Fabrication of microwell arrays
The polydimethylsiloxane (PDMS) stamps are formed through replica molding using microstructured silicon wafers. Wafers are processed via deep reactive ion etching as we previously described in several publications 9–11. This fabrication process requires the use of a clean room and special equipment accessible in numerous universities. Once the PDMS stamps are produced, these are used to routinely imprint hydrogel microwell arrays.
Microwell arrays (1000 microwells) are produced in 6 well-plates and then cut and inserted into 12-well plates. Alternatively, microwell arrays are directly imprinted into 96-well plates (400 microwells). Microwells have a diameter and a height of 200 μm. The geometry of the interspace is shown in Figure 1a and allows to extrapolate all dimensions of the microwell array.
Culture of embryonic stem cells.
The ESCs are maintained on a gelatin coating in medium #1 (2i medium) for 11 days. A bank of 50 vials containing 0.5 million cells per vial is prepared. When a blastoid experiment is started, a vial is defrosted and plated on gelatin in medium #1. After two days of culture, the cells are passaged, seeded at 15.000 cells/cm2 and cultured for two days. In total, ESCs are thus cultured for 15 days in medium #1 before starting the blastoid experiment. This leads to the formation of tightly packed, medium-sized colonies with clear edges (see Figure 1b). Note that different ESCs lines can grow at different speed and that the initial seeding density must be adjusted accordingly to obtain such colonies.
Culture of trophoblast stem cells.
The TSCs are maintained on Matrigel® coatings in medium #3 (TX medium5). The 10 ml bottle of Matrigel® is defrosted by standing overnight in a fridge and aliquoted in 1 ml Eppendorf tubes. Note that both the tubes and the pipet tips must be previously cooled down in a freezer and the aliquoting done on ice to prevent the premature gelification of the Matrigel. The stock of tubes containing Matrigel® is frozen. One tube is defrosted, the day before starting the TSC culture, by standing overnight in a fridge. The tube can then be maintained defrosted in a fridge for several weeks. Matrigel® is then resuspended 1/90 v/v in ice-cold DMEM/F12. The suspension is added to the cell culture dish (1ml/10cm2) and incubated for one hour inside a lamina flow hood, at room temperature. The Matrigel® suspension is then removed and the TSCs are seeded in medium #3. TSCs previously culture with serum12 are maintained in medium #3 for 3 weeks before making a bank. A bank of 50 vials containing 0.5 million cells per vial is then prepared. When a blastoid experiment is started, a vial is defrosted and plated on Matrigel® in medium #3. After two days of culture, the cells are passaged, seeded at 15.000 cells/cm2 and cultured for four days. Note that, similar to ESCs, different TSCs lines can grow at different speed and that the initial seeding density must be adjusted accordingly to obtain colonies as represented in Figure 1b.
Formation of blastoids.
The ESCs are washed with PBS, trypsinized for three minutes to form a single cell suspension. The trypsin is inhibited using the soya bean solution and the cells are carefully suspended in medium #2 (serum medium). Cell concentration is counted using a counting chamber. Cells are then seeded on top of the microwell arrays with a cell concentration resulting in the pooling of a mean of five cells per microwell. One ml of medium #2 is used for the aggregation of the ESCs. Within 24–36 hours, the cells form tight, round aggregates. All cells participate to the formation of the aggregate (see Figure 1b).
After 24-36 hours, the TSCs are then washed with PBS, trypsinized for three minutes to form a single cell suspension. The trypsin is inhibited using the soya bean solution and the cells are carefully suspended in medium #4 (blastoid medium). Cell concentration is counted using a counting chamber. The medium #2 is largely removed from the microwell arrays and the TSCs are then seeded on top of the microwell arrays containing the ESCs aggregates, with a cell concentration resulting in the pooling of a mean of 12 cells per microwell. Upon settling of the cells within the microwells (15 minutes), one ml of medium #4 is added.
Nota Bene: The difficulty is to synchronize the two cell cultures so that both cultures are ready, in a perfect state, for the experiment. The initial state of the cells (e.g. confluency, morphology of the colonies) is crucial for the success of the experiment.
The time of TSCs seeding is considered as the starting point (0 hours). Within 24 hours, the TSCs aggregate with the ESCs and initiate cavitation. At 24 hours, 1 mM 8Br-cAMP in a 10 ul of F12 medium is added to each microwell array. Within 48 hours, 2–10% of the aggregates formed a blastoid, which expanded and stabilized by 65 hours. A blastoid is defined based on morphological parameters of E3.5 blastocysts, as a cystic structure with an outer circularity superior to 0.9 (circularity = 4pi(area/perimeter2), and a diameter comprised between 70 and 110 micrometers, including a single regular cavity lined by a single layer of TSCs and including ESCs (Figure 1c).
Trophospheres are obtained by seeding TSCs (mean of 12 cells per microwell) on top of microwell arrays that do not contain a ESCs aggregate, in the same medium as used for blastoids (Figure 1c).
Manipulation and dissociation for single cell analysis
Upon formation, blastoids can be harvested using a mouth pipet holding a glass capillary, just like blastocysts. Under a stereomicroscope, specific blastoids can be harvested from individual microwells and pooled into round-bottom 96 well-plates for fixation or dissociation.
Blastoid dissociation:
- Prepare dissociation capillaries of different sizes (75-150 um).
- Warm up the incubator/shaker to 37deg.C and set up at ~350RPM. Warm up the hotplate.
- Defreeze and warm up the Collagenase IV, TrypleE, RNA-free BSA and maintain them in the water-bath.
- Prepare 15ml of PBS + 2% FBS (PBS+)
- Prepare 1mL of PBS + 1uL of Hoechst
- Add 150uL PBS in two rows of the 96 well plate; 50uL of collagenase IV 600U/ml in a third row; and 100uL of RNA-free BSA in a fourth row (These wells will be used for TrypleE dissociation)
- Take 150 blastoids / EBs / Trophospheres out of the microwells and wash them with 2x in PBS-0 (without Ca/Mg)
- Deposit blastoids / EBs / Trophospheres in 50 uL of Collagenase IV 600U/ml. Tape the plate at the bottom of a plastic box and place the box in the bacteria shaker, at 350RPM for 30 minutes.
Nota Bene: using bigger volume (> 50uL) reduces the mixing as it is the surface of the liquid, down to a certain depth, that moves. The 96 well-plate must be placed into a plastic box to prevent contamination. The speed of shaking can be tested using 40 uL of PBS + 10 uL of Laemmli buffer (blue ladder for SDS page): the buffer forms a disc at the bottom of the well that, above a certain speed, mixes with the PBS.
- During that time, trypsinize ESC and TSC from the 6 well-plate 2D culture. Block with the trypsine inhibitor. Spin down and re-suspend in 3mL of PBS+. Add 1mL to each FACS tube. Place on ice while waiting to FACS.
- Transfer the blastocysts to TrypleE 10X (no dilution) and shake for 20 minutes
Nota Bene: Upon collagenase treatment, blastoids will stay intact. However, they will collapse after TrypleE treatment. Blastoids / EBs / Trophospheres will gather into one cluster, in the center of the 96 well.
- Bring to the stereomicroscope and use a large capillary to dissociate the large cluster, and a small capillary to dissociate into single cells
Nota Bene 1: Capillaries of different diameters should be available. EBs necessitate large dissociation capillaries only. Blastoids and trophospheres necessitate capillaries with intermediate diameter between the ones used for EBs and for single cells.
Nota Bene 2: EBs will give a lot more cells than blastoids. Blastoids will give more cells than trophospheres, which are the most difficult to dissociate.
- Prepare a 1mL Eppendorf with 200uL of Trypsine inhibitor.
- Harvest the cells with a P200 and place in the Eppendorf. Wash each well 3 times with PBS+ and also add in the Eppendorf.
- Harvest with a P1000 and push through the filter of a FACS tube
- Add 30 uL of pre-diluted Hoechst (1/400)
- Place on ice and FACS as soon as possible.