Human pluripotent stem cell (hPSC) culture:
· hiPSCs are maintained in the pluripotency medium mTeSR Plus supplemented with 1x penicillin-streptomycin (P-S) on hESC-qualified Matrigel (1:100 dilution; Corning) at 37oC, 5% CO2. Cultures are passaged 1:6 in 6-well plates every 4-7 days depending on confluency and growth rate of the cell line in question using Gentle Cell Dissociation Reagent (GCDR). For EMLO formation, low passage number is ideal.
· hPSC lines are cryopreserved in mFreSR cryopreservation medium according to manufacturer’s instructions.
· In general, 6 well plates are used hPSC culture, passaged 1 well to 6 wells (1:6) at ~75%
Establishing hPSC cultures for EMLO induction:
1. Prepare hESC-qualified Corning Matrigel-coated 6-well plates by diluting Matrigel 1:100 in ice-cold DMEM/F-12.
a. Add 1 ml per well to pre-chilled tissue culture-treated plates and incubate at 37oC in a humidified incubator with 5% CO2 for 1.5-2 h prior to passaging.
2. Bring mTeSR Plus medium to room temperature without using a water bath.
3. Establish 2D hiPSC cultures for induction.
a. Passage the stem cells with GCDR: aspirate the cell culture medium, add 1 ml of GCDR to the well(s) to be passaged. Note, this well is passaged 1:6, so depending on the experiment multiple wells can be passaged for scale-up. However, a substantial number of EMLOs can be generated from a small starting culture batch (1-4 wells of hPSCs).
b. Incubate cells in GCDR for 3 min at room temperature. This incubation time requires cell line-specific optimization according to the manufacturer’s instructions.
c. While the cells are incubating in GCDR, use a 5 ml serological pipette to aspirate the Matrigel-DMEM/F12 solution from the coated 6-well plate and add 1 ml room temperature mTeSR Plus medium to each well.
d. At the end of GCDR incubation, aspirate the reagent and add 3 ml mTeSR Plus to the well that is being passaged. The additional wells containing hPSC colonies can be stored, passaged for continued hPSC culture, or used for other parallel experiments being performed. In the well containing 3 ml mTeSR Plus, use a 5 ml serological pipette to gently dislodge the treated colonies. Orient the serological pipette orthogonally to the plane of the plate and perform a forward/backward scraping motion over the entire area of the well, then rotate the plate 90 degrees and repeat to ensure all cells are dislodged from the substrate.
e. Using a P-1000 blue tip, pipette the suspension up and down 2-3 times before transferring 0.5 ml to each well of the 6 well plate already containing 1 ml/well. Avoid over-pipetting in order to maintain small aggregates. Return the coated plates with cell suspensions to the incubator and incubate overnight allowing colonies to adhere.
f. The next day, visually inspect cultures for small colony adherence and, if positive, aspirate the 1.5 ml mTeSR, rinse 2x in 1 ml DMEM/F-12 to remove non-adherent colony debris and add 2 ml fresh room temperature mTeSR Plus to each well. Return the plate to the incubator and maintain this culture with fresh media changes when necessary until the cultures are approximately 50-60% confluent. Since the colonies are not dissociated and reseeded as single cells on Matrigel prior to 2D induction, this confluency has been found to be optimal.
2D induction of hPSC colonies for EMLO formation:
4. When cultures reach ~50-60% confluency, they are ready for induction in N2B27 medium supplemented with 3 μM CHIR 99021, 40 ng/mL FGF2. Note that the CHIR 99021 concentration is critical for induction and the optimal concentration varies within the tight window of 3-3.5 μM. In our study, we applied 3 μM CHIR 99021 to all cell lines without line-specific optimization, but formation efficiencies may be further improved at this step. Prepare 10 ml of N2B27 with these added factors and warm to room temperature without a water bath.
5. At the time of induction, aspirate mTeSR Plus medium and rinse 2x with DMEM/F-12. Add 2 ml N2B27 + 3 μM CHIR 99021 + 40 ng/mL FGF2 (Induction Medium) per well depending on how much material is required for downstream experiments. One well can generate hundreds of EMLOs. Other wells can be maintained in mTeSR for further passaging or other experiments.
6. Return the plates to the incubator and repeat this step 24 h later after visual inspection. Depending on the cell line, the CHIR 99021 induces a rounding-off of colonies as transcriptional programs begin to transition away from pluripotency maintenance. For EMLOs, we use a 48 h induction duration. Ensure that FOXA2 expression is not lost by IF. In addition to the rounding-off of colonies during induction, there is also a loss of the well-demarcated hPSCs within the colony into a more “blended” appearance (Figure 2). If maintained under these conditions, the edges of the colonies will begin to develop a raised character around the perimeter along with cells that begin to migrate away from the colony border. The typical ideal point at which to proceed to the next step (dissociation and transition to orbital shaking culture) is between these two time/morphological benchmarks.
Transition to orbital shaking culture and EMLO maturation:
At 2 days (48 hr) after induction with N2B27 + CHIR/FGF2 as adherent 2D colonies, cultures are ready to generate single cell suspensions for direct transition to orbital shaking culture.
Prior to dissociation:
7. Prepare 10 (or 20 ml) N2B27 freshly supplemented with 10 ng/ml FGF2, 2 ng/ml HGF, 2 ng/ml IGF-1, and 50 μM ROCK inhibitor (Y-27632). Note, this is 5x the typical concentration of ROCK inhibitor used for routine cell passaging. The higher ROCK inhibitor concentration is useful to ensure cell survival, promote aggregation, and to induce the neural crest cell lineage. Warm freshly prepared medium to room temperature.
8. Dilute Accutase 1:1 in HBSS (CM-free) for enzymatic dissociation and warm to room temperature.
9. Pre-treat tissue culture-treated 6-well plates with Anti-Adherence Rinse Solution (STEMCELL Technologies) for 10 min. Alternatively, use ultra-low adhesion 6-well plates.
10. After 10 min, aspirate the rinsing solution and rinse twice with 2 ml HBSS. The second rinse can be kept in the wells while cells are dissociated to prevent drying out.
11. Aspirate medium from the induced wells and rinse 2x in 1 ml DPBS or HBSS, then add 1 ml Accutase diluted 1:1 in the HBSS CM-free and return to the incubator for 5 min.
12. At 5 min, if the cells remain adherent, aspirate the enzyme solution and add 1 ml N2B27 basal medium (no supplements) per well. Perform the same serological pipette scraping technique as described above for passaging hPSCs in GCDR. If cells detach during the 5 min incubation time with Accutase, add 1 ml of culture medium and perform a centrifugation step in 2 ml tubes at 350 x g for 5 min to remove the enzyme and collect the cells.
13. Using a P-1000 blue tip, pipette gently to generate a single cell suspension. Dissociation to single cells occurs quickly and can be monitored by visual inspection under a tissue culture microscope. Combine the wells and add the entire volume to a 15 ml conical tube, centrifuge at 350 x g for 5 min.
14. Aspirate the N2B27 basal medium from the cell pellet and dilute in the appropriate volume of N2B27 supplemented with the ROCK inhibitor, FGF2/HGF/IGF-1 so that each well of the 6-well plate contains 2 x 106 cells in 2 ml medium (1 x 106 cells/ml). The cell density can be optimized for a particular cell line in question to include ~2 to 4 x 106 cells per well (1 - 2 x 106 cells/ml). We used this approach as described by Trujillo et al. (2019)  for generating cerebral organoids. An alternative to 15 ml centrifugation is to perform the spin steps in 2 ml epp tubes at a higher (450 x g) speed to ensure all cells are retained.
Orbital shaking (day 0 = 0 h, dissociation):
15. Place the plates on an orbital shaker set to the clockwise direction. The rotational speed of orbital shaking is key to achieving the initial size of the starting aggregates. Orbital shaker speed may require optimization per cell line behavior. For aggregation, cultures are shaken at 80 rpm. This speed is reduced to 70-75 rpm later in the EMLO protocol as elongation occurs to prevent shearing.
Validating aggregate cell number (day 1 = 24 h post-dissociation):
16. The next day, validate aggregation by visual inspection using a microscope.
a. One direct method to determine cell number (300-400 cells at this stage) is by fixation of a subset of aggregates 24 h after transitioning to orbital shaker, staining with DAPI and counting cells from imaging data using confocal microscope-generated Z-stacks.
b. One indirect bulk method is to determine the concentration of aggregates from a subset of the population by hemocytometer, dilute in buffer to achieve a known absolute number of aggregates, dissociate to single cells, and count again with hemocytometer or an automated cell counting system. Divide the total number of cells by the number of aggregates used to determine an average value at this stage.
Changing medium (day 1 = 24 h, one-half volume)
17. Perform one-half volume media changes.
a. Depending on the density of the aggregates produced, it may be advantageous to split the wells 1:2. If splitting, prepare additional low-adhesion dishes in advance. This will be a function of the cell line used, the initial density of single cells added to the wells, and the orbital shaker speed.
b. At this time point (24 h post-aggregation), one-half volume of medium should be replaced with fresh N2B27 without ROCK inhibitor. Prepare freshly supplemented N2B27 medium so that the final concentration of FGF2, HGF, and IGF-1 after adding the new one-half volume remains the same. Change medium by pooling the aggregates in a 15 ml conical tube and allow them to settle by gravity before aspirating. Add fresh medium and return the aggregates to low adhesion wells. Fresh plates can be used at this step to reduce non-viable cell debris.
c. Return the cultures to the orbital shaker and maintain until day 3.
18. Visually inspect the shaking cultures on the intervening day 2. Even by phase contrast microscopy, the polarization into two relatively distinct domains may be visible.
19. On day 3, aspirate the entire volume of media and replace with fresh N2B27 + 2 ng/ml HGF, 2 ng/ml IGF-1 (no FGF2 this time) and return to the orbital shaker once more, overnight to day 4.
Visual inspection and exclusion of growth factors (day 4)
20. At day 4, visually inspect the cultures. Morphological signs of polarization and elongation may be evident at this stage by phase contrast microscopy, such as protrusions from the main body of the aggregate (Figure 3). Replace the entire volume with fresh N2B27 basal medium only (no growth factors added).
a. Transfer the aggregates from a single well 1:1 to 100 mm dishes pre-treated with Anti-Adherence Rinsing Solution and 2x HBSS rinses. Dilute one well of the 6-well plate to a final volume of 7-8 ml N2B27 in the pre-treated 100 mm dish.
b. Return the dish to the orbital shaker and reduce the speed to 70 rpm to prevent shearing of compartments as elongation proceeds. Maintain the developing EMLOs in this manner for the remainder of the protocol.
21. Replenish EMLO culture media using a 15 or 50 ml conical tube with N2B27 basal every 3 to 5 days as necessary. For our study, EMLOs were maintained to 40 days. The continued orbital shaking method allows the transition of the small gastruloids with three germ layers to larger organoid-like multi-compartment structures linked by the gut tube and neurons.