TITLE: Engineering brain assembloids to interrogate human neural circuits
AUTHORS: Yuki Miura, Min-Yin Li, Omer Revah, Se-Jin Yoon, Genta Narazaki, Sergiu   Pasca

[DESCRIPTION]
The development of neural circuits involves wiring of neurons locally following their generation and migration, as well as establishing long-distance connections between brain regions. Studying these developmental processes in the human nervous system remains difficult because of limited access to the tissue that can be maintained as functional over time in vitro. We have previously developed a method to convert human pluripotent stem cells into regionalized neural organoids that can be fused and integrated to form assembloids and study neuronal migration. In this protocol, we describe approaches to model long-range neuronal connectivity in human brain assembloids. We present how to generate 3D spheroids resembling specific domains of the nervous system and then how to integrate them physically to allow axonal projections and synaptic assembly. In addition, we describe a series of assays including viral labeling and retrograde tracing, 3D live imaging of axon projection and optogenetics combined with calcium imaging and electrophysiological recordings to probe and manipulate the circuits in assembloids. We anticipate that these approaches will be useful in deciphering human-specific aspects of neural circuit assembly and in modeling neurodevelopmental disorders with patient-derived cells.

[BEFORE_START]
CRITICAL: Before starting hPS cells and the long-term organoid/assembloid cultures, carefully cleaning the incubator is important to avoid potential contamination. Because most culture plates will be in the incubator for several hundred days, the surface of the incubator and culture dishes should be regularly cleaned. The amount of water in the incubator should be monitored frequently to maintain the required humidity. All cell culture-related procedures should be performed in a sterile environment with biological safety cabinets and sterilized materials. The biological safety cabinets should be regularly certiﬁed.

[GUIDELINES]
Troubleshooting
 
            Step  Problem  Possible reason  Solution    Throughout  Cultures were contaminated  Inadequate sterile technique  Improve sterile technique. Wear   laboratory coat. Use 70% ethanol to spray your hands and any equipment or   reagents that go into the safety cell culture cabinet. Autoclave all   non-sterile equipment. Change HEPA ﬁlters in ncubators regularly. Autoclean   incubators regularly. Identify sources of contamination in the incubator or   cell culture room. Clean the water bath in the incubator and in the cell   culture room on a weekly basis.    29  Individual spheroids do not form  Overﬁlled individual wells of   the AggreWell plate  Count cell number carefully and   add the required amount of cells to each well. Centrifuge the plate with   optical speed    31  Spheroids do not form  hPS cells are differentiating   Always check hPS cell quality   before use          hPS cell colonies are too small  Allow hPS cell colonies to grow   larger. Do not use until the optimal day for splitting (80–85% conﬂuency)          Over-crowding of hPS cell   Colonies  Start differentiation by using   hPS cell colonies with the optimal conﬂuency (80–85%)          Mycoplasma or other contamination  Perform mycoplasma testing   regularly (ideally, once per month) and discard the cells and use new hPS   cell stock if mycoplasma positive    38  Difﬁculty in inducing forebrain fate (low FOXG1 expression)  hPS cells are challenging to differentiate into forebrain   cells  Consider adding XAV-939 (1.25   µM) from days 1–6 of differentiation       Spheroids stick to one another  Too many spheroids cultured per plate  Transfer some spheroids to a new or larger low-attachment plate. If   already attached, separate them by gently pipetting up and down by using a   10-or 25-ml pipette (for spheroids up to day 25) or two P20 pipette tips at   later stages       Spheroids are too small  hPS cells are differentiating  See solutions for Step 31          hPS cell colonies are too small  See solutions for Step 31          Mycoplasma or other   contamination  See solutions for Step 31       Spheroids are disintegrated  Excessive pipetting  Be gentle when handling   spheroids at all times    38 and 39  Growth of spheroids is fast  Abnormal genome integrity in the   hPS cell line  Check the genome integrity of   the hPS cell line by using SNP array, and conﬁrm that there is no de novo   large duplication or deletion    39 and 40  Medium turns acidic overnight  Too many spheroids cultured per   plate  In the initial stages of differentiation, 12 ml of medium per 10-cm   plate is sufﬁcient. As spheroids grow, use 12–15 ml of medium per plate.   Maintaining <30 spheroids per 10-cm plate is recommended    40A  Sparse or no viral labeling  The viral titer was too low  Make sure that viral titers are   sufficiently high          Insufﬁcient time between   infection and ﬂuorescent protein expression  Wait ≥1–2 weeks for viral gene   expression          Incorrect serotype  Use optimal AAV serotypes    40B  Unable to assemble hCS and   hStrS   hCS or hStrS at late stages of   differentiation  Try to fuse smaller or   early-stage spheroids (days 47–80). After day 80, the success rate is lower    40F  Unable to ﬁnd GCaMP6+ cells inassembloids   under a confocal microscope  The signal of GCaMP6 is weak  Check expression of GCaMP6 by using an epiﬂuorescence microscope (e.g.,   EVOS or REVOLVE). Increase laser intensity          Imaging only a few z-slices  Increase thickness of slices by   changing the pinhole size and/or image more slices    40G  Cells are hard to find under   bright field  Cell bodies are not always easy   to identify by using bright field  Identify cells by using   fluorescence imaging       Cells are blown away by pressure  The pressure is too high  Apply minimal positive pressure         Pipette tips are too large  Use a pipette with smaller tips      It is dif ficult to form a tight   seal. Cells cannot form a fully tight seal even though cells are partially   sucked into the pipette  Pipette tips are too large  Use a pipette with smaller tips      Photobleaching of ﬂuorescence when checking cells  The LED power is too high  Lower the LED power and increase   gain and/or exposure time in the camera software setting      Cells are hard to break in  For some cells, it is hard to   break in with just suction  Try ‘zap’ in the 700B panel for   multiple trials; start from short durations such as 50 μs and then increase   durations step by step; try strong suctions immediately after zaps. Use   pipettes with slightly bigger tips      Cells get significantly resealed after break-in  Pipette tips are too small  Use pipettes with slightly bigger tips        Osmolarity of the external   solution and/or the internal solution is not appropriate  Check osmolarity of both the   external solution and the internal solution        The relative position of the   pipette tip and the cell changes in whole-cell mode  Slightly adjust the position of pipette tips  

Media
CRITICAL:  When ready for use, distribute aliquots of the required volume of medium into50 mL sterile tubes and pre-warm in a water bath at 37 °C or at 37 Room temperature. Avoid multiple cycles of refrigeration and warming. Add growth factors and small molecules before use and only after pre-warming aliquots of media.


[STEPS]
SECTION: Maintenance of hPS cells in feeder-free conditions Timing 4–6 d
1. Culture hPS cells on human recombinant vitronectin with Essential 8 medium. When hPS cell cultures reach ~80% conﬂuency, passage hPS cultures by using0.5 millimolar (mM) EDTA in PBS.  
 

 

SECTION: Maintenance of hPS cells in feeder-free conditions Timing 4–6 d
2. Regularly check hPS cell cultures for mycoplasma (e.g., PCR) and chromosomal abnormalities  (e.g., SNP or comparative genomic hybridization arrays).
SECTION: Preparing vitronectin-coated dishes
3. Prepare the vitronectin-coated plates at 37 Room temperature and pre-warm the required volume of Essential 8 medium at 37 Room temperature.

 

SECTION: Preparing vitronectin-coated dishes
4. To coat a well of a six-well plate, dilute 60 µLof vitronectin in 6 mL (1:100) of DPBS (without calcium and magnesium), add 1 mLof diluted vitronectin solution to the well and incubate at 37 Room temperature for 60 min.
 


SECTION: Passaging hPS cells
5.  Aspirate culture medium from the hPS cells plate and rinse with2 mL of DPBS (without calcium and magnesium).

SECTION: Passaging hPS cells
6.  Add 1 mLof 0.5 millimolar (mM)EDTA in DPBS to the well of a six-well plate and incubate at  37 Room temperature for 7 min. 
 


SECTION: Passaging hPS cells
7.  Aspirate the EDTA solution and add 1 mL of pre-warmed complete Essential 8 medium per well of a six-well plate.

SECTION: Passaging hPS cells
8. Remove cells from the well by gently squirting medium and pipetting the colonies with either a 5 mL serological pipette or P1000 tips with a pipette.  
 

SECTION: Passaging hPS cells
9. Aspirate the residual vitronectin solution from the pre-coated plate.
 

SECTION: Passaging hPS cells
10. Add 2 mL of pre-warmed Essential 8 medium to each well of a coated six-well plate. Mix the cell suspensions by pipetting and transfer the appropriate volume of cell suspension into each well containing pre-warmed complete Essential 8 medium according to the desired split ratio (1:6 to 1:20 ratio, depending on the conﬂuency and growth rate of individual hPS cell lines).

SECTION: Passaging hPS cells
11.  Move the plate in several quick, ﬁgure-of-eight-like movements to disperse cells.
SECTION: Passaging hPS cells
12. Gently place the plate in a 37 °C, 5% CO2 incubator.

SECTION: Passaging hPS cells
13. Perform a medium change after every passage (every1080 minto1440 min), except for the day after passage,  when conﬂuency of cells is low. 

SECTION: Preparation of hPS cells for 3D neural differentiation
14. Passage hPS cells from a six-well plate to 10-cm plates and grow to ~80–85% conﬂuency before the day of aggregation.

SECTION: Preparation of hPS cells for 3D neural differentiation
15.  2880 min and 1440 minbefore the aggregation, pre-treat cells with 1% DMSO (100 µLper 10 mL of Essential 8 medium for a 10-cm dish) to improve differentiation of hPS cell lines that are more challenging to differentiate.


SECTION: Generation of 3D spheroids by using an AggreWell plate
16. Add 500 µLof anti-adherence rinsing solution in a well of a new AggreWell plate and incubate the plate for 5 min at 37 Room temperature If you notice (under the microscope) bubbles in any of the wells of the plate, centrifuge the AggreWell plate at1300 x g, 5 min in a swinging bucket rotor. Aspirate the rinsing solution and wash with 1 mL of PBS.

SECTION: Generation of 3D spheroids by using an AggreWell plate
17. Pre-warm Essential 8, accutase and DMEM/F12 medium at 37 Room temperature. Supplement Essential 8 medium with ROCK inhibitor (Y-27631; 1:1,000) to a ﬁnal concentration of 10 micromolar (µM).

SECTION: Generation of 3D spheroids by using an AggreWell plate
18.  Add 0.5 mLof Essential 8 medium supplemented with Y-27632 to each well of an Aggrewell plate. Before starting aggregation, an Aggrewell plate can be centrifuged at 2000 x g, 5 min in a swinging bucket rotor that is ﬁtted with a plate holder to remove any air bubbles in the microwells. 
 


SECTION: Generation of 3D spheroids by using an AggreWell plate
19. Set the plate aside in an incubator while preparing a single-cell suspension of hPS cells for aggregation.
SECTION: Generation of 3D spheroids by using an AggreWell plate
20. Aspirate the maintenance medium from the hPS cell culture plates and rinse cells twice with DPBS (without calcium and magnesium).
SECTION: Generation of 3D spheroids by using an AggreWell plate
21. Add 4 mL of accutase per 10-cm plate and incubate at 37 °Cfor 7 min in a 5% CO2 incubator until cells detach by gentle shaking from the plate.

SECTION: Generation of 3D spheroids by using an AggreWell plate
22. Gently pipette the cell suspension two to three times with a serological pipette to ensure that any remaining clumps are fully dissociated.
SECTION: Generation of 3D spheroids by using an AggreWell plate
23. Transfer the cells to a 50 mL conical tube.
 


SECTION: Generation of 3D spheroids by using an AggreWell plate
24.  Add 6 mL of DMEM/F12 to rinse any remaining cells off the plate, transfer to a conical tube, and mix the well with the cell suspension in a total volume of 10 mL.

SECTION: Generation of 3D spheroids by using an AggreWell plate
25. Count cells and centrifuge the conical tube containing the cell suspension at 200 x g, 4 min.

SECTION: Generation of 3D spheroids by using an AggreWell plate
26. Resuspend the cells in Essential 8 medium supplemented with Y-27632 by adjusting the concentration of the single-cell suspension to 2.5–3 million in1 mL, and transfer the 1 mLof cell suspension into a well of an AggreWell plate to achieve a ﬁnal volume of 1.5 mL per well  .
     

SECTION: Generation of 3D spheroids by using an AggreWell plate
27. Pipette gently several times to re-establish an even distribution of cells throughout the well. 
SECTION: Generation of 3D spheroids by using an AggreWell plate
29.  Check under a microscope to verify that cells are evenly distributed among the microwells.    
  
 



SECTION: Generation of 3D spheroids by using an AggreWell plate
30.  Incubate the cells at 37 °C, 5% CO2 for 1440 min.
 


SECTION: Dislodging 3D spheroids from the AggreWell plate ● Timing 1 h
31. Pre-warm DMEM/F12 and Essential 6 medium at 37 Room temperature for 30 min to60 minbefore dislodging.

SECTION: Dislodging 3D spheroids from the AggreWell plate ● Timing 1 h
32. Collect the spheroids from the microwells by gently pipetting the medium in the well up and down two to three times with a micropipetter with a 1000 µL tip that was cut with scissors to allow a wider opening.
   
 

SECTION: Dislodging 3D spheroids from the AggreWell plate ● Timing 1 h
35. Invert the strainer and place it over a new 50 mL conical tube. Collect the spheroids by adding 12 mL of Essential 6 medium on top of the inverted strainer. Observe the AggreWell plate under the microscope to ensure that all aggregates have been removed from the wells. Repeat the wash if necessary.  
 

SECTION: Dislodging 3D spheroids from the AggreWell plate ● Timing 1 h
36. Place spheroids in ultra-low-attachment 10-cm plates in Essential 6 medium supplemented with dorsomorphine (5 micromolar (µM)), SB-431542 (10 micromolar (µM)).
 
   
 

SECTION: Neural induction
37. Add dorsomorphine and SB-431542 to Essential 6 medium for the ﬁrst 7200 min. XAV-939 (1.25 micromolar (µM)) can also be added for the ﬁrst 7200 min of differentiation if forebrain differentiation is not highly efﬁcient.

SECTION: Neural induction
38. Perform a medium change daily (but not on day 1) at approximately the same time of the day by gently transferring the neural spheroids to a50 mL Falcon tube (if they are too small) and aspirating the medium once they sit at the bottom of the tube. When spheroids are large, perform the medium change by gently tilting the plate (until spheroids settle at the bottom) and removing the remaining medium. This can minimize the transferring time of the spheroids in and out of the plate at this critical differentiation period.  
 


SECTION: Dislodging 3D spheroids from the AggreWell plate ● Timing 1 h
34. Pipette 1 mL of DMEM/F-12 medium across the entire surface of the well to dislodge any remaining spheroids. Collect washes and pass over the strainer used in the previous step.

SECTION: Dislodging 3D spheroids from the AggreWell plate ● Timing 1 h
33. Place a 40 μm cell strainer on top of a 50 mL conical tube and pass the suspension of spheroids through the strainer. The aggregates will remain on the ﬁlter of the strainer.  

SECTION: Generation of 3D spheroids by using an AggreWell plate
28.   Centrifuge the AggreWell plate at 100 x g, 3 min to distribute the cells in the micro-wells.   
  

SECTION: Patterning and differentiation
39. From day 6, replace the medium daily with NM with supplements listed in option A for hCSs and option B for hStrSs. 
 TROUBLESHOOTING 
SECTION: (B)   Generation of hStrSs
0.129. From day 6, replace the medium daily with NM supplemented with 50 ng/ml activin A and 2.5 micromolar (µM) IWP-2. Change the medium daily from day 6 to day 11.	
 
SECTION: Viral labeling of neural spheroids ● Timing 3 d
0.129. For labeling neural spheroids, dilute 0.5 µL of virus solution (recommended virus particle volumes are listed in Table 1) in 200 µL of NM and add mixed NM to a 1.5 mL Eppendorf tube. Then, transfer one to three spheroids in the tube containing NM and viruses. We note that transduction efﬁciency depends on the target cell types, the promoter, stage of differentiation and brain region. The tube can be placed   in an incubator at 37 °C, 5% CO2.
 

SECTION: Generating cortico-striatal assembloids ● Timing 3–4 d
0.129. (C)	Cryosection and immunostaining ● Timing 6–7 d 

(i)	When ready to perform immunocytochemistry, transfer spheroids to a 1.5 mLmicrocentrifuge tube by using a cut P1000 tip. Gently remove the medium and add 500 µL to 1 mLof cold 4% PFA/PBS (or warm 4% PFA/4% sucrose/PBS). Leave spheroids at4 °C 1440 min.
(ii)	After ﬁxation, gently remove PFA and wash with PBS at 4 Room temperature.
(iii)	Add 1 mL of 30% (wt/vol) sucrose/PBS for cryopreservation. Leave spheroids or assembloids at 4 °C until they sink to the bottom of tube (≥1440 min to 2880 min).
(iv)	Prepare square disposable molds that ﬁt with the sample size and number and ﬁll the molds with embedding solution (1:1, OCT/30% sucrose; see Reagent setup).
 


(v)	Transfer spheroids from tubes into the mold by using a P1000 cut pipette tip. Place 4–10 spheroids in the mold. If spheroids move while embedding, use a P20 tip to gently place them in the embedding solution.
 


(vi)	Place the spheroids in a cryomold on ice for 20 min and allow the spheroids to sink to the bottom of the mold.
(vii)	Snap-freeze the spheroids by placing the mold directly on dry ice. Once completely frozen, store molds at-80 °C.
 


(viii)	When ready for sectioning, transfer the mold from -80 °C to the cryostat chamber at -20 °C for ~30 min before sectioning.
(ix)	Remove the block from the mold, paste the specimen on the stage by using OCT and section by using standard techniques. Use a brush to prevent crumbling of the sections. Spheroids and assembloids can be cryo-sectioned at   to   thickness, depending on the purpose of the experiments.
(x)	Collect sections on Superfrost Plus slides. Slides can be stored at -20 °Cto -80 °Cuntil ready to immunostain.
(xi)	For immunostaining, remove the slide from the freezer and leave at 80 Room temperature to thaw for 5 min to 10 min.
(xii)	Wash the slide three times with PBS to remove excess OCT/sucrose on the slides.
(xiii)	Remove PBS and block for 60 minat80 Room temperature with 10% normal donkey serum (NDS), 0.3% Triton- X and 0.1% BSA in PBS. Paraﬁlm can be placed on top of the slide glass to avoid drying of the antibody solution. Alternatively, a hydrophobic PAP pen can be used to draw circles around the sections, and staining can be performed inside these circles.
(xiv)	Perform the immunostaining steps in a humidiﬁed chamber to avoid drying of sections.
(xv)	Add the primary antibodies diluted in antibody solution containing 2% NDS and 0.1% Triton-X in PBS and keep at 4 °C60 min.
(xvi)	The next day, perform three 5 min PBS washes at 4 Room temperature.
(xvii)	Add secondary antibodies diluted in antibody solution for 60 min at 4 Room temperature Paraﬁlm can be placed on top of the slide glass to avoid drying.
(xviii)	Perform three 5 min washes at4 Room temperature with PBS.
(xix)	Add Hoechst in PBS (1:10,000 dilution) for 5 minto 7 minat 4 Room temperature.
(xx)	Aspirate the Hoechst solution and wash twice with PBS.
(xxi)	Aspirate PBS and mount the sections by adding one drop of Aquamount solution on top of sections and by placing a coverglass on top.
(xxii)	Use a ﬂuorescent microscope to image (Fig. 2j, k and Fig. 3f–i).
 

.
SECTION: Patterning and differentiation
40. (A) Generation of hCSs


SECTION: Patterning and differentiation
40.1. From day 6, replace the medium daily with NM supplemented with 20 ng/ml  FGF2 and  20 ng/ml   EGF for generating hCSs. Change the medium daily from day 6 to day 15.
SECTION: Patterning and differentiation
40.3. From day 22 to day 46, replace FGF-2 and EGF with  20 ng/ml  BDNF,  20 ng/ml  NT-3,  200 micromolar (µM) AA, 50 micromolar (µM)  dibutyryl cAMP sodium salt and 10 micromolar (µM) DHA. Perform medium changes with 12 mL to 15 mL of supplemented NM per 10-cm plate every 2 d.
SECTION: Patterning and differentiation
40.2. From day 16 onward, change the medium every other day until day 22. Continue to use NM supplemented with   20 ng/ml  FGF2 and  20 ng/ml  Q29 EGF.
SECTION: Patterning and differentiation
41. (B) Generation of hStrSs

SECTION: Patterning and differentiation
41.1. From day 6, replace the medium daily with NM supplemented with 50 ng/ml activin A and 704 2.5 micromolar (µM)  IWP-2. Change the medium daily from day 6 to day 11.

SECTION: Patterning and differentiation
41.2. From day 12 to day 15, supplement NM with  50 ng/ml activin A, 2.5 μm  IWP-2 and 100 nanomolar (nM)  SR11237. Perform medium changes with 12 mL to 15 mL  of NM per 10-cm plate every day.

SECTION: Patterning and differentiation
41.4. From day day 22 to day 41, supplement NM with  20 ng/ml BDNF,   20 ng/ml  NT-3, 200 micromolar (µM)  AA, 50 micromolar (µM)  dibutyryl cAMP sodium salt and 10 micromolar (µM) DHA. Perform medium changes with  12 mL to 15 mL  of NM per 10-cm plate every 2 d.

SECTION: Patterning and differentiation
41.3. From day 16 onward, change the medium every other day until day 22. Continue to use NM supplemented with  50 ng/ml activin A,  2.5 μm IWP-2 and  100 nanomolar (nM) SR11237. Perform medium changes with  12 mL to 15 mL of NM per 10-cm plate every 2 d.
SECTION: Patterning and differentiation
41.5. From day 42 to day 46, supplement NM with  20 ng/ml  BDNF,   20 ng/ml  NT-3, 200 micromolar (µM) AA, 50 micromolar (µM) dibutyryl cAMP sodium salt, 10 micromolar (µM)  DHA and   2.5 micromolar (µM) DAPT. Perform medium changes with 12 mL  to15 mL of NM per 10-cm plate every 2 d.

SECTION: Long-term culture and phenotyping ● Timing indefinite
42. After day 46, perform medium changes with 12–15 ml of NM without growth factors every 4–5 d.
 

SECTION: Long-term culture and phenotyping ● Timing indefinite
43. (A) Viral labeling of neural spheroids ● Timing 3 d
SECTION: Long-term culture and phenotyping ● Timing indefinite
43.1. For labeling neural spheroids, dilute 0.5 µL of virus solution (recommended virus particle volumes are listed in Table 1) in 200 µL of NM and add mixed NM to a 1.5 mL Eppendorf tube. Then, transfer one to three spheroids in the tube containing NM and viruses. The tube can be placed overnight in an incubator at 37 °C, 5% CO2.
 


SECTION: Long-term culture and phenotyping ● Timing indefinite
43.3. On day 3, wash the spheroids with 1 mL of fresh NM (two to three times) and then transfer the spheroids back to their culture plate.
 

SECTION: Long-term culture and phenotyping ● Timing indefinite
43.2. The next day, add an extra 800 µLof fresh NM to the tube and incubate for 1440 min.

SECTION: Long-term culture and phenotyping ● Timing indefinite
44. (B) Generating cortico-striatal assembloids ● Timing 3–4 d
SECTION: Long-term culture and phenotyping ● Timing indefinite
44.1. To generate cortico-striatal assembloids, transfer one hCS and one hStrS into a 1.5 mL Eppendorf tube containing 1 mL of NM.  
  
 



SECTION: Long-term culture and phenotyping ● Timing indefinite
44.3. After spheroids are assembled, transfer them into an ultra-low-attachment plate by using a P1000 pipette with the tip cut for a larger bore opening. Assembly of hCS and hStrS between days 46 and 80 is more efficient.
 

SECTION: Long-term culture and phenotyping ● Timing indefinite
44.2. Incubate the spheroids at 37 °C for 3–4 d, completely replacing the medium on day 2.
 


SECTION: Long-term culture and phenotyping ● Timing indefinite
45. (C) Cryosection and immunostaining ● Timing 6–7 d
SECTION: Long-term culture and phenotyping ● Timing indefinite
45.1. When ready to perform immunocytochemistry, transfer spheroids to a 1.5 mL microcentrifuge tube by using a cut P1000 tip. Gently remove the medium and add 500 µL to 1 mL of cold 4% PFA/PBS (or warm 4% PFA/4% sucrose/PBS). Leave spheroids at 4 °C 1440 min.
SECTION: Long-term culture and phenotyping ● Timing indefinite
45.2. After fixation, gently remove PFA and wash with PBS at 4 Room temperature.
SECTION: Long-term culture and phenotyping ● Timing indefinite
45.3. Add 1 mLof 30% (wt/vol) sucrose/PBS for cryopreservation. Leave spheroids or assembloids at 4 °C until they sink to the bottom of tube (≥24–48 h).
SECTION: Long-term culture and phenotyping ● Timing indefinite
45.4. Prepare square disposable molds that fit with the sample size and number and fill the molds with embedding solution (1:1, OCT/30% sucrose; see Reagent setup).
 

SECTION: Long-term culture and phenotyping ● Timing indefinite
45.5. Transfer spheroids from tubes into the mold by using a P1000 cut pipette tip. Place 4–10 spheroids in the mold. If spheroids move while embedding, use a P20 tip to gently place them in the embedding solution.
 

SECTION: Long-term culture and phenotyping ● Timing indefinite
45.6. Place the spheroids in a cryomold on ice for 20 min and allow the spheroids to sink to the bottom of the mold.

SECTION: Long-term culture and phenotyping ● Timing indefinite
45.7. Snap-freeze the spheroids by placing the mold directly on dry ice. Once completely frozen, store molds at -80 °C.
 

SECTION: Long-term culture and phenotyping ● Timing indefinite
45.8. When ready for sectioning, transfer the mold from -80 °C to the cryostat chamber at -20 °C for ~30 min before sectioning.

SECTION: Long-term culture and phenotyping ● Timing indefinite
45.9. Remove the block from the mold, paste the specimen on the stage by using OCT and section by using standard techniques. Use a brush to prevent crumbling of the sections. Spheroids and assembloids can be cryo-sectioned at  –  thickness, depending on the purpose of the experiments.

SECTION: Long-term culture and phenotyping ● Timing indefinite
45.16. The next day, perform three 5 min PBS washes at 4 Room temperature (1/3).

SECTION: Long-term culture and phenotyping ● Timing indefinite
45.15. Add the primary antibodies diluted in antibody solution containing 2% NDS and 0.1% Triton-X in PBS and keep at 4 °C 60 min.

SECTION: Long-term culture and phenotyping ● Timing indefinite
45.14. Perform the immunostaining steps in a humidified chamber to avoid drying of sections.
SECTION: Long-term culture and phenotyping ● Timing indefinite
45.13. Remove PBS and block for 60 min at 80 Room temperature with 10% normal donkey serum (NDS), 0.3% Triton- X and 0.1% BSA in PBS.
 

SECTION: Long-term culture and phenotyping ● Timing indefinite
45.12. Wash the slide three times with PBS to remove excess OCT/sucrose on the slides.
SECTION: Long-term culture and phenotyping ● Timing indefinite
45.11. For immunostaining, remove the slide from the freezer and leave at 80 Room temperature to thaw for 5 min–10 min

SECTION: Long-term culture and phenotyping ● Timing indefinite
45.10. Collect sections on Superfrost Plus slides. Slides can be stored at -20 °C to -80 °C until ready to immunostain.

SECTION: Long-term culture and phenotyping ● Timing indefinite
45.19. Add secondary antibodies diluted in antibody solution for 60 min at 4 Room temperature. 
 

SECTION: Long-term culture and phenotyping ● Timing indefinite
45.26. Use a fluorescent microscope to image.    
 
   
 



SECTION: Long-term culture and phenotyping ● Timing indefinite
45.25. Aspirate PBS and mount the sections by adding one drop of Aquamount solution on top of sections and by placing a coverglass on top.

SECTION: Long-term culture and phenotyping ● Timing indefinite
45.24. Aspirate the Hoechst solution and wash twice with PBS.

SECTION: Long-term culture and phenotyping ● Timing indefinite
45.23. Add Hoechst in PBS (1:10,000 dilution) for 5 min–7 min at4 Room temperature.

SECTION: Long-term culture and phenotyping ● Timing indefinite
45.20. Perform three 5 min washes at 4 Room temperature with PBS (1/3).

SECTION: Long-term culture and phenotyping ● Timing indefinite
45.17. The next day, perform three 5 min PBS washes at 4 Room temperature (2/3).
SECTION: Long-term culture and phenotyping ● Timing indefinite
45.18. The next day, perform three 5 min PBS washes at 4 Room temperature (3/3).
SECTION: Long-term culture and phenotyping ● Timing indefinite
45.22. Perform three 5 min washes at 4 Room temperature with PBS (3/3).
SECTION: Long-term culture and phenotyping ● Timing indefinite
45.21. Perform three 5 min washes at 4 Room temperature with PBS (2/3).
SECTION: Long-term culture and phenotyping ● Timing indefinite
46. (D) CUBIC clearing and imaging ● Timing 4–7 d

SECTION: Long-term culture and phenotyping ● Timing indefinite
46.1. Transfer spheroids to a 1.5 mL microcentrifuge tube by using a cut P1000 tip. Gently remove the medium and add 500 µL to 1 mL of cold 4% PFA/PBS (or add to 1 mL of warm 4% PFA/4% sucrose/PBS and leave at 4 Room temperature for 20 min). Leave spheroids at 4 °C 20 min.

SECTION: Long-term culture and phenotyping ● Timing indefinite
46.16. Once assembloids become transparent, transfer them to a well of a 96-well imaging- compatible plate filled with 150 µL of CUBIR-R+ solution and image them with a confocal microscope   . 

SECTION: Long-term culture and phenotyping ● Timing indefinite
46.15. For refractive index–matching, remove PBS from the 1.5 mL tubes, add 1 mL of CUBIC-R in the 1.5 mL tube and leave for 120 min at 37 Room temperature.

SECTION: Long-term culture and phenotyping ● Timing indefinite
46.12. Wash with PBS three times at 37 Room temperature for 120 min (1/3).

SECTION: Long-term culture and phenotyping ● Timing indefinite
46.11. Add secondary antibodies diluted in solution containing 0.2% Triton-X100 and 3% NDS in PBS and incubate assembloids at 37 °C for 120 min.

SECTION: Long-term culture and phenotyping ● Timing indefinite
46.8. Wash with PBS three times at 37 °C for 120 min (1/3).

SECTION: Long-term culture and phenotyping ● Timing indefinite
46.7. Add a combination of primary antibodies diluted in solution containing 0.2% Triton-X100, 3% NDS in PBS and incubate assembloids at 37 °C for 120 min.

SECTION: Long-term culture and phenotyping ● Timing indefinite
46.4. Wash with PBS three times at 37 Room temperature for 120 min (1/3).

SECTION: Long-term culture and phenotyping ● Timing indefinite
46.3. Add 500 µL to 1 mL of CUBIC-L for de-lipidation and incubate at 37 °C for 2880 min.

SECTION: Long-term culture and phenotyping ● Timing indefinite
46.2. After fixation, wash samples with PBS twice at 4 Room temperature.

SECTION: Long-term culture and phenotyping ● Timing indefinite
46.9. Wash with PBS three times at 37 °C for 120 min (2/3).
SECTION: Long-term culture and phenotyping ● Timing indefinite
46.10. Wash with PBS three times at 37 °C for 120 min (3/3).
SECTION: Long-term culture and phenotyping ● Timing indefinite
46.13. Wash with PBS three times at 37 Room temperature for 120 min (2/3).
SECTION: Long-term culture and phenotyping ● Timing indefinite
46.14. Wash with PBS three times at 37 Room temperature for 120 min (3/3).
SECTION: Long-term culture and phenotyping ● Timing indefinite
46.6. Wash with PBS three times at 37 Room temperature for 120 min (3/3).
SECTION: Long-term culture and phenotyping ● Timing indefinite
46.5. Wash with PBS three times at 37 Room temperature for 120 min (2/3).
SECTION: Long-term culture and phenotyping ● Timing indefinite
47. (E) Live imaging of axon growth ● Timing 2–8 h
 



SECTION: Long-term culture and phenotyping ● Timing indefinite
47.1. Using a cut P1000 tip, transfer assembloids to a well of a 96-well imaging-compatible plate in 150 µL–200 µL of NM.
    
 






SECTION: Long-term culture and phenotyping ● Timing indefinite
47.4. Process fluorescent images with Fiji and quantify the percentage of eYFP coverage in hStrSs and the percentage of mCherry coverage in hCSs.

  

SECTION: Long-term culture and phenotyping ● Timing indefinite
47.3. Set up imaging positions with z-stacks and take images by using an ×10 objective lens at a depth of 0–500 µm.
  

SECTION: Long-term culture and phenotyping ● Timing indefinite
47.2. Place the 96-well plate into an inverted microscope with a motorized stage under environmentally controlled conditions (37 °C, 5% CO2), and keep the assembloids for 15 min–30 min before experiments to let assembloids settle at the bottom of the plate.

SECTION: Long-term culture and phenotyping ● Timing indefinite
48. (F) Optogenetic stimulation and calcium imaging ● Timing 2–6 h
 

SECTION: Long-term culture and phenotyping ● Timing indefinite
48.1. Transfer an assembloid onto a   glass coverslip in a   plate in NM and image by using an ×10 objective on a Leica TCS SP8 confocal microscope.
SECTION: Long-term culture and phenotyping ● Timing indefinite
48.2. For optogenetic stimulation, activate ChrimsonR-tdTomato+ cells in hCSs with   light by using an optical ﬁber-coupled LED. Our stimulation experiments included 1,500 frames, and one   pulse of LED light (68 ms) was applied every 150 frames as generated by a Cyclops LED connected to the LEICA TCS SP8 microscope. 

     

SECTION: Long-term culture and phenotyping ● Timing indefinite
48.3. Image GCaMP6 at a frame rate of 14.7 frames/s. 

     

SECTION: Long-term culture and phenotyping ● Timing indefinite
48.4. Process raw GCaMP6 signals with Fiji.
SECTION: Long-term culture and phenotyping ● Timing indefinite
48.5. After registration of ROIs, transform raw time series to relative changes in ﬂuorescence by using the following formula: ΔF/F = (Ft – F0)/F0.
SECTION: Long-term culture and phenotyping ● Timing indefinite
48.7. For quantiﬁcation of time-locked ΔF/F values, calculate the amplitude of ΔF/F values from each cell as the maximum ΔF/F values within 20–30 frames (1,360–2,040-ms window) after LED stimulation (minus the mean of the baseline 1 s before stimulation).

   
 
TROUBLESHOOTING
SECTION: Long-term culture and phenotyping ● Timing indefinite
48.6. To verify whether responses are time-locked to LED stimulation, compare ΔF/F responses to ΔF/F values obtained at randomly selected time points.
SECTION: Long-term culture and phenotyping ● Timing indefinite
49. (G)	Whole-cell electrophysiological recording ● Timing 2–6 h
 

SECTION: Long-term culture and phenotyping ● Timing indefinite
49.1. Add 0.2 g of low-melt agarose into a   plate containing 5 mLof aCSF and mix well (4% agarose). Dissolve low-melt agarose by using a microwave (avoid spillover due to overheating).
 

SECTION: Long-term culture and phenotyping ● Timing indefinite
49.5. Once cells are identiﬁed through ﬂuorescence, patch under differential interference contrast (DIC) or ﬂuorescence. For DIC, the microscope is switched to a bright ﬁeld. For patching under ﬂuorescence, there are at least two approaches:
Increase the bright ﬁeld exposure under epiﬂuorescence so that both ﬂuorescence (to visualize the cell) and bright ﬁeld (to visualize the pipette tip) can be seen simultaneously. Once a ‘dimple’ is seen under ﬂuorescence or increased pipette resistance is achieved, release positive pressure and form a tight seal.
Use ﬂuorophore-coated pipettes, patching under epiﬂuorescence.  Back-ﬁll glass pipettes by dipping the tips in the BSA-Alexa Fluor (0.02% in PBS) in a 1.5-ml Eppendorf tube for 5 s to 10 s and, after drying for 10 min, ﬁll pipettes with internal solution and patch.      





SECTION: Long-term culture and phenotyping ● Timing indefinite
49.4. Patch-clamping on spheroid slices is usually performed on ﬂuorescently labeled neurons. Record labeled neurons with glass pipettes with small tips (8–10 MΩ with K-gluconate internal solution, 7–9 MΩ with Cs internal solution)
 
.
SECTION: Long-term culture and phenotyping ● Timing indefinite
49.3. Cut a block of agarose out of the culture plate by using a blade and glue the block on the plate of the vibratome. Slice spheroids at a   to   thickness with cold aCSF in a vibratome. Transfer slices into aCSF (at 4 Room temperature bubbled with 95% O2/5% CO2).
SECTION: Long-term culture and phenotyping ● Timing indefinite
49.2. Wait for a few minutes for the agarose to cool down to 37 °C and transfer spheroids or assembloids from the medium into agarose with a cut P1000 pipette tip.

    
 

 

SECTION: Long-term culture and phenotyping ● Timing indefinite
49.8. Make recordings at 30 °C to 37 °Cor37 Room temperature within 300 min to360 minafter sectioning.
TROUBLESHOOTING
SECTION: Long-term culture and phenotyping ● Timing indefinite
49.6. Once the pipette is approaching the target cell, provide minimal positive pressure. Once the tight seal is formed, strong suction is required to break cells.
 

SECTION: Long-term culture and phenotyping ● Timing indefinite
49.7. For recording optically evoked responses in assembloids, apply light at the maximal power through the 40× objective by using a CoolLED (  for ChR2 and   for Chrimson). CoolLED is controlled by a protocol in pClamp.