Apr 21, 2022
Engineering brain assembloids to interrogate human neural circuits
- Yuki Miura1,2,
- Min-Yin Li1,2,
- Omer Revah1,2,
- Se-Jin Yoon1,2,
- Genta Narazaki1,
- Sergiu Pasca1,2
- 1Department of Psychiatry and Behavioral Sciences, Stanford University, Stanford, CA, USA;
- 2Stanford Brain Organogenesis, Wu Tsai Neurosciences Institute, Stanford University, Stanford, CA, USA.
Protocol Citation: Yuki Miura, Min-Yin Li, Omer Revah, Se-Jin Yoon, Genta Narazaki, Sergiu Pasca 2022. Engineering brain assembloids to interrogate human neural circuits. protocols.io https://dx.doi.org/10.17504/protocols.io.36wgq4xxkvk5/v1
Manuscript citation:
https://www.nature.com/articles/s41596-021-00632-z
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: November 01, 2021
Last Modified: April 21, 2022
Protocol Integer ID: 54690
Keywords: human neural circuits, brain assembloids, viral labeling, retrograde tracing, 3D live imaging of axon projection, optogenetics
Disclaimer
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Abstract
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.
Guidelines
Troubleshooting
| A | B | C | D | |
| 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 optimal 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 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.
Materials
Biological materials
hPS cells
REAGENT
DMEM/F-12, HEPESThermo Fisher ScientificCatalog #11330032
Essential 8™ MediumThermo FisherCatalog #A1517001
Essential 6™ MediumThermo Fisher ScientificCatalog #A1516401
Neurobasal™-A MediumThermo FisherCatalog #10888022
B-27™ Supplement (50X), minus vitamin AThermo FisherCatalog #12587010
CRITICAL The B-27 supplement must be without vitamin A.
B-27™ Plus Supplement (50X)Thermo FisherCatalog #A3582801
GlutaMAX™ SupplementThermo Fisher ScientificCatalog #35050061
Penicillin-Streptomycin (10,000 U/mL)Thermo Fisher ScientificCatalog #15140122
CRITICAL: Differentiations can also be performed without penicillin-streptomycin, but the risk of contamination, especially in long-term cultures, will be higher.
●ROCK inhibitorY-27632SelleckchemCatalog #S1049
●SMAD inhibitorDorsomorphinSigma AldrichCatalog #P5499-5MG
SB 431542TocrisCatalog #1614
XAV 939TocrisCatalog #3748
Recombinant Human EGF Protein CFR&D SystemsCatalog #236-EG
Recombinant Human FGF basic/FGF2/bFGF (146 aa) ProteinR&D SystemsCatalog #233-FB
IWP-2SelleckchemCatalog #S7085
Recombinant Human/Murine/Rat Activin A (CHO derived)peprotechCatalog #120-14P
SR 11237TocrisCatalog #3411
Recombinant Human/Murine/Rat BDNFpeprotechCatalog #450-02
Recombinant Human NT-3peprotechCatalog #450-03
L-Ascorbic Acid 2-Phosphate Trisodium SaltFUJIFILM Wako Pure Chemical CorporationCatalog # 323-44822
Db-cAMP (dibutyryl-cyclic AMP)SigmaCatalog #D0627
cis-4710131619-Docosahexaenoic acidSigma AldrichCatalog #D2534-25MG
DAPT 5 mg
Stemcell TechnologiesCatalog #72082
Cell culture reagents
Vitronectin (VTN-N) Recombinant Human Protein, TruncatedThermo FisherCatalog #A14700
DPBS (no Ca, no Mg)ThermofisherCatalog #14190144
0.5M EDTA solutionThermo Fisher ScientificCatalog #15575020 )
Dimethyl sulfoxideSigma AldrichCatalog #D2650
Anti-Adherence Rinsing SolutionStemcell TechnologiesCatalog #07010
Accutase (cell detachment solution) Innovative Cell Technologies, IncCatalog #AT-104
Corning® 500 mL Cell Culture Grade Water Tested to USP Sterile Water for Injection SpecificationsCorningCatalog #25-055-CV
● Ethyl ethanol 200 proof (Gold Shield, cat. no. 412804)
Safety information
Ethyl ethanol is flammable and should be stored in a flame-protective cabinet.
For cryosection and immunostaining only
16% ParaformaldehydeElectron Microscopy SciencesCatalog #15710
Safety information
PFA should be stored in a safety cabinet and should be disposed of according to institutional guidelines after use.
SucroseSigma AldrichCatalog #S9378
Tissue-Plus™ O.C.T. CompoundFisher ScientificCatalog #23-730-571
Triton X-100Sigma AldrichCatalog #T8787-50ML
Donkey SerumEmd MilliporeCatalog #S30-100ML
Recombinant Anti-DARPP32 antibody [EP720Y] (ab40801)AbcamCatalog #ab40801
Anti-GAD67 Antibody clone 1G10.2Merck MilliporeCatalog #MAB5406
Anti-Ctip2 antibody [25B6] (ab18465)AbcamCatalog #ab18465
Anti-NeuN antibody [1B7] - Neuronal MarkerAbcamCatalog #ab104224
GFP antibodyGenetexCatalog #GTX13970
mCherry antibodyGenetexCatalog #GTX128508
MAP 2 antibody - 188 004Synaptic SystemsCatalog #188 004
Anti-SATB2 antibody [SATBA4B10] - C-terminal (ab51502)AbcamCatalog #ab51502
Donkey anti-Rabbit IgG (H L) Highly Cross-Adsorbed Secondary Antibody Alexa Fluor 488Thermo Fisher ScientificCatalog #A-21206
Donkey anti-Mouse IgG (H L) Highly Cross-Adsorbed Secondary Antibody Alexa Fluor 568Thermo Fisher ScientificCatalog #A10037
Alexa Fluor® 647 AffiniPure Donkey Anti-Rat IgG (H L)Jackson ImmunoresearchCatalog #712-605-153
Alexa Fluor® 488 AffiniPure Donkey Anti-Chicken IgY (IgG) (H L)Jackson ImmunoresearchCatalog #703-545-155
Donkey anti-Rabbit IgG (H L) Highly Cross-Adsorbed Secondary Antibody Alexa Fluor 568Thermo Fisher ScientificCatalog #A10042
Alexa Fluor® 647 AffiniPure Donkey Anti-Guinea Pig IgG (H L) (min X Bov Ck Gt Sy Hms Hrs Hu MsJackson ImmunoresearchCatalog #706-605-148
AF647 Donkey anti-mouse IgGThermofisherCatalog #A-31571
Hoechst 33258Thermo Fisher ScientificCatalog #H3569
Aqua-Poly/MountPolysciencesCatalog #18606-100
For clearing and 3D staining of assembloids only
Tissue-Clearing Reagent CUBIC-L [for Animals]TCI ChemicalsCatalog #T3740
Tissue-Clearing Reagent CUBIC-R (M) [for Animals]TCI ChemicalsCatalog #T3741
For viral labeling only (see Table 1 for full details)
- AAV-DJ-hSyn1::eYFP (Stanford University Neuroscience Gene Vector and Virus Core, cat. no. GVVC-AAV-16)
- AAV-DJ-hSyn1::mCherry (Stanford University Neuroscience Gene Vector and Virus Core, cat. no. GVVC-AAV-17)
- Rabies-ΔG-Cre-eGFP (Salk Institute Viral Vector Core)
- pAAV-EF1a-CVS-G-WPRE-pGHpAaddgeneCatalog #67528 )
- AAV-DJ-EF1-DIO-mCherry (Stanford University Neuroscience Gene Vector and Virus Core, cat. no. GVVC-AAV-14)
- AAV1-Syn::ChrimsonR-tdT (Addgene, cat. no. 59171-AAV1)
- AAV-DJ-Ple94 (GPR88)-iCre (Addgene, cat. no. 49125)
- AAV-DJ-EF1a-DIO-GCaMP6s (Stanford University Neuroscience Gene Vector and Virus Core, cat. no. GVVC-AAV-91)
Table 1:
| A | B | C | D | E | F | |
| Virus and serotype | Gene of interest | Target cell type | Stock concentration | Working concentration | ||
| AAV-DJ-Syn1::eYFP | AAV-DJ | eYFP | Neuron | 4.56 × 10^12 Vg/ml | 1.14 × 10^10 Vg/ml | |
| AAV-DJ-Syn1::mCherry | AAV-DJ | mCherry | Neuron | 1.50 × 10^13 Vg/ml | 3.75 × 10^10 Vg/ml | |
| AAV-DJ-EF1-DIO-mCherry | AAV-DJ | mCherry | Any | 1.60 × 10^13 Vg/ml | 4.00 × 10^10 Vg/ml | |
| AAV-DJ-EF1a::G | AAV-DJ | Rabies glycoprotein | Any | 3.00 × 10^12–2.08 × 10^13 Vg/ml | 7.50 × 10^9-5.20 × 10^10 Vg/ml | |
| AAV1-hSyn-ChrimsonR-tdT | AAV1 | ChrimsonR-tdTomato | Neuron | 2.10 × 10^13 Vg/ml | 5.25 × 10^10 Vg/ml | |
| AAV-DJ-Ple94-iCre | AAV-DJ | iCre | Neuron | 1.09 × 10^12 Vg/ml | 2.73 × 10^9 Vg/ml | |
| AAV-DJ-EF1a-DIO-GCaMP6s | AAV-DJ | GCaMP6s | Any | 1.60 × 10^13 Vg/ml | 4.00 × 10^10 Vg/ml | |
| ΔG-Rabies-Cre-eGFP | G-deletedrabies virus | Cre-eGFP | Any | 6.52 × 10^8 TU/ml | 1.63 × 10^5 TU/ml | |
For whole-cell patch-clamp recording only
LOW MELT AGAROSEIBI ScienticCatalog #IB70051 )
Sodium chloride Sigma AldrichCatalog #S7653
KClMillipore SigmaCatalog #P9333
Sodium phosphate monobasic monohydrateSigma AldrichCatalog #S9638 )
Magnesium sulfate heptahydrate (MgSO4)Millipore SigmaCatalog #63138
CaCl2 solution Sigma AldrichCatalog #21115
Sodium BicarbonateSigma AldrichCatalog #S6297
D-( )-glucose Sigma AldrichCatalog #G7528 )
Potassium gluconate Sigma AldrichCatalog #P1847
Adenosine 5′-triphosphate magnesium saltSigma AldrichCatalog #A9187
Sodium GTPSigma AldrichCatalog #G8877 (MilliporeSigma, cat. no. G8877)
HEPESSigma AldrichCatalog #H3375
Ethylene glycol-bis(2-aminoethylether)-NNN′N′-tetraacetic acidSigma AldrichCatalog #03777
Albumin from Bovine Serum (BSA) Alexa Fluor™ 488 conjugateThermo Fisher ScientificCatalog #A13100
BrainPhys™ Neuronal MediumStem Cell TechnologiesCatalog #05790
BrainPhys without phenol red Stemcell TechnologiesCatalog # 05791
EQUIPMENT
Equipment
AggreWell™800
NAME
Microwell Culture Plates
TYPE
AggreWell
BRAND
34815
SKU
LINK
Equipment
Corning® Primaria™ 100 mm x 20 mm Standard Cell Culture Dish
NAME
Cell Culture Dish
TYPE
Corning® Primaria
BRAND
353803
SKU
LINK
Equipment
Corning® 15 mL PP Centrifuge Tubes
NAME
Corning® 15 mL PP Centrifuge Tubes, Bulk Packed with CentriStar™ Cap, Sterile, 50/Sleeve, 500/Case
TYPE
Corning®
BRAND
430791
SKU
LINK
Equipment
Corning® 50 mL Centrifuge Tubes
NAME
Corning® 50 mL PP Centrifuge Tubes, Conical Bottom with CentriStar™ Cap, Bulk Packed, Sterile, 25/Pack, 500/Case
TYPE
Corning®
BRAND
430829
SKU
LINK
Equipment
Corning® 100 mm Ultra-Low Attachment Culture Dish
NAME
Culture Dish
TYPE
Corning®
BRAND
Discontinued: 3262; Suggested Replacement: 4615
SKU
LINK
;
Equipment
Corning® 60 mm Ultra-Low Attachment Culture Dish
NAME
Ultra-Low Attachment Culture Dish
TYPE
Corning®
BRAND
3261
SKU
LINK
Equipment
Corning® Costar® Ultra-Low Attachment Multiple Well Plate
NAME
size 6 well, flat bottom clear Ultra-Low Attachment Multiple Well Plate
TYPE
Corning®
BRAND
CLS3471-24EA
SKU
LINK
Equipment
Costar® 24-well Clear Flat Bottom Ultra-Low Attachment Multiple Well Plates
NAME
24-well Clear Flat Bottom Ultra-Low Attachment Multiple Well Plates
TYPE
Corning®
BRAND
3473
SKU
LINK
Equipment
Fisherbrand™ Premium Microcentrifuge Tubes: 1.5mL
NAME
Microcentrifuge Tubes
TYPE
Fisherbrand™
BRAND
05408129
SKU
LINK
Note
Autoclave tubes before use.
Equipment
5 mL Serological Pipet, Polystyrene, 0.1 Increments, Individually Packed, Sterile, 50/Bag, 200/Case
NAME
Serological Pipet, Polystyrene
TYPE
Falcon®
BRAND
356543
SKU
LINK
Equipment
10 mL Serological Pipet, Polystyrene, 0.1 Increments
NAME
Serological Pipet, Polystyrene
TYPE
Falcon®
BRAND
356551
SKU
LINK
Equipment
Falcon® 25 mL Serological Pipet, Polystyrene, Space Saver, 0.25 Increments, Individually Packed, Sterile, 50/Bag, 200/Case
NAME
Serological Pipet, Polystyrene
TYPE
Falcon®
BRAND
356525
SKU
LINK
Equipment
Falcon® 50 mL Serological Pipet, Polystyrene, 1.0 Increments, Individually Wrapped, Sterile, 25/Pack, 100/Case
NAME
50 mL Serological Pipet, Polystyrene, 1.0 Increments
TYPE
Falcon®
BRAND
356550
SKU
LINK
Equipment
Low Retention Aerosol Barrier Pipette Tip MBP® ART® SoftFit-L™ 1,000 µL Sterile
NAME
TIP, PIPETTE ART SOFTFIT-L HINGED RACK 1000UL
TYPE
MBP® ART® SoftFit-L™
BRAND
2779-HR
SKU
LINK
Equipment
Aerosol Barrier Pipette Tip MBP® ART® SoftFit-L™ 200 µL Sterile
NAME
TIP, PIPETTE W/FILTER HINGED COVER 200UL-200L (5PK/CS)
TYPE
MBP® ART® SoftFit-L™
BRAND
2769-HR
SKU
LINK
Equipment
Aerosol Barrier Pipette Tip MBP® ART® SoftFit-L™ 20 µL Sterile
NAME
TIP, PIPETTE W/FILTER HINGED COVER 200UL-20L (5PK/CS)
TYPE
MBP® ART® SoftFit-L™
BRAND
2749-HR
SKU
LINK
● Sterilized scissors
Equipment
VWR® Air Jacketed CO2 Incubators
NAME
Air Jacketed CO2 Incubator
TYPE
VWR®
BRAND
10810-902
SKU
LINK
● Biological safety cabinet (Labconco, cat no. 97000-862)
● Water bath
● Culture microscope (Olympus, cat. no. CKX41)
● EVOS FL cell imaging system (Life Technologies, model AMF4300)
● REVOLVE 4 upright, inverted, bright field, fluorescent microscope (Echo Laboratories, cat. no. FJSD2001)
● Confocal microscope (Leica, model TCS SP8)
● Stainless steel surgical blade, sterile, no. 10 (Medicon, cat. no. 01.22.10)
●
Equipment
Falcon® 40 µm Cell Strainer, Blue, Sterile, Individually Packaged, 50/Case
NAME
Cell Strainer
TYPE
Falcon®
BRAND
352340
SKU
LINK
●
Equipment
Corning® non-treated culture dishes
NAME
culture dishes
TYPE
Corning®
BRAND
CLS430589-500EA
SKU
LINK
● Hemocytometer
Equipment
Nylon Syringe Filter, 0.22μm, 30mm, Bulk Packed, Non-sterile
NAME
Syringe Filter
TYPE
Nylon
BRAND
229775
SKU
LINK
● Centrifuge 5810 R (Eppendorf, cat. no. 022628102)
For viral labeling and assembloid formation only
● Sterile 1.5-ml microcentrifuge
● Scissors and/or razor blades
For cryosection and immunostaining only
Equipment
VWR® Superfrost® Plus Micro Slide, Premium
NAME
Superfrost slides
TYPE
VWR® Superfrost®
BRAND
48311-703
SKU
LINK
Equipment
Fisherbrand™ Disposable Base Molds
NAME
Disposable Base Molds
TYPE
Fisherbrand™
BRAND
22363553
SKU
LINK
● Humidified chamber
Equipment
Elite PAP Pen
NAME
PAP Pen
TYPE
Elite
BRAND
K039
SKU
LINK
● Cover glasses, rectangles (Fisherbrand, cat. no. 22-266882)
●
Equipment
Leica CM1860 - Routine Histopathology Applications
NAME
Cryostats
TYPE
Leica
BRAND
CM1860
SKU
LINK
Equipment
Red Sable Brushes
NAME
Sable Brushes
TYPE
Sable
BRAND
11812
SKU
LINK
For optogenetics experiments only
Equipment
M625F2 - 625 nm, 13.2 mW (Min) Fiber-Coupled LED, 1000 mA, SMA
NAME
Fiber-Coupled LED
TYPE
M625F2
BRAND
M625F2
SKU
LINK
● CYCLOPS LED driver 3.6 (Open Ephys)
For whole-cell patch-clamp recording only
Equipment
Leica VT1200 - Semi-Automatic Vibrating Blade Microtome
NAME
Semi-Automatic Vibrating Blade Microtome
TYPE
Leica VT1200
BRAND
VT1200
SKU
LINK
● Microwave
● Upright SliceScope microscope (Scientifica)
● MultiClamp 700B amplifier (Molecular Devices)
● Digidata 1550B digitizer (Molecular Devices)
Equipment
P-1000 Next Generation Micropipette Puller
NAME
Micropipette Puller
TYPE
P-1000
BRAND
P-1000
SKU
LINK
● Thick/standard wall borosilicate glass with filament (Sutter Instrument, cat. no. BF150-86-10)
Equipment
pE-4000
NAME
universal Illumination System for fluorescence microscopy
TYPE
CoolLED
BRAND
pE-4000
SKU
LINK
● Infinity2 CCD camera (Teledyne Lumenera)
SOFTWARE
● Fiji (ImageJ, version 2.1.0, National Institutes of Health)
● MATLAB (version R2018a, 9.4.0, MathWorks)
● pCLAMP software (version 10.6, Molecular Devices)
● Mini Analysis software (version 6.0.3, Synaptosoft)
● Infinity Capture software (Teledyne Lumenera) compatible with the CCD camera
REAGENT SETUP
Growth factors and chemical stock solutions
BiologyResuspend 25 mg of dorsomorphin in 12.52 mL of sterile DMSO to obtain a 5 millimolar (mM) stock solution and use at a final concentration of 2.5 micromolar (µM) (1:2,000). Resuspend 10 mg of SB-431542 in 2.60 mL of 100% ethanol to obtain a 10 millimolar (mM) stock solution and use at a final concentration of 10 μM (1:1,000). Resuspend EGF, FGF2, BDNF and NT-3 growth factors in sterile 0.1% (wt/vol) BSA/PBS to 20 μg/ml stock concentration and use at 20 ng/ml (1:1,000). Resuspend 10 mg of IWP-2 in 8.58 mL of sterile DMSO to obtain a 2.5 millimolar (mM) stock solution; use at a final concentration of 2.5 micromolar (µM) (1:1,000). Resuspend 100 µg of activin A in2 mL of cell culture-grade water to obtain a50 µg/ml stock solution; use at a final concentration of 50 ng/ml (1:1,000). Resuspend 10 mg of SR11237 in 5.26 mL of sterile DMSO to obtain a5 millimolar (mM) stock solution and further dilute the stock solution to obtain a 5 micromolar (µM) solution and use at a final concentration of 5 nanomolar (nM) (1:1,000). Resuspend 322 mg of AA in 5 mL of cell culture-grade water to obtain a 200 millimolar (mM) stock solution; use at a final concentration of 200 micromolar (µM) (1:1,000). Resuspend 250 mg of dibutyryl cAMP sodium salt in 5.088 mL of cell culture–grade water to obtain a 100 millimolar (mM) stock solution and use at a final concentration of 50 micromolar (µM) (1:2,000). Resuspend 5 mg of DAPT in 2.31 mL of sterile DMSO to obtain a5 millimolar (mM) stock solution and use at a final concentration of 2.5 Milimolar (mM) (1:2,000). Aliquots of stock solutions are stored at either -20 °C or-80 °C for 2 months. Once thawed, stocks can be kept at4 °C for ≤336:02:00 .
Essential 8 medium (for use in Steps 1 –30). Prepare the medium as shown in the table below. 314 This medium can be stored for 2 weeks at 4 °C.
| A | B | C | |
| Composition | Volume (500 ml total) (ml) | Final concentration | |
| Essential 8 medium | 490 | – | |
| Essential 8 supplement | 10 | 2% (vol/vol) |
Essential 6 medium (for use in Steps 31–38). Prepare the medium as shown in the table below. This medium can be stored for 2 weeks at4 °C .
| A | B | C | D | |
| Composition | Volume (500 ml total) (ml) | Final concentration | Comments | |
| Essential 6 medium | 495 | – | ||
| Penicillin-streptomycin | 5 | 1% (vol/vol) | Optional | |
| Dorsomorphin | – | 2.5 μM (1:2,000) | Add just before use | |
| SB-431542 | – | 10 μM (1:1,000) | Add just before use | |
| XAV-939 (optional) | – | 1.25 µM (1:2,000) | Add just before use |
Neural differentiation medium (NM) for hCS (for use in Steps 39 and 40, on days 6 and onward of differentiation). Prepare the medium as detailed in the table below. Store stock for ≤168:02:00 at 4 °C and add growth factors just before use if required for the culture.
| A | B | C | D | |
| Composition | Volume (500 ml total) (ml) | Final concentration | Comments | |
| Neurobasal A | 480 | – | ||
| B-27 supplement | 10 | 2% (vol/vol) | Needed from day 6 to day 46 | |
| B-27 Plus supplement | 10 | 2% (vol/vol) | Needed from day 46 onward | |
| GlutaMax | 5 | 1% (vol/vol) | – | |
| Penicillin-streptomycin | 5 | 1% (vol/vol) | – | |
| EGF (20-µg/ml 411 Q25 stock) | – | 20 ng/ml (1:1,000) | Needed from day 6 to day 22. Add just before use | |
| FGF2 (20-µg/ml stock) | – | 20 ng/ml (1:1,000) | Needed from day 6 to day 22. Add just before use | |
| BDNF (20-µg/ml stock) | – | 20 ng/ml (1:1,000) | Needed from day 22 to day 46. Add just before use | |
| NT-3 (20-µg/ml stock) | – | 20 ng/ml (1:1,000) | Needed from day 22 to day 46. Add just before use | |
| AA (200 mM stock) | – | 200 µM (1:1,000) | Needed from day 22 to day 46. Add just before use | |
| Dibutyryl cAMP sodium salt (100 mM stock) | – | 50 µM (1:2,000) | Needed from day 22 to day 46. Add just before use | |
| DHA (10 mM stock) | – | 10 µM (1:1,000) | Needed from day 22 to day 46. Add just before use |
NM for hStrSs (for use in Steps 39 and40, on days 6 and onward of differentiation). Prepare the medium as detailed in the table below. Store the stock for ≤1 week at 4 °C and add growth factors just before use if required for the culture.
| A | B | C | D | |
| Composition | Volume (~500 ml total) (ml) | Final concentration | Comments | |
| Neurobasal A | 480 | – | – | |
| B-27 supplement | 10 | 2% (vol/vol) | Needed from day 6 to day 46 | |
| B-27 Plus supplement | 10 | 2% (vol/vol) | Needed from day 46 onward | |
| GlutaMax | 5 | 1% (vol/vol) | – | |
| Penicillin-streptomycin | 5 | 1% (vol/vol) | Needed from day 6 to day 22. Add just before use | |
| IWP-2 (2.5 mM stock) | – | 2.5 μM (1:1,000) | Needed from day 6 to day 22. Add just before use | |
| Activin A (50-µg/ml stock) | – | 50 ng/ml (1:1,000) | Needed from day 6 to day 22. Add just before use | |
| SR11237 (100 µM stock) | – | 100 nM (1:1,000) | Needed from day 22 to day 46. Add just before use | |
| BDNF (20-µg/ml stock) | – | 20 ng/ml (1:1,000) | Needed from day 22 to day 46. Add just before use | |
| NT-3 (20-µg/ml stock) | – | 20 ng/ml (1:1,000) | Needed from day 22 to day 46. Add just before use | |
| AA (200 mM stock) | – | 200 µM (1:1,000) | Needed from day 22 to day 46. Add just before use | |
| Dibutyryl cAMP sodium salt (100 mM stock) | – | 50 µM (1:2,000) | Needed from day 22 to day 46. Add just before use | |
| DHA (10 mM stock) | – | 10 µM (1:1,000) | Needed from day 22 to day 46. Add just before use | |
| DAPT (5 mM stock) | – | 2.5 µM (1:2,000) | Needed from days 42 to 46. Add just before use |
PFA
Prepare 4% (vol/vol) PFA by mixing 10 mL of 16% PFA with 30 mL of DPBS and store at4 °C for
≤1 week.
Embedding solution
Prepare a 30% (wt/vol) sucrose solution by mixing 15 g of sucrose with PBS to a final volume of 50 mL . Store the 30% (wt/vol) sucrose/PBS solution at 4 °C for ≤2 weeks. Add 25 mL of OCT compound to 25 mL of 30% (wt/vol) sucrose/PBS to make a 1:1 embedding solution. Shake the solution atRoom temperature until the OCT compound and sucrose are completely mixed. Leave the mixed solution overnight at 4 °C to remove bubbles formed from the mixing process. This solution can be stored at4 °C for ≤2 weeks.
Blocking solution for immunostaining
Prepare the blocking solution for immunostaining by mixing 10% (vol/vol) normal donkey serum with 0.1% (wt/vol) BSA and 0.3% (vol/vol) Triton-X100 in PBS. Keep this solution at 4 °C for ≤1 week.
Antibody solution for immunostaining
Prepare the antibody solution for immunostaining by mixing 2% (vol/vol) normal donkey serum with 0.1% (vol/vol) Triton-X100 in PBS. Keep this solution at4 °C for ≤1 week.
Artificial cerebrospinal fluid (aCSF)
Prepare concentrated (10×) aCSF stock solution containing 1.26 Molarity (M) NaCl, 25 millimolar (mM) KCl, 12.5 millimolar (mM) NaH2PO4, 260 millimolar (mM) NaHCO3 and100 millimolar (mM) D-(+)-glucose. Keep this solution at4 °C for ≤1 month. Dilute this solution and add MgSO4 and CaCl2 to prepare aCSF working solution containing 126 millimolar (mM) NaCl, 2.5 millimolar (mM) KCl, 1.25 millimolar (mM) NaH2PO4, 1 millimolar (mM) MgSO4,2 millimolar (mM) CaCl2, 26 millimolar (mM) NaHCO3 and 10 millimolar (mM) D-(+)-glucose before use.
Internal solution for whole-cell recording
Prepare internal solution containing 127 millimolar (mM) K-gluconate,8 millimolar (mM) NaCl, 4 millimolar (mM) MgATP, 0.3 millimolar (mM) Na2GTP, 10 millimolar (mM) HEPES and 0.6 millimolar (mM) EGTA, pH 7.2, adjusted with KOH (290 mOsm). Keep this solution at -20 °C for ≤1 year.
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.
Maintenance of hPS cells in feeder-free conditions Timing 4–6 d
Maintenance of hPS cells in feeder-free conditions Timing 4–6 d
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.
Note
CRITICAL: To ensure consistency and obtain high-quality differentiation, the hPS cell culture should not grow denser than 90%. Ideally, passages can be performed at 80% confluency
Representative image of hiPS cell culture.
.
Regularly check hPS cell cultures for mycoplasma (e.g., PCR) and chromosomal abnormalities (e.g., SNP or comparative genomic hybridization arrays).
Preparing vitronectin-coated dishes
Preparing vitronectin-coated dishes
1h 30m
1h 30m
Prepare the vitronectin-coated plates at Room temperature and pre-warm the required volume of Essential 8 medium at Room temperature .
Note
CRITICAL: According to the manufacturer’s recommendation, Essential 8 medium should not be warmed in a 37 °C water bath.
To coat a well of a six-well plate, dilute 60 µL of vitronectin in 6 mL (1:100) of DPBS (without calcium and magnesium), add 1 mL of diluted vitronectin solution to the well and incubate at Room temperature for 01:00:00 .
Note
Vitronectin can be aspirated just before transferring the cell suspension to the well.
1h
Passaging hPS cells
Passaging hPS cells
30m
30m
Aspirate culture medium from the hPS cells plate and rinse with2 mL of DPBS (without calcium and magnesium).
Add 1 mL of 0.5 millimolar (mM) EDTA in DPBS to the well of a six-well plate and incubate at Room temperature for 00:07:00 .
Note
Cells are ready to be removed when colonies start to separate.
7m
Aspirate the EDTA solution and add 1 mL of pre-warmed complete Essential 8 medium per well of a six-well plate.
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.
Note
CRITICAL: Do not pipette too many times; three to five times should be enough. Difficulty with detaching cells suggests differentiation of hPS cells.
Aspirate the residual vitronectin solution from the pre-coated plate.
Note
It is not necessary to wash culture plates after the removal of vitronectin.
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).
Move the plate in several quick, figure-of-eight-like movements to disperse cells.
Gently place the plate in a 37 °C , 5% CO2 incubator.
Perform a medium change after every passage (every18:00:00 to24:00:00 ), except for the day after passage, when confluency of cells is low.
1d 18h
Preparation of hPS cells for 3D neural differentiation
Preparation of hPS cells for 3D neural differentiation
3d
3d
Passage hPS cells from a six-well plate to 10-cm plates and grow to ~80–85% confluency before the day of aggregation.
48:00:00 and 24:00:00 before the aggregation, pre-treat cells with 1% DMSO (100 µL per 10 mL of Essential 8 medium for a 10-cm dish) to improve differentiation of hPS cell lines that are more challenging to differentiate.
3d
Generation of 3D spheroids by using an AggreWell plate
Generation of 3D spheroids by using an AggreWell plate
3d
3d
Add 500 µL of anti-adherence rinsing solution in a well of a new AggreWell plate and incubate the plate for 00:05:00 at Room temperature If you notice (under the microscope) bubbles in any of the wells of the plate, centrifuge the AggreWell plate at1300 x g, 00:05:00 in a swinging bucket rotor. Aspirate the rinsing solution and wash with 1 mL of PBS.
10m
Pre-warm Essential 8, accutase and DMEM/F12 medium at Room temperature . Supplement Essential 8 medium with ROCK inhibitor (Y-27631; 1:1,000) to a final concentration of 10 micromolar (µM) .
Add 0.5 mL of 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, 00:05:00 in a swinging bucket rotor that is fitted with a plate holder to remove any air bubbles in the microwells.
Note
CRITICAL: Remaining air bubbles in the Aggrewell plate interrupt the aggregation of hPS cells.
5m
Set the plate aside in an incubator while preparing a single-cell suspension of hPS cells for aggregation.
Aspirate the maintenance medium from the hPS cell culture plates and rinse cells twice with DPBS (without calcium and magnesium).
Add 4 mL of accutase per 10-cm plate and incubate at 37 °C for 00:07:00 in a 5% CO2 incubator until cells detach by gentle shaking from the plate.
7m
Gently pipette the cell suspension two to three times with a serological pipette to ensure that any remaining clumps are fully dissociated.
Transfer the cells to a 50 mL conical tube.
Note
CRITICAL: The use of50 mL conical tubes for cell culture (instead of 15 mL conical tubes) is highly recommended to avoid the risk of contamination by accidentally attaching the side of the pipette to a wall of the conical tube when pipetting.
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 .
Count cells and centrifuge the conical tube containing the cell suspension at 200 x g, 00:04:00 .
4m
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 mL of cell suspension into a well of an AggreWell plate to achieve a final volume of 1.5 mL per well
Image of detached cells in an Aggrewell plate during the aggregation process.
.
Pipette gently several times to re-establish an even distribution of cells throughout the well.
Centrifuge the AggreWell plate at 100 x g, 00:03:00 to distribute the cells in the micro-wells.
Centrifugation of an Aggrewell plate.
3m
Check under a microscope to verify that cells are evenly distributed among the microwells.
hiPS cells in the Aggrewell plate after centrifugation.
Note
CRITICAL: Overfilled individual wells of the AggreWell plate after centrifugation will facilitate aggregation across wells and prevent the formation of individual spheroids.
Incubate the cells at 37 °C , 5% CO2 for 24:00:00 .
Note
TROUBLESHOOTING
1d
Dislodging 3D spheroids from the AggreWell plate ● Timing 1 h
Dislodging 3D spheroids from the AggreWell plate ● Timing 1 h
1w 0d 1h 30m
1w 0d 1h 30m
Pre-warm DMEM/F12 and Essential 6 medium at Room temperature for 00:30:00 to01:00:00 before dislodging.
1h 30m
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.
Aggregated hiPS cells days after aggregation in the Aggrewell plate.
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.
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.
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.
Note
CRITICAL: Difficulty with detaching cells from the Aggrewell plate suggests differentiation of hPS cells.
Place spheroids in ultra-low-attachment 10-cm plates in Essential 6 medium supplemented with dorsomorphine (5 micromolar (µM) ), SB-431542 (10 micromolar (µM) ).
Note
If forebrain induction (e.g., low FOXG1 expression) is challenging with spheroids derived from a specific hPS cell line, we recommend testing whether addition of XAV-939 (1.25 micromolar (µM) ) from days24:00:00 to 144:00:00 of differentiation may facilitate this process.
Representative images of spheroids.
Neural induction
Neural induction
1w 3d
1w 3d
Add dorsomorphine and SB-431542 to Essential 6 medium for the first 120:00:00 . XAV-939 (1.25 micromolar (µM) ) can also be added for the first 120:00:00 of differentiation if forebrain differentiation is not highly efficient.
1w 3d
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.
Note
CRITICAL: The time that spheroids are kept outside of the incubator should be minimized at all stages of differentiation. After they reach 4 mm in diameter, keep no more than 30 spheroids per 10-cm dish.
TROUBLESHOOTING
Patterning and differentiation
Patterning and differentiation
From day 6, replace the medium daily with NM with supplements listed in option A for hCSs and option B for hStrSs.
TROUBLESHOOTING
(A) Generation of hCSs
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.
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.
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.
(B) Generation of hStrSs
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.
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.
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.
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.
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.
Long-term culture and phenotyping ● Timing indefinite
Long-term culture and phenotyping ● Timing indefinite
18h 40m
18h 40m
After day 46, perform medium changes with 12–15 ml of NM without growth factors every 4–5 d.
Note
Examples of additional experiments that can be carried out by using these spheroids include viral labeling of spheroids (option A), generation of cortico-striatal assembloids (option B), 3D clearing with CUBIC (option C), axon projection imaging (option D), retrograde labeling with ΔG rabies virus (option E), optogenetics coupled with calcium imaging (option F) and electrophysiological recording (option G)
(A) Viral labeling of neural spheroids ● Timing 3 d
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.
Note
We note that transduction efficiency depends on the target cell types, the promoter, stage of differentiation and brain region. CRITICAL The optimal serotype and titer of each virus depends on the gene construct of interest, as well as the target cell type. AAV-1, AAV-8, and AAV-DJ serotypes have been more extensively tested in hCSs and hStrSs. Virus at a titer higher than 1.0 × 10^12–1.0 × 10^13 genome copies/ml was obtained from the Stanford Neurosciences Institute Gene Vector and Virus Core or from Addgene and stored at -80 °C . ΔG-rabies virus at a titer higher than 1.0 × 100.8 transduction units/ml was obtained from the Salk Institute, diluted to 1:10 by using NM and stored at -80 °C .
The next day, add an extra 800 µL of fresh NM to the tube and incubate for 24:00:00 .
1d
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.
Note
Expression of the gene of interest is usually observed 7–14 d after AAV infection and 3–5 d after ΔG-rabies virus infection. ? TROUBLESHOOTING
(B) Generating cortico-striatal assembloids ● Timing 3–4 d
To generate cortico-striatal assembloids, transfer one hCS and one hStrS into a 1.5 mL Eppendorf tube containing 1 mL of NM.
Scheme illustrating viral labeling and assembly of an hCS and an hStrS, and images showing the process of assembly.
Note
CRITICAL The size of the spheroids for assembly should be <3 mm. Once in the tube, each spheroid should be placed horizontally next to each other at the bottom of the tube (but not stacked vertically) (Fig. 2m).
Incubate the spheroids at 37 °C for 3–4 d, completely replacing the medium on day 2.
Note
CRITICAL The medium change should be performed gently to avoid breaking the fusion.
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.
Note
Assembly is usually complete after 3–4 d. ? TROUBLESHOOTING
(C) Cryosection and immunostaining ● Timing 6–7 d
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 Overnight .
1d
After fixation, gently remove PFA and wash with PBS at Room temperature .
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 (≥24–48 h).
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).
Note
CRITICAL To avoid creating bubbles while filling the mold, use a P1000 pipette in which the tip has been slightly cut to widen the opening.
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.
Note
CRITICAL Avoid carrying sucrose when transferring spheroids.
Place the spheroids in a cryomold On ice for 00:20:00 and allow the spheroids to sink to the bottom of the mold.
20m
Snap-freeze the spheroids by placing the mold directly on dry ice. Once completely frozen, store molds at -80 °C .
Note
Frozen molds can be stored indefinitely at -80 °C until ready to cryo-section.
When ready for sectioning, transfer the mold from -80 °C to the cryostat chamber at -20 °C for ~00:30:00 before sectioning.
30m
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 10 µm –30 µm thickness, depending on the purpose of the experiments.
Collect sections on Superfrost Plus slides. Slides can be stored at -20 °C to -80 °C until ready to immunostain.
For immunostaining, remove the slide from the freezer and leave at Room temperature to thaw for 00:05:00 –00:10:00
15m
Wash the slide three times with PBS to remove excess OCT/sucrose on the slides.
Remove PBS and block for 01:00:00 at Room temperature with 10% normal donkey serum (NDS), 0.3% Triton- X and 0.1% BSA in PBS.
Note
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.
1h
Perform the immunostaining steps in a humidified chamber to avoid drying of sections.
Add the primary antibodies diluted in antibody solution containing 2% NDS and 0.1% Triton-X in PBS and keep at 4 °C Overnight .
1h
The next day, perform three 00:05:00 PBS washes at Room temperature (1/3).
5m
The next day, perform three 00:05:00 PBS washes at Room temperature (2/3).
5m
The next day, perform three 00:05:00 PBS washes at Room temperature (3/3).
5m
Add secondary antibodies diluted in antibody solution for 01:00:00 at Room temperature .
Note
Parafilm can be placed on top of the slide glass to avoid drying.
1h
Perform three 00:05:00 washes at Room temperature with PBS (1/3).
5m
Perform three 00:05:00 washes at Room temperature with PBS (2/3).
5m
Perform three 00:05:00 washes at Room temperature with PBS (3/3).
5m
Add Hoechst in PBS (1:10,000 dilution) for 00:05:00 –00:07:00 atRoom temperature .
12m
Aspirate the Hoechst solution and wash twice with PBS.
Aspirate PBS and mount the sections by adding one drop of Aquamount solution on top of sections and by placing a coverglass on top.
Use a fluorescent microscope to image.
Left: Image showing immunostaining for DARPP32 (yellow), GAD67 (magenta) and CTIP2 (cyan) in an hStrS at day 85. Right: Image showing immunostaining for DARPP32 (green), NeuN (cyan) and CTIP2 (magenta) in an hStrS at day 85.
Scheme illustrating retrograde viral labeling of cortical projecting neurons in cortico-striatal assembloids, and representative retrograde viral images of Daf21 cortico-striatal assembloids.
Note
CRITICAL Ideally, cryosections should be sampled from several layers of the spheroids or assembloids
(D) CUBIC clearing and imaging ● Timing 4–7 d
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 Room temperature for 00:20:00 ). Leave spheroids at 4 °C Overnight .
40m
After fixation, wash samples with PBS twice at Room temperature .
Add 500 µL to 1 mL of CUBIC-L for de-lipidation and incubate at 37 °C for 48:00:00 .
2d
Wash with PBS three times at Room temperature for 02:00:00 (1/3).
2h
Wash with PBS three times at Room temperature for 02:00:00 (2/3).
2h
Wash with PBS three times at Room temperature for 02:00:00 (3/3).
2h
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 02:00:00 .
2h
Wash with PBS three times at 37 °C for 02:00:00 (1/3).
2h
Wash with PBS three times at 37 °C for 02:00:00 (2/3).
2h
Wash with PBS three times at 37 °C for 02:00:00 (3/3).
2h
Add secondary antibodies diluted in solution containing 0.2% Triton-X100 and 3% NDS in PBS and incubate assembloids at 37 °C for 02:00:00 .
2h
Wash with PBS three times at Room temperature for 02:00:00 (1/3).
2h
Wash with PBS three times at Room temperature for 02:00:00 (2/3).
2h
Wash with PBS three times at Room temperature for 02:00:00 (3/3).
2h
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 02:00:00 at Room temperature .
2h
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
Scheme illustrating 3D clearing and imaging, and images of cleared assembloids and 3D stained cortico-striatal assembloids (eYFP: green; mCherry: magenta; MAP2: blue). RI, refractive index.
.
(E) Live imaging of axon growth ● Timing 2–8 h
Note
CRITICAL For live imaging of axon growth in assembloids, viral labeling and fusion are performed at days 60–65, and assembloids are imaged at 8, 14 and 21 d after fusion.
Using a cut P1000 tip, transfer assembloids to a well of a 96-well imaging-compatible plate in 150 µL –200 µL of NM.
Scheme and images illustrating viral labeling, assembly and live imaging of cortico-striatal assembloids after their transfer into 96-well plates.
Note
CRITICAL Avoid adding bubbles at the bottom of the wells. The distance from the lens to the sample is critical for imaging.
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 00:15:00 –00:30:00 before experiments to let assembloids settle at the bottom of the plate.
45m
Set up imaging positions with z-stacks and take images by using an ×10 objective lens at a depth of 0–500 µm.
Process fluorescent images with Fiji and quantify the percentage of eYFP coverage in hStrSs and the percentage of mCherry coverage in hCSs.
Representative images of Daf8–21 cortico-striatal assembloids labeled with eYFP and mCherry.
(F) Optogenetic stimulation and calcium imaging ● Timing 2–6 h
Note
CRITICAL: For optogenetic stimulation and calcium imaging in assembloids, viral labeling and fusion are performed at days 60–65, and assembloids are imaged at 30–90 d after fusion.
Transfer an assembloid onto a 200 mm glass coverslip in a 35 mm plate in NM and image by using an ×10 objective on a Leica TCS SP8 confocal microscope.
For optogenetic stimulation, activate ChrimsonR-tdTomato+ cells in hCSs with 625 nm light by using an optical fiber-coupled LED. Our stimulation experiments included 1,500 frames, and one 625 nm pulse of LED light (68 ms) was applied every 150 frames as generated by a Cyclops LED connected to the LEICA TCS SP8 microscope.
Scheme illustrating viral labeling and assembly for optogenetics coupled with calcium imaging, representative images of imaging setup during optogenetic stimulation using a 625-nm LED.
Image GCaMP6 at a frame rate of 14.7 frames/s.
Representative image and trace of GCaMP6s signal in response to LED stimulation.
Process raw GCaMP6 signals with Fiji.
After registration of ROIs, transform raw time series to relative changes in fluorescence by using the following formula: ΔF/F = (Ft – F0)/F0.
To verify whether responses are time-locked to LED stimulation, compare ΔF/F responses to ΔF/F values obtained at randomly selected time points.
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).
Quantification of stimulation-dependent response.n=180 cells from 10 assembloids with three hiPS cell lines; two-tailed Wilcoxon test. ***P=0.0002. Stim, stimulation.
TROUBLESHOOTING
(G) Whole-cell electrophysiological recording ● Timing 2–6 h
Note
CRITICAL For whole-cell electrophysiological recording in assembloids, viral labeling and fusion are performed at days 60–65, and recordings are performed at 30–60 d after fusion.
Add 0.2 g of low-melt agarose into a 35 mm plate containing 5 mL of aCSF and mix well (4% agarose). Dissolve low-melt agarose by using a microwave (avoid spillover due to overheating).
Note
CRITICAL:
- Usually 10 s is sufficient.
- STEP BrainPhys can be used in place of aCSF.
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.
Images showing the process of agarose embedding and slicing of cortico-striatal assembloids.
Note
CRITICAL: Avoid transferring the medium to prevent changes in the local concentration of agarose. Cool down agarose at 4 °C for 00:10:00 until it becomes solid.
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 200 µm to 250 µm thickness with cold aCSF in a vibratome. Transfer slices into aCSF (at Room temperature bubbled with 95% O2/5% CO2).
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)
Note
CRITICAL: Use glass pipettes with small tips instead of large tips.
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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 00:00:05 to 00:00:10 and, after drying for 00:10:00 , fill pipettes with internal solution and patch.
DIC (top) andfluorescent (bottom) images of sliced cortico-striatal assembloids expressing eYFP in an hStrS.
Representative image of patching from hSyn::eYFP+neurons with a non-fluorescently labeled pipette, and image showing afluorophore-coated patch pipette.
10m 15s
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.
Note
CRITICAL: It is not always necessary to form a ‘dimple’ to get a Giga-seal; in most cases, an increase in the pipette resistance is suggestive of contact of the pipette tip with the cell membrane.
For recording optically evoked responses in assembloids, apply light at the maximal power through the 40× objective by using a CoolLED (480 nm for ChR2 and 550 nm for Chrimson). CoolLED is controlled by a protocol in pClamp.
Representative traces of oEPSC, oEPSP and spikes from striatal neurons in cortico-striatal assembloids. oEPSC, optically evoked excitatory postsynaptic current; oEPSP, optically evoked excitatory postsynaptic potential.
Note
CRITICAL: Always check opsin expression before recording.
Make recordings at 30 °C to 37 °C orRoom temperature within 05:00:00 to06:00:00 after sectioning.
TROUBLESHOOTING
11h