Before starting gastruloid protocol
2D cell culture
Mouse embryonic stem cells (mESCs) are grown in standard conditions, on Gelatin (0.1%) coated 6-well plates, in ESLIF and without MEFs in a humidified incubator at 37 °C and 5% CO2. For efficient gastruloid formation, it is important that the cells are split and fed regularly, and are never grown too confluent or too sparse. We generally split our cultures ~1:5 every two days to keep the cultures between ~20 and 100% confluency, and feed the cells on days on which they don’t need splitting.
Aliquoting of N2B27:
1. Thaw the bottle(s) of N2B27 at 37 °C in the water bath, shake occasionally, and remove them from the water bath as soon as the last piece of ice has melted to prevent the formation of protein aggregates.
2. Vigorously shake the bottle and store at 4 °C overnight.
3. The next day, shake the bottle vigorously and check if there are any precipitates (if desired, take a sample and check for protein aggregates under the microscope).
4. Aliquot in 50 ml tubes (max. 35 ml per tube to prevent medium from leaking out upon freezing) and store at -20 °C.
Note: if the medium is thawed incorrectly, protein aggregates (visible as strings in the medium) will form. These aggregates do not inhibit gastruloid formation, but can be inconvenient during gastruloid imaging experiments.
Preparing N2B27 aliquots for gastruloid culture:
1. Thaw the required number of aliquots 2 days prior to gastruloid formation by placing them on a rotator at 4 °C overnight.
2. Transfer the thawed medium to a T175 cell culture flask 1 day prior to gastruloid formation, and place the flask in the cell culture incubator with the lid slightly open to allow the pH of the N2B27 to adjust overnight.
Note: make sure that the cells are at ~80-85% confluency when starting gastruloid formation.
1. Pre-warm PBS, ESLIF medium, and Trypsin to 37 °C in a water bath.
2. Aspirate the medium from the cells and wash the cells 2x with PBS.
3. Add 1 ml pre-warmed Trypsin to the well of the 6-well plate and incubate for approx. 3 minutes in the incubator to dissociate the cells.
4. Dissociate the cells into a single-cell suspension using a P1000 pipette, and transfer to a 50 ml Falcon tube.
5. Add 4 ml of ESLIF medium to neutralize the Trypsin.
6. Spin down at 200 g for 3 mins.
7. Remove the supernatant and wash the cell pellet with 5 ml PBS.
8. Spin down at 200 g for 3 mins.
9. Carefully remove PBS and add another 5 ml PBS for a second wash.
Note: don’t skip this step – the second wash step is essential for the generation of reproducible aggregates.
10. Spin down at 200 g for 3 mins.
11. Carefully remove all PBS without disturbing the cell pellet.
Note: Leftover PBS can inhibit the formation of aggregates. We typically use a P200 pipette to remove the last few μl of PBS.
12. Resuspend the cell pellet in 1 ml N2B27 (from incubator, see “Preparing N2B27 aliquots for gastruloid culture”-instructions above) using a P1000 pipette.
13. Add a few ml of N2B27 (from the incubator) to dilute the cells and simplify counting. We typically dissolve the cells from 1 well of a 6-well plate in a total of 5 ml of N2B27.
Note: the pH of N2B27 quickly changes once it is taken out of the incubator. It is therefore important to try to work as quickly as possible once the cells are resuspended in N2B27. Place the T175-flask with N2B27 in the incubator in between steps to prevent the pH (and colour) from changing too much.
14. Count cells to determine the cell concentration. We use a Bürker-Türk (Marienfeld) counting chamber for this.
15. Dilute the cells to the required concentration in N2B27 medium in a new Falcon tube.
Our aggregates are typically made using 300 cells, meaning that we add 300 cell to every well of the 96-wells plate. To achieve this, for every 96-well plate we add 37.500 cells to 5 ml of medium (to obtain a cell concentration of 7.5 cells/μl), of which 40 μl (with 300 cells) is then added to each well of the 96-well plate.
16. Distribute 40 μl of the cell suspension into U-bottomed, non-adherent 96-well plates. To this end, transfer the solution into a sterile tray and distribute using a multichannel and DNA LoBind tips. Cover the plate with its corresponding lid.
Note that this pipetting step is more accurate (less variation in volume per tip) when DNA LoBind tips instead of regular tips are used.
17. Make sure that the drops are in the centre of the wells and not sticking to the walls instead (gently tapping the side of the plate might be needed to achieve this), confirm the presence of cells with a microscope and incubate the plate in an incubator (37 °C, 5 % CO2) for 48 hours.
Chiron pulse (48 hours after aggregation)
18. For each 96-well plate, prepare 16 ml of N2B27 supplemented with 4.8 μl 10 mM Chiron (final concentration of 3 μM) in a Falcon tube.
19. Pour the medium-Chiron mix into a tray and add 150 μl of this mix to all wells using a multichannel and DNA LoBind tips. Hold the pipette at an angle, push the pipette tips to the side of the wells and pipette the medium out with some force (without spilling over) to dislodge the gastruloids from the plate. This is important for reliable gastruloid formation. Place the plate in the incubator for an additional 24 hours.
Note: if the gastruloids are not dislodged from the bottom of the place properly, try to add 75 μl twice instead of 150 μl once, so that more force can be applied without spilling.
Medium change (72 hours after aggregation)
20. For each plate, you will need 16 ml of N2B27.
21. Carefully remove 150 μl medium per well using a multichannel pipette. Hold the pipette at an angle and slightly press against the wall of the wells to prevent loss of gastruloids.
22. Add 150 μl fresh N2B27 per well using the multichannel pipette, and place the plate back in the incubator for an additional 24 hours. Again, use some force to dislodge the gastruloids (you may want to add two times 75 μl if the aggregates are difficult to dislodge from the plate).
To generate gastruloids that do not form somite-like structures:
Second Medium change (96 hours after aggregation)
23. Repeat steps 21 and 22.
This will result in “standard gastruloids”. The protocol to generate such gastruloids is very similar to previously published gastruloid protocols5. If the protocol is performed successful, these gastruloids will be similar to E8.5 mouse embryos at 120 hours after aggregation1,4,5. Such gastruloids do however not generate somite-like structures.
To generate gastruloids that do form somite-like structures:
To induce somite formation, gastruloids are embedded in Matrigel at day 4 (96 hours after aggregation). In our hands, somite formation works best with gastruloids that are embedded in low percentages (10-25%) of Matrigel (option 1). However, somite-like structures were also observed in gastruloids embedded in higher (50-100%) percentages of Matrigel (option 2). For additional details regarding the effect of various concentrations of Matrigel, see Extended Data Fig. 9c of associated manuscript, van den Brink et al., Nature, 20207).
Option 1: Embed gastruloids in 10-25% Matrigel (96 hours after aggregation)
23. Thaw Matrigel on ice.
24. Pool the gastruloids in a 5 ml low binding Eppendorf tube on ice. We typically pool one plate of 96 gastruloids into three 5 ml tubes.
25. Replace the N2B27 medium in the tube with fresh cold N2B27 (from incubator, as described in the “Preparing N2B27 aliquots for gastruloid culture”-instructions above, and then placed on ice in a Falcon tube for few minutes). Add 3.6 ml N2B27 per tube when embedding in 10% Matrigel, and 3 ml per tube when embedding in 25% Matrigel.
26. Add the correct volume of Matrigel to the tube (400 μl per tube when embedding in 10% Matrigel; 1 ml per tube when embedding in 25% Matrigel).
27. Mix suspension well by pipetting up and down a few times with a P1000 pipette from which ~3mm of the tip is cut off with sterile scissors, and distribute 500 μl of this mix per well over 8 wells of a 24-well plate (see “REAGENTS section” for two examples of suitable plate types) using the P1000 pipette with the tip cut off. Mix suspension before pipetting into each well to prevent gastruloids from settling at the bottom of the tube. When this procedure is performed correctly, there will be approximately 4 gastruloids in every well.
28. Leave the plate on the bench for ~1 minute so that gastruloids settle to the bottom.
Note: if the plate is moved before the gastruloids are settled, gastruloids will clump together in the centre of the well. To avoid this, make sure that the plate is left on a bench untouched for 1 minute before putting the plate in the incubator.
29. Place the plate in the incubator for an additional 24 hours.
Note: if the gastruloids are still all located in the centre of the well, gently agitate the plate to distribute them over the well before putting the plate into the incubator.
Option 2: Embed gastruloids in 50-100% Matrigel (96 hours after aggregation)
23. Thaw Matrigel on ice, and place a falcon tube with N2B27 (from incubator) on ice.
24. Mix the Matrigel with the required amount of cold N2B27 medium (none for embedding in 100% Matrigel; 50% of the required volume for embedding in 50% Matrigel).
25. Add 60 μl of the Matrigel-N2B27 mix to each well of a 24-well plate on ice.
We typically put a metal block on ice and place the plate on top of this cold metal block. Make sure that the plate is cold before adding Matrigel.
26. Transfer gastruloids one by one into the Matrigel using a P20 pipette. To make sure that only a single gastruloid is added with as little medium as possible, use a dissection microscope when taking gastruloids from the plate.
27. After adding all gastruloids to the Matrigel, incubate the plate at 37 °C for 10 mins to solidify the Matrigel.
28. When the Matrigel is solid, add 500 μl N2B27 medium (37 °C; pre-incubated in the incubator for pH equilibration as described in the “Preparing N2B27 aliquots for gastruloid culture”-instructions above) to each well.
29. Place the plate in the incubator for an additional 24 hours.
Fixation of gastruloids grown in Matrigel (120 hours after aggregation)
30. Add PBS0 to the wells without removing the medium/Matrigel.
31. Remove the medium/PBS0 and wash again with PBS0.
Note: do not yet try to actively remove gastruloids from the Matrigel during this step.
32. Incubate in 4% PFA/PBS overnight at 4 °C by adding ~500 μl 4% PFA/PBS to every well of the 24-well plate.
33. Wash 3 times for 5 min in PBS-Tween (0.1% Tween-20 (v/v)).
34. Transfer gastruloids to 1.5 ml DNA LoBind tubes using a P1000 pipette from which ~3mm of the tip is cut off with sterile scissors. For gastruloids grown in 10-25% Matrigel: some gastruloids might still be embedded in the Matrigel and/or attached to the bottom of the plate in this stage of the protocol. If this is the case, pipette PBS next to the gastruloids with some force in order to dislodge them. If this doesn’t work, use a spatula to remove the gastruloids from the plate. For gastruloids grown in 100% Matrigel: use a spatula to scrape the gastruloids from the plate bottom. Leftover pieces of 100% Matrigel that will that might still be attached to the gastruloids after this step should dissolve after the proteinase K digestion and the overnight incubation step in hybridization buffer at 68 °C in the staining protocol.