TITLE: Generation of hCS from hiPSC maintained in feeder-free conditions
AUTHORS: Sejin Yoon, Sergiu P. Pașca

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Here, we show the generation of 3D human cortical spheroids (hCS) from human induced pluripotent stem cells (hiPSC) maintained in </span><span style = "font-weight:bold;">feeder-free, xeno-free conditions</span><span>. This protocol describes hiPSC maintenance, generation of 3D cell aggregates in microwells, and cortical differentiation into hCS.</span></div></div>

[STEPS]
?. [Maintenance and long-term culture]
Incubate intact spheroids with  Fluo-4 diluted in NM for  at  and 5% CO2, followed by a  wash.
37 °C
?. [Maintenance and long-term culture]
Using an appropriate imaging system, record spontaneous calcium activity for several minutes (one frame every 8–10 s) in one 10-μm z-stack plane. Maintain the spheroids in NM during the recordings. We do not perfuse during calcium-imaging sessions, in order to minimize drift.
?. [Maintenance and long-term culture]
Export fluorescence intensity (F) as mean gray values in the ImageJ software (https://imagej.nih.gov/ij/). Signal decay should be controlled by subtracting the mean fluorescence of the background (Fb). To estimate changes in intracellular calcium, ΔF is computed as (Fell-Fb)/F0, where F0 represents the minimum F value per cell across the entire duration of recording from which Fb was subtracted.
?. [Live imaging of cell migration and calcium dynamics]
Transfer a fused hCS–hSS to a well of a 96-well plate (glass-bottom plate) in  of neural medium.
200 µl
Owing to the limited amount of medium that can be included in a 96-well plate, avoid putting more than one brain assembloid into a well.
?. [Live imaging of cell migration and calcium dynamics]
Place a 96-well plate into an inverted microscope with a motorized stage under environmentally controlled conditions (37 °C, 5% CO2).
?. [Live imaging of cell migration and calcium dynamics]
Maintain the assembled spheroids in an environmentally controlled chamber for  –  before imaging to let them acclimatize and settle. Movement in the room should be kept to a minimum.
?. [Live imaging of cell migration and calcium dynamics]
Set up the imaging positions so that up to ~8 fields with z stacks (corresponding to eight assembled hCS–hSSs) can be imaged in a sequential manner, with each position or frame being imaged at a rate of 15–20 min per frame. Our z stacks normally span a depth of 50–150 μm and have a step size of 1.5–2.5 μm. We have found that hCS–hSSs can be imaged for up to ~18 h, after which time imaging must be paused, so the medium should be carefully changed before continuing imaging. On our live-imaging system (Leica SP8 confocal microscope), we use The laser power density should be empirically determined in other imaging systems.
?. [Live imaging of cell migration and calcium dynamics]
If spheroids drift and this impacts image analysis, correct the images for drift computationally. We have found that the image registration plug-ins from ImageJ (e.g., linear stack alignment with SIFT) are generally sufficient to correct for minor-to-moderate drift. However, we discard sessions with major drift due to handling. Occasional drifting of spheroids may occur, as they are not embedded during imaging sessions.
?. [Maintenance and long-term culture]
Fix the spheroids with 4% (vol/vol) PFA for at .
4 °C
Note that this incubation time was optimized for 50- to 150-d-old hCSs, hSSs, or forebrain assembloids. However, given that the size of the spheroids after this age does not substantially change, this fixation time is probably suitable for older spheroids as well. If using younger spheroids (up to 25 d old), incubation times may be shortened. Keep in mind, however, that iDISCO clearing tends to result in a shrinking of tissue, so using early spheroids may make handling challenging.
?. [Maintenance and long-term culture]
Perform three  washes at  with DPBS.
0 Room temperature
?. [Maintenance and long-term culture]
Transfer the spheroids to 4-ml glass vials.
?. [Maintenance and long-term culture]
Dehydrate the spheroids with a day-long methanol (MeOH) dilution series at  (20%, 40%, 60%, 80%, and 100% MeOH/DPBS,  per step).
0 Room temperature
?. [Maintenance and long-term culture]
Incubate the spheroids in 5% H2O2/95% MeOH overnight at .
4 °C
?. [Maintenance and long-term culture]
The following day, rehydrate with a reverse MeOH dilution series (80% MeOH/DPBS, 80%, 60%, 40%, 20%, 0%, and 0% MeOH/0.2% Triton X-100/DPBS,  per step).
?. [Maintenance and long-term culture]
Incubate the spheroids overnight in 0.2% (vol/vol) Triton X-100, 20% DMSO, and 0.3 M glycine/DPBS at .
37 °C
?. [Maintenance and long-term culture]
Block the spheroids with 0.2% (vol/vol) Triton X-100, 10% DMSO, and 6% goat serum/ DPBS at  for .
37 °C
Critical Step: Perform this and all the following steps in closed tubes filled to the topto prevent oxidation of tissue.
?. [Maintenance and long-term culture]
Incubate the spheroids with 0.2% Tween 20 for .
?. [Maintenance and long-term culture]
Incubate the spheroids in 10 μg/ml heparin/DPBS (PTwH) for .
?. [Maintenance and long-term culture]
Incubate the spheroids with the desired primary antibodies (e.g., anti-GFAP, 1:1,000 dilution; anti-GFP, 1:1,000; and anti-SOX2, 1:500) for  in PTwH with 5% DMSO and 3% goat serum at .
37 °C
?. [Maintenance and long-term culture]
Undertake a day-long wash series with PTwH ( to  steps for at least 5 h: : PTwH; : PTwH; : PTwH; : PTwH; and : PTwH).
?. [Maintenance and long-term culture]
Dilute the secondary antibodies in PTwH and 3% goat serum and incubate for an additional  at .
37 °C
?. [Maintenance and long-term culture]
Wash off excess secondary antibody by washing for  with PTwH. Repeat three times so spheroids have been washed for at least .
?. [Maintenance and long-term culture]
Incubate the spheroids in 50% THF/H2O overnight at .
0 Room temperature
?. [Maintenance and long-term culture]
Perform a three-step THF series ( 80%/H2O,   100% THF,   100% 1016 THF) at .
0 Room temperature
?. [Maintenance and long-term culture]
Clear the spheroids by incubating in benzyl ether. Neural spheroids should be cleared within only a few minutes of incubation.
?. [Maintenance and long-term culture]
Image cleared tissue. When imaging cleared tissue, it is critical to use a chamber that is resistant to the organic solvents used in Step 42 and 43). For this purpose, we 3D-printed a chamber using semi-clear acrylic-type plastic from ProJet at the Stanford 3D Printing Core and followed the instructions that can be found at https://idisco.info/idisco-protocol/. If using this chamber, use silicone sealant to fix a rectangular coverslip on the bottom of the 3D-printed chamber. When spheres are cleared, add them to the chamber with benzyl ether.
Critical Step: Make sure the chamber is completely sealed to the cover glass to avoid any benzyl ether leaking onto the microscope. We recommend visiting https://idisco.info/ for additional troubleshooting tips.
?. [Maintenance of feeder-free (FF) hiPSC]
Human induced pluripotent stem cells (hiPSC) are cultured on vitronectin in Essential 8™ (E8) medium and are passaged every 4–5 days using EDTA.
?. [Maintenance of feeder-free (FF) hiPSC]
For passaging hiPSC, coat wells of a 6-well plate by diluting  in  (1:100 dilution) and adding  of diluted vitronectin solution per well.
[vitronectin]
[DPBS]
1 ml
?. [Maintenance of feeder-free (FF) hiPSC]
Aspirate medium and rinse with – per well.
3 ml
[DPBS]
?. [Maintenance of feeder-free (FF) hiPSC]
Aspirate the EDTA solution and add .
[pre-warmed E8]
?. [Maintenance of feeder-free (FF) hiPSC]
Remove cells by gently squirting medium and pipetting the colonies with a 5 ml serological pipette.
Avoid the generation of bubbles.
?. [Maintenance of feeder-free (FF) hiPSC]
Aspirate the residual vitronectin solution from the pre-coated dish and add .
[pre-warmed E8 medium]
?. [Maintenance of feeder-free (FF) hiPSC]
Mix the cell suspensions by gently inverting several times, then transfer the appropriate volume into each well containing pre-warmed E8 medium according to the desired split ratio.
?. [Maintenance of feeder-free (FF) hiPSC]
Gently place the plate into a , 5 % CO2 incubator.
37 °C
?. [Maintenance of feeder-free (FF) hiPSC]
No media change should be performed after the day of passage. Afterwards, replace medium daily (– per well).
2 ml
2.5 ml
Cultures should be checked regularly for Mycoplasma contamination and the presence of genomic abnormalities.
?. [[Optional step] Differentiation day –2: DMSO pre-treatment]
Two days prior to starting neural differentiation, and one day prior to spheroid formation, pre-treat hiPSC with  (120 μl per 12 ml of E8 medium for one 100 mm culture dish).
[DMSO]
This stage is optional and based on reference:Chetty, S. et al. A simple tool to improve pluripotent stem cell differentiation. Nature Methods 10, 553-556, doi:10.1038/nmeth.2442 (2013).
?. [Differentiation day –1: Generation of 3D spheroids from hiPSC maintained in FF]
To generate spheroids, passage hiPSC from a 6-well to a 100 mm culture dish and culture them to 80–90 % confluency.
?. [Differentiation day –1: Generation of 3D spheroids from hiPSC maintained in FF]
Pre-warm E8 medium, Accutase, and DMEM/F-12 at .
37 Room temperature
?. [Differentiation day –1: Generation of 3D spheroids from hiPSC maintained in FF]
Pre-treat wells with  to each well and centrifuge at  for  in a swinging bucket rotor fitted with plate holders.
[Anti-Adherence Rinsing Solution]
Centrifuge: 1300 34
?. [Differentiation day –1: Generation of 3D spheroids from hiPSC maintained in FF]
Aspirate maintenance medium from the hiPSC plates and rinse cells twice with DPBS (no calcium, no magnesium).
?. [Differentiation day –1: Generation of 3D spheroids from hiPSC maintained in FF]
Add  per 100 mm culture plate.
[Accutase]
?. [Differentiation day –1: Generation of 3D spheroids from hiPSC maintained in FF]
Add pre-warmed E8 medium up to 10 ml volume.
?. [Differentiation day –1: Generation of 3D spheroids from hiPSC maintained in FF]
Centrifuge the cell suspension at  for .
Centrifuge: 200 34
?. [Differentiation day –1: Generation of 3D spheroids from hiPSC maintained in FF]
Add  of this cell suspension to the previously prepared AggreWell plate, which contains 1 ml of E8 medium supplemented with Y-27632. Each well of AggreWell™800 plate contains 300 microwells, and one microwell will have 10,000 cells.
1 ml
?. [Differentiation day –1: Generation of 3D spheroids from hiPSC maintained in FF]
Centrifuge the AggreWell™800 plate at  for  to distribute the cells in the microwells.
Centrifuge: 100 34
?. [Differentiation day 0: Dislodging and harvesting aggregated spheroids]
Harvest the hiPSC-derived spheroids from the microwells by firmly pipetting medium in the well up and down with a 1 ml disposable tip that has been cut.
?. [Differentiation day 0: Dislodging and harvesting aggregated spheroids]
Place a 40 μm strainer on a 50 ml conical tube and pass the suspension of spheroids through the strainer.
?. [Differentiation day 0: Dislodging and harvesting aggregated spheroids]
Invert the strainer, and place over a new 50 ml conical tube. Collect the spheroids by washing with Essential 6™ (E6) medium for neural induction.
?. [Differentiation day 0: Dislodging and harvesting aggregated spheroids]
Observe the AggreWell™800 plate under the microscope to ensure that all aggregates have been removed from the wells. Repeat wash if necessary.
?. [Neural Differentiation]
Harvested spheroids are placed in ultra-low attachment 100 mm plates in E6 medium supplemented with  and .
[Dorsomorphin (DM)]
[SB-431542 (SB)]
Optionally,   can be added for the first five days. Media changes are performed daily, except for day 1.
?. [Neural Differentiation]
On day 6, E6 medium containing DM and SB is replaced with neural medium (NM) supplemented with EGF2 () and FGF2 () for the 19 days with daily medium change in the first 10 days, and every other day medium changes for the subsequent 9 days.   AB1Neural Medium (NM) CompositionVolume (∼ 500 ml)2Neurobasal™-A Medium480 ml3B-27™ Supplement (50X), minus vitamin A10 ml4GlutaMAX™ Supplement (1:100)5 ml5Penicillin-Streptomycin (10,000 U/mL)5 ml
AB1Neural Medium (NM) CompositionVolume (∼ 500 ml)2Neurobasal™-A Medium480 ml3B-27™ Supplement (50X), minus vitamin A10 ml4GlutaMAX™ Supplement (1:100)5 ml5Penicillin-Streptomycin (10,000 U/mL)5 ml
?. [Neural Differentiation]
To promote differentiation of the neural progenitors into neurons, FGF2 and EGF are replaced with  and  starting at day 25 (with media changes every other day).
[BDNF]
[NT-3]
?. [Neural Differentiation]
From day 43 onwards only NM without growth factors is used for medium changes every four days or as needed.
?. [Differentiation day –1: Generation of 3D spheroids from hiPSC maintained in FF]
Supplement E8 medium with the ROCK inhibitor (Y-27632, 1:1000) to a final concentration of .
?. [Differentiation day –1: Generation of 3D spheroids from hiPSC maintained in FF]
Check under a microscope to ensure that bubbles have been removed from microwells and rinse each well with .
[warm DMEM/F-12 medium]
?. [Differentiation day –1: Generation of 3D spheroids from hiPSC maintained in FF]
Remove the wash medium, and add  per well of E8 supplemented with Y-27632. Set plate aside in an incubator while preparing the single cell suspensions of hiPSC.
1 ml
?. [Differentiation day –1: Generation of 3D spheroids from hPSC maintained in FF]
?. [Differentiation day –1: Generation of 3D spheroids from hiPSC maintained in FF]
Incubate for  at , 5 % CO2 incubator.
37 °C
?. [Differentiation day –1: Generation of 3D spheroids from hiPSC maintained in FF]
Resuspend the pellet with E8 medium and count cell number.
?. [Differentiation day –1: Generation of 3D spheroids from hiPSC maintained in FF]
Centrifuge the cell suspension at  for .
Centrifuge: 200 34
?. [Differentiation day –1: Generation of 3D spheroids from hPSC maintained in FF]
?. [Differentiation day –1: Generation of 3D spheroids from hiPSC maintained in FF]
Resuspend the pellet with pre-warmed E8 medium supplemented with Y-27632 to obtain 3 million cells per 1 ml of medium.
?. [Differentiation day –1: Generation of 3D spheroids from hiPSC maintained in FF]
Incubate for  at , in a 5 % CO2 incubator.
37 °C
?. [Maintenance of feeder-free (FF) hiPSC]
Keep it at  for .
0 Room temperature
?. [Maintenance of feeder-free (FF) hiPSC]
Add  of .
1 ml
[EDTA]
?. [Maintenance of feeder-free (FF) hiPSC]
Incubate for  at .
0 Room temperature
?. [Differentiation day 0: Dislodging and harvesting aggregated spheroids]
Pipette  across the entire surface of the well to dislodge any remaining spheroids. Collect washes and pass over the strainer until every spheroids are recovered by checking under a microscope.
[DMEM/F-12 medium]