPBS |
Table 1: Solution recipes.
15 ml High-Clarity Polypropylene Conical Tube (BD Falcon) | Fisher Scientific | 352096 | |
24 well ultra-low attachment plate (Corning) | Fisher Scientific | CLS3473-24EA | |
35 mm TC-treated Easy-Grip Style Cell Culture Dish (BD Falcon) | Fisher Scientific | 353001 | |
50 ml High-Clarity polypropylene conical tube (BD Falcon) | Fisher Scientific | 352098 | |
60 mm TC-treated Easy-Grip Style Cell Culture Dish (BD Falcon) | Fisher Scientific | 353004 | |
70 µM nylon cell strainer (Corning) | Fisher Scientific | 08-771-2 | |
Antibiotic-Antimycotic (100X) | Thermo Fisher Scientific | 15240062 | Pen/Strep also works |
B27 supplement without vitamin A (50x) | Thermo Fisher Scientific | 12587010 | |
B6 ACTb-EGFP mice | The Jackson Laboratory | 003291 | |
BD Insulin syringe 0.5 mL | Thermo Fisher Scientific | 14-826-79 | |
Class 2 Dispase (Roche) | Millipore Sigma | 4942078001 | |
Class 3 Collagenase | Worthington Biochemical | LS004206 | |
Corning Cell Recovery solution | Fisher Scientific | 354253 | Follow the guidelines for use – Extraction of Three-Dimensional Structures from Corning Matrigel Matrix |
Corning Costar Ultra-Low Attachment 6-well | Fisher Scientific | CLS3471 | |
Dexamethasone | Millipore Sigma | D4902-25MG | |
DMEM/F12, no phenol red | Thermo Fisher Scientific | 11039-021 | |
DNase (Deoxyribonuclease I) | Worthington Biochemical | LS002007 | |
Donkey anti-Goat 647 | Thermo Fisher Scientific | A21447 | Use at 1:500, Lot: 1608641, stock 2 mg/mL, RRID:AB_2535864 |
Donkey anti-Mouse 647 | Jackson ImmunoResearch | 715-606-150 | Use at 1:1000, Lot: 140554, stock 1.4 mg/mL |
Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F12) | Thermo Fisher Scientific | 11330-057 | |
Dulbecco's phosphate-buffered saline (DPBS) | Thermo Fisher Scientific | 14190-250 | Without Mg2+/Ca2+ |
EGF | Fisher Scientific | AF-100-15-100ug | |
Fetal Bovine Serum | VWR | 97068–085 | 100% US Origin, premium grade, Lot: 059B18 |
Fluoromount-G (Southern Biotech) | Fisher Scientific | 0100-01 | Referred to as mounting media in text |
Gentamicin | Thermo Fisher Scientific | 15710064 | |
Glycine | Fisher Scientific | BP381-5 | |
Goat anti-WAP | Santa Cruz Biotech | SC-14832 | Use at 1:250, Lot: J1011, stock 200 µg/mL, RRID:AB_677601 |
Hoechst 33342 | AnaSpec | AS-83218 | Use 1:2000, stock is 20mM |
Insulin | Millipore Sigma | I6634-100mg | |
KCl | Fisher Scientific | P217-500 | |
KH2PO4 | Fisher Scientific | P285-500 | |
KRT14–CreERtam | The Jackson Laboratory | 5107 | |
Matrigel Growth Factor Reduced (GFR); Phenol Red-Free; 10 mL | Fisher Scientific | CB-40230C | Lot: 8204010, stock concentration 8.9 mg/mL |
MillexGV Filter Unit 0.22 µm | Millipore Sigma | SLGV033RS | |
Millicell EZ SLIDE 8-well glass, sterile | Millipore Sigma | PEZGS0816 | These chamber slides are great for gasket removal but other brands can work well (e.g. Lab Tek II). |
Mouse anti-SMA | Millipore Sigma | A2547 | Use at 1:500, Lot: 128M4881V, stock 5.2 mg/mL, RRID:AB_476701 |
N-2 Supplement (100x) | Thermo Fisher Scientific | 17502048 | |
NaCl | Fisher Scientific | S671-3 | |
NaH2PO4 | Fisher Scientific | S468-500 | |
Nrg1 | R&D | 5898-NR-050 | |
Ovine Pituitary Prolactin | National Hormone and Peptide Program | Purchased from Dr. Parlow at Harbor-UCLA Research and Education Institute | |
Paraformaldahyde | Millipore Sigma | PX0055-3 | |
Pentobarbital | Millipore Sigma | P3761 | |
R26R-EYFP | The Jackson Laboratory | 6148 | |
Rho inhibitor Y-27632 | Tocris | 1254 | |
R-spondin | Peprotech | 120-38 | |
Sodium Hydroxide | Fisher Scientific | S318-500 | |
Sterile Filtered Donkey Serum | Equitech-Bio Inc. | SD30-0500 | |
Sterile Filtered Donkey Serum | Equitech-Bio Inc. | SD30-0500 | |
Triton X-100 | Millipore Sigma | x100-500ML | Laboratory grade |
Trypsin EDTA 0.05% | Thermo Fisher Scientific | 25300-062 |
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.
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.
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.