3D culture: preparation and differentiation
The preparation should be done inside a sterile biosafety cabinet. Ensure that all media is warmed to 37 °C especially when using thermoreversible gel.
1. Place aliquots of Matrigel, hydrogel (e.g. Mebiol gel) and an empty 2 mL tube on ice.
2. Dissociate hESCs with Accutase as single cells (Stem Cell Technologies). Spin cells at 300 g for 3 min, and resuspended with hESC media to 1–2x106 cells/mL. We used mouse embryonic fibroblast conditioned media (MEF-CM) supplemented with 20 ng/mL bFGF whose preparation is described in10.
3. Add ~30 µL of cell suspension to the empty tube on ice. Mix in 30 µL Matrigel and 150 µL hydrogel (in that order) and aspirate-release a few times with the P1000 pipette tip to disperse the cells throughout the gel until the mix appears homogenous. Ensure to minimize bubbling.
4. Quickly transfer the cell-gel mix to the bottom of a 35-mm dish in a snake-like pattern, covering most or the entire surface. If doing live-cell imaging, ensure to use cell culture imaging dish, such as the Ibidi 35-mm µ-dish (Ibidi #81156).
5. Transfer the dish to the incubator at 37 °C and 5% CO2 for ~5 minutes to solidify the gel.
6. Add 1.5 mL of hESC media (in our case, MEF-CM + 20ng/mL bFGF) supplemented with 10 μM ROCK inhibitor Y27632 and optionally with pen/strep or equivalent antibiotic for ~24 h. The media should completely cover the gel.
7. 24h after seeding, replace medium with hESC medium (in our case, MEF-CM + 20ng/mL bFGF) optionally supplemented with pen/strep and let grow for additional 1–2 days. The gel should be solid, but take care when aspirating the medium. In case the gel appears loose or pieces of it have broken off, collect the medium using a wide bore tip or serological pipette, spin down at 100 g for 90 s and resuspend in new media.
8. Refresh media and add morphogen. For example, to treat with intermediate BMP4 concentration, as the majority of the work in the accompanying manucsript9, add 1 ng/mL BMP4 for 24–48h. If the results will be assessed with immunofluorescence (IF) staining, place the dish in the incubator and do not disturb it until the end of the experiment. To perform live-cell imaging, place the dish in the environmental chamber atop a microscope and commence imaging at a desired time. It is best to image 3D colonies suspended above but as close to the surface as possible.
3D culture: IF staining
The IF staining can be performed at the bench, except when handling PFA which should be done in a chemical hood.
1. Wash dish once with DPBS +/+ then add 4% PFA. Typically at this stage the gel is still solid, however if it is loose or broken off, collect media, spin down at 100 g for 90 s, resuspend in DPBS+/+ then repeat and resuspend in 4% PFA. Keep in PFA for 30–60 min.
2. Remove PFA and wash once in DPBS+/+. Repeat the spin down cycle if gel loosens. Block with 2% BSA (m:v) in PBST (PBST: 0.1% Triton-X in DPBS+/+). Keep in block for at least 1h in the fridge. The sample can stay in the block solution in the fridge for at least two weeks until ready for staining.
3. By this point, most of the gel will loosen and in the case of thermoreversible gel like Mebiol, all the gel will liquefy. Perform the spin down cycle in between each wash step from this point onward. Do not aspirate the supernatant between spin downs with a vacuum aspirator; instead, gently remove it with a P1000 pipette. Replace media with primary antibodies dissolved in the block solution and keep overnight in the fridge. The staining time can be adjusted based on the primary antibody.
4. Wash twice in PBST.
5. Resuspend in secondary antibody and DAPI dissolved in block solution for 30 min at room temperature in a dark chamber.
6. Wash secondary antibody twice in PBST, move the sample to an imaging dish and keep in PBST.