TITLE: Protocol to generate Gastruloids (LSCB, EPFL)
AUTHORS: Stefano Vianello, Mehmet Girgin, Giuliana Rossi, Matthias Lutolf

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Gastruloid generation protocol as performed in the Lutolf Lab, EPFL.</div><div class = "text-block">For previously published protocols, see:</div><div class = "text-block"><span>Baillie-Johnson, Peter, et al. "Generation of aggregates of mouse embryonic stem cells that show symmetry breaking, polarization and emergent collective behaviour in vitro."</span><span style = "font-style:italic;">JoVE (Journal of Visualized Experiments)</span><span>105 (2015): e53252. </span></div><div class = "text-block">and</div><div class = "text-block"><span>Mehmet, G., et al. "Generating Gastruloids from Mouse Embryonic Stem Cells."</span><span style = "font-style:italic;">Protocol Exchange </span><span>(2018).</span></div></div>

[STEPS]
?. [Preparation of the cell suspension]
Using a vacuum line+glass pasteur pipette, aspirate out the culture medium and replace with  PBS-/- , for a short wash
3 ml
When removing liquid during washes, do not completely dry out the cells. The surface of the well should still look glossy.
?. [Preparation of the cell suspension]
Aspirate out the PBS, and replace with  Accutase.
500 µl
?. [Preparation of the cell suspension]
Let Accutase act for ~, , tapping the sides of the plate to ease dissociation,  and until most cells are floating (as clumps or single cells)
0 Room temperature
?. [Preparation of the cell suspension]
Slightly tilt the plate, and use a P1000 to pipette the suspension up and down to further break down cell clumps. Use each ejection to wash the surface of the plate, so to collect as many cells as possible.
?. [Preparation of the cell suspension]
Transfer the Accutase-cell suspension to a clean 15mL Falcon tube labelled "cells"
?. [Preparation of the cell suspension]
Use ~  DMEM-10%Serum to further wash the surface of the well, again, pipetting up and down and hoping to collect any previously missed cells. Transfer this secondary cell suspension in the same tube as before (total volume  )
4.5 ml
5 ml
?. [Preparation of the cell suspension]
Centrifuge  ( )  min
Centrifuge: 200 34
Centrifuge: 1000 33
4 °C
?. [Preparation of the cell suspension]
Using a vacuum line+glass pasteur pipette, aspirate the supernatant without disturbing the pellet,  redissolve the pellet in  PBS-/-, and centrifuge again   () min
10 ml
Centrifuge: 200 34
Centrifuge: 1000 33
4 °C
?. [Preparation of the cell suspension]
Repeat  for a second PBS-/- wash
?. [Preparation of the cell suspension]
Using a vacuum line+glass pasteur pipette, aspirate the supernatant without disturbing the pellet, and resuspend the pellet in  N2B27
1 ml
It is important to thoroughly resuspend the pellet at this point, as a single cell suspension is needed for accurate counting later on. Resuspend the pellet by pipetting several times (~20) with a P1000, then switching to a P200, and then to a P20
?. [Cell counting and Gastruloid seeding]
Load  of the N2B27-cell suspension into a manual cell counter (Neubauer chamber/haemocytometer), and calculate the concentration of cells in your suspension.
10 µl
Each well of the 96well plate will receive one   drop of N2B27. Each drop should contain 300 cells, i.e. a concentration of 300 cells /   = 7.5 cells/uLTo fill an entire 96well plate with   drops,     x 96 well =   N2B27 are needed.  For simplicity,   N2B27 suspension are prepared: at 7.5 cells/uL, this requires 37500 cells.
At this point, cells can also be used to seed a new culture and keep propagating the line. We plate from 65000 to 75000 cells in a well of a tissue-culture-treated 6well plate, filled with 2mL prewarmed DMEM/10%Serum+2i/LIF
?. [Cell counting and Gastruloid seeding]
To   fresh N2B27, add the volume of cell suspension carrying 37500 cells, as calculated before. (If you want to prepare more than one plate of gastruloids, scale the volume of N2B27 and of cells accordingly)
5 ml
?. [Cell counting and Gastruloid seeding]
Load the  N2B27-cell suspension into a multichannel pipette reservoir, and dispense  of this solution to each well of an ultra-low-adhesion 96well plate
5 ml
40 µl
Make sure that the cell suspension in the reservoir is always well mixed, to ensure homogeneous dispensing in the wells. At each transfer, pipette up and down several time with the multichannel, and slightly agitate the reservoir from time to time.
?. [Cell counting and Gastruloid seeding]
Lightly tap the plate against the surface of the hood to make sure all drops are at the very bottom of each well, doublecheck under the microscope for the presence of cells in the drops, and leave the plate in a humidified incubator, 5%CO2, undisturbed  hThis is considered the beginning of Day 1 (D1), t=0h-24h
?. [Growing Gastruloids]
Beginning of Day 2 (D2), t=24h-48hNo intervention needed at this timepoint. The Gastruloids will keep developing in their  of N2B27. Leave to grow  h more
40 µl
Gastruloids should look like a small (  ) cluster, still in the process of aggregating. Individual cells might still be visibile in the surroundings
?. [Growing Gastruloids]
Beginning of Day 3 (D3), t=48h-72hPrepare  N2B27, adding CHIR99021 to a final concentration of  . Load this into a multichannel pipette reservoir, and dispense  of this solution to each well of the plate (i.e. total volume of  per well ). Place back in the incubator for  h
16 ml
150 µl
190 µl
Gastruloids should look like a small, clean, translucent sphere of around   diameter. No additional (satellite) spheres should be seen around it.
?. [Growing Gastruloids]
Beginning of Day 4 (D4), t=72h-96hUsing a multichannel pipette, remove  of the medium from each well and replace with  fresh N2B27. Place back in the incubator for  h
150 µl
150 µl
Gastruloids are round spheres of around   in diameter. Extensive cell shedding is expected as a consequence of the CHIR pulse.
?. [Growing Gastruloids]
Beginning of Day 5 (D5), t=96h-120Using a multichannel pipette, remove  of the medium from each well and replace with  fresh N2B27. Place back in the incubator for  h
150 µl
150 µl
Gastruloids morphology is no longer symmetrical: they look like a ovoid with length of around  . The protruding part is more translucent than the rest, and this is the part that will elongate in the next 24h.At t=120h, the Gastruloid looks like a bowling pin with length of around   -  . A thinner extension protrudes at the "posterior", more translucent than the denser anterior (spherical).
Day 6 (D6) and Day 7 (D7), t=144h, 168hCulture can be extended up to 168h (i.e. 7 days total). This can be done by continuing the daily medium changes as above, or by transferring individual Gastruloids to separate wells of a 24well plate, in   fresh N2B27, and replacing half of the medium one day later. The plate is kept shaking on an orbital shaker, in the incubator for both additional days. If keeping the Gastruloids in the original 96well plate, no shaking is required but there is an increased risk of the Gastruloids adhering to the sides of the well and degenerating.