Culture Conditions Prior to Aggregation:
Maintain mESCs in ESLIF medium (see Reagents & Equipment) on 0.1% gelatin-precoated tissue culture-treated plastic (e.g. 25cm2 flasks) in a humidified incubator (37°C, 5% CO2).
Passage the mESCs to new flasks every other day; exchange 50-66% of the culture medium for fresh, pre-warmed medium on alternating days.
Culture the mESCs for at least two passages post-thawing before experimental use. Stocks that have been maintained in vitro for more than 30 passages may produce more variable results.
Culture cells to 40-60% confluence at the point of passaging or experimental use. A density of 1.8x104 cells/cm2 is recommended, but this should be optimised for each cell line.
Routinely test all cell lines for Mycoplasma contamination and maintain stocks under antibiotic free conditions to quickly identify microbial infections.
0 hours: Preparation of Gastruloids from mESCs and miPSCs (one 96-well plate)
Note 1: The following protocol describes generation of gastruloids from mESCs and miPSCs. Gastruloids can be reproducibly generated with mESC and miPSC lines from genetic backgrounds that include the 129 strain. The culture requirements may differ for mESCs and miPSCs (e.g. the use of ES+LIF Medium1 or 2, respectively). Such differences can also be observed between different mESC lines from different genetic backgrounds.
Note 2: Gastruloids derived from miPSCs generally require higher starting cell numbers (e.g. 600-800 cells/well) compared to mESC-derived gastruloids (e.g. 300 cells/well). The optimum starting cell number should be defined empirically.
Note 3: Gastruloids can be formed successfully from cells cultured in 2i conditions (N2B27 + 3µM Chi + 1µM PD0325901 + LIF), although the process of elongation is slightly delayed with respect to cells from ESL medium.
1. Pre-warm PBS, ESLIF, N2B27 and Trypsin-EDTA in a 37°C waterbath.
Optional: If maintaining the cell stock, pre-coat a T25 tissue culture flask with 5mL 0.1% Gelatin in PBS.
2. Aspirate the medium from the T25 tissue culture flask and rinse gently with 5mL PBS, twice.
3. Aspirate the PBS and add 1mL of pre-warmed Trypsin-EDTA.
4. Rock the flask to detach the colonies of cells. If required, strike the wall of the flask gently a few times to aid detachment, or incubate the flask at 37°C for 30 seconds.
5. Dissociate the colonies into a single cell suspension with a P1000 micropipette tip by ejecting the suspension forcefully against a wall of the flask.
CRUCIAL: A single cell suspension is essential for accurate cell counting. Errors in counting affect the size of the gastruloids, which is known to affect the level of axial organisation (see ).
6. Neutralise the Trypsin-EDTA with 5mL ESLIF and transfer to a centrifuge tube.
7. Centrifuge the suspension for 3 minutes at approximately 170 x g.
8. Aspirate the supernatant and add 5mL warm PBS.
Tip: The pellet should become resuspended by the addition of the PBS. In order to avoid the loss of cells through transfer errors, drawing the suspension into the pipette is discouraged.
9. Centrifuge the suspension for 3 minutes at approximately 170 x g.
10. Aspirate the supernatant and add 5mL warm PBS.
11. Centrifuge the suspension for 3 minutes at approximately 170 x g.
12. Aspirate the PBS, minimising carry-over by tilting the tube to remove as much as possible while leaving the pellet intact.
13. Fully resuspend the pellet in 1mL N2B27 using a P1000 micropipette.
Optional: If required, dilute this suspension further in N2B27 to facilitate cell counting. Work with the newly diluted suspension for subsequent steps.
14. Load the haemocytometer (or the counting slide of an automated cell counter) with 10μL of cell suspension and determine the density of the suspension.
15. Determine the volume of suspension required to produce a cell concentration of 7.5 mESCs/μL or 20 miPSCs/μL in N2B27.
E.g. 3.75x104 mESCs or 10x104 miPSCs in a final volume of 5mL N2B27 is sufficient for a single 96-well plate, plus a small amount of dead volume; this gives 300 mESCs or 800 miPSCs per 40μL drop after plating.
Tip: The number of cells per aggregate is adjusted empirically for each cell line to give aggregates of around 150μm diameter at the 48 hour time point. Tables for commonly used cell lines can be found in  and .
16. Add the calculated volume of suspension to the required amount of N2B27, mix well and transfer to a sterile reservoir.
Tip: Vortexing the suspension prior to plating or pipetting it up and down within the reservoir can ensure that the cells are well mixed.
17. Pipette 40μL of plating suspension into each well of a sterile, U-bottomed non-tissue culture-treated 96-well plate with a multichannel micropipette.
Tip: Take care to position the droplets in the bottom of each well and not clinging to the walls, as U-shaped droplets are required for efficient aggregation.
18. Confirm that cells can be seen within each well using the inverted benchtop microscope.
19. Return the plate to the incubator for 48 hours.
Optional: If maintaining the cell stock, aspirate the Gelatin from the T25 and add 6mL pre-warmed ESLIF medium. Calculate the required volume of the cell suspension for 4.5x105 cells and add this to the ESLIF medium (there is a small carry-over of N2B27).
48 hours: Addition of Secondary Medium
20. Pre-warm N2B27 in the 37°C waterbath.
21. Prepare secondary medium as a 3μM Chiron solution in N2B27. 16mL is ample for a single plate.
Tip: Using secondary media with different compositions will yield different results that are often apparent as changes in morphology (see  and ).
22. Transfer 150μL of secondary medium to each well using the multichannel micropipette and a sterile plastic reservoir.
23. Return the plate to the incubator for a further 24 hours.
72 hours: Removal of Secondary Medium
24. Pre-warm N2B27 in the 37°C waterbath.
25. Carefully remove 150μL of the secondary medium from each well using the multichannel micropipette, holding it at an angle to aspirate slowly from the side of each well.
26. Add 150μL of fresh, pre-warmed N2B27 to each well using the multichannel micropipette.
Tip: Add the fresh medium with sufficient force to move the gastruloids within the well, thereby avoiding adhesion to the plastic.
27. Return the plate to the incubator for a further 24 hours.
96 hours: Change of Medium
28. Repeat steps 24-27 to exchange 150μL culture medium for fresh, pre-warmed N2B27.
120 hours: Change of Medium
29. If maintaining the culture, repeat steps 24-27 to exchange 150μL culture medium for fresh, pre-warmed N2B27.
Extended Culture (120-168h):
30. To prolong the culture, transfer the gastruloids individually into low-attachment 24-well plates in 700μL volumes of fresh N2B27 at 120 hours.
Tip: Cut a P1000 micropipette tip approximately 5mm from the end and use this to collect and transfer the gastruloids with minimal damage.
31. Incubate on an incubator-compatible orbital shaker for 48 hours at 40 RPM.
32. Replenish the medium after 24 hours (144 hours) by exchanging 400μL medium for fresh N2B27.