May 26, 2020
Generation of hCS from hiPSC maintained in feeder-free conditions
This protocol is a draft, published without a DOI.
- Sejin Yoon1,2,3,
- Sergiu P. Pașca1,2,3
- 1Department of Psychiatry and Behavioral Sciences, Stanford University School of Medicine, Stanford, CA, USA;
- 2Human Brain Organogenesis Program, Stanford University, Stanford, CA, USA;
- 3e-mail: spasca@stanford.edu
- Neurodegeneration Method Development CommunityTech. support email: ndcn-help@chanzuckerberg.com
Protocol Citation: Sejin Yoon, Sergiu P. Pașca 2020. Generation of hCS from hiPSC maintained in feeder-free conditions. protocols.io https://protocols.io/view/generation-of-hcs-from-hipsc-maintained-in-feeder-bbxuipnw
Manuscript citation:
Yoon, S., Elahi, L.S., Pașca, A.M. et al. Reliability of human cortical organoid generation. Nat Methods 16, 75–78 (2019). https://doi.org/10.1038/s41592-018-0255-0
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: January 29, 2020
Last Modified: May 26, 2020
Protocol Integer ID: 32468
Keywords: Three-dimensional Culture, 3D Culture, Brain, Human, hCSs, hSSs, hiPSC
Abstract
Here, we show the generation of 3D human cortical spheroids (hCS) from human induced pluripotent stem cells (hiPSC) maintained in feeder-free, xeno-free conditions. This protocol describes hiPSC maintenance, generation of 3D cell aggregates in microwells, and cortical differentiation into hCS.
Attachments
Guidelines
Figure 1. Schematic showing the protocol for generating hCS-FF from hiPSC
Materials
MATERIALS
DPBS (no Ca, no Mg)ThermofisherCatalog #14190144
Penicillin-Streptomycin (10,000 U/mL)Thermo Fisher ScientificCatalog #15140122
Accutase®, 100 mlInnovative Cell Technologies, IncCatalog #AT104
Essential 8™ MediumGibco - Thermo Fisher ScientificCatalog #A1517001
Recombinant Human/Murine/Rat BDNFpeprotechCatalog #450-02
Recombinant Human NT-3peprotechCatalog #450-03
DMEM/F-12, HEPESThermo Fisher ScientificCatalog #11330032
Vitronectin (VTN-N) Recombinant Human Protein, TruncatedThermo FisherCatalog #A14700
Neurobasal™-A MediumThermo ScientificCatalog #10888022
Essential 6™ MediumThermo Fisher ScientificCatalog #A1516401
UltraPure™ 0.5 M EDTA pH 8.0Thermo Fisher ScientificCatalog #15575020
B-27™ Supplement (50X) minus vitamin AThermo Fisher ScientificCatalog #12587010
GlutaMAX™ SupplementThermo Fisher ScientificCatalog #35050061
DorsomorphinMerck MilliporeSigma (Sigma-Aldrich)Catalog #P5499-5MG
SB 431542TocrisCatalog #1614
Y-27632SelleckchemCatalog #S1049
Recombinant Human EGF Protein CFR&D SystemsCatalog #236-EG
Recombinant Human FGF basic/FGF2/bFGF (146 aa) ProteinR&D SystemsCatalog #233-FB
XAV 939TocrisCatalog #3748
Anti-Adherence Rinsing SolutionSTEMCELL Technologies Inc.Catalog #07010
Stock Solutions
| Growth Factors and small molecules | ||
| Dorsomorphin (2.5 μM); dissolved in DMSO | Sigma P5499-5MG | |
| SB-431542 (10 μM); dissolved in Ethanol | Tocris 1614 | |
| Y-27632 (10 μM) | Selleckchem S1049 | |
| EGF (20 ng/mL) | R&D 236-EG | |
| FGF2 (20 ng/mL) | R&D 233-FB | |
| BDNF (20 ng/mL) | Peprotech 450-02 | |
| NT-3 (20 ng/mL) | Peprotech 450-03 | |
| XAV 939 (1.2 μM); dissolved in DMSO | Tocris 3748 | |
Equipment
Cell culture dishes and plates
Equipment
AggreWell™800
NAME
Microwell culture plate
TYPE
AggreWell™
BRAND
34811
SKU
LINK
Equipment
Falcon® 40 µm Cell Strainer
NAME
Cell Strainer
TYPE
Falcon
BRAND
352340
SKU
LINK
Equipment
100 mm Ultra-Low Attachment Culture Dish
NAME
Treated Culture Dishes
TYPE
Corning®
BRAND
3262
SKU
LINK
Equipment
Primaria™ 100 mm x 20 mm Standard Cell Culture Dish
NAME
Enhanced Tissue Culture Surfaces
TYPE
Corning®
BRAND
353803
SKU
LINK
Equipment
6-well Clear TC-treated Multiple Well Plates
NAME
96 Well Microplates
TYPE
Costar®
BRAND
3506
SKU
LINK
Safety warnings
See SDS (Safety Data Sheet) for safety warnings and hazards.
Before start
Maintenance of feeder-free (FF) hiPSC
Maintenance of feeder-free (FF) hiPSC
Human induced pluripotent stem cells (hiPSC) are cultured on vitronectin in Essential 8™ (E8) medium and are passaged every 4–5 days using EDTA.
For passaging hiPSC, coat wells of a 6-well plate by diluting 60 µL vitronectin in 6 mL DPBS (1:100 dilution) and adding 1 mL of diluted vitronectin solution per well.
Keep it at Room temperature for 01:00:00 .
Aspirate medium and rinse with 3 mL –4 mL DPBS per well.
Add 1 mL of 0.5 millimolar (mM) EDTA .
Incubate for 00:07:00 at Room temperature .
Aspirate the EDTA solution and add 2 mL pre-warmed E8 .
Remove cells by gently squirting medium and pipetting the colonies with a 5 ml serological pipette.
Note
Avoid the generation of bubbles.
Aspirate the residual vitronectin solution from the pre-coated dish and add 2 mL pre-warmed E8 medium .
Mix the cell suspensions by gently inverting several times, then transfer the appropriate volume into each well containing pre-warmed E8 medium according to the desired split ratio.
Gently place the plate into a 37 °C , 5 % CO2 incubator.
No media change should be performed after the day of passage. Afterwards, replace medium daily (2 mL –2.5 mL per well).
Note
Cultures should be checked regularly for Mycoplasma contamination and the presence of genomic abnormalities.
[Optional step] Differentiation day –2: DMSO pre-treatment
[Optional step] Differentiation day –2: DMSO pre-treatment
Two days prior to starting neural differentiation, and one day prior to spheroid formation, pre-treat hiPSC with 1 % volume DMSO (120 μl per 12 ml of E8 medium for one 100 mm culture dish).
Note
This stage is optional and based on reference:Chetty, S. et al. A simple tool to improve pluripotent stem cell differentiation. Nature Methods 10, 553-556, doi:10.1038/nmeth.2442 (2013).
Differentiation day –1: Generation of 3D spheroids from hiPSC maintained in FF
Differentiation day –1: Generation of 3D spheroids from hiPSC maintained in FF
To generate spheroids, passage hiPSC from a 6-well to a 100 mm culture dish and culture them to 80–90 % confluency.
Pre-warm E8 medium, Accutase, and DMEM/F-12 at Room temperature .
Supplement E8 medium with the ROCK inhibitor (Y-27632, 1:1000) to a final concentration of 10 micromolar (µM) .
Pre-treat wells with 500 µL Anti-Adherence Rinsing Solution to each well and centrifuge at 1300 x g for 00:05:00 in a swinging bucket rotor fitted with plate holders.
Check under a microscope to ensure that bubbles have been removed from microwells and rinse each well with 2 mL warm DMEM/F-12 medium .
Remove the wash medium, and add 1 mL per well of E8 supplemented with Y-27632. Set plate aside in an incubator while preparing the single cell suspensions of hiPSC.
Aspirate maintenance medium from the hiPSC plates and rinse cells twice with DPBS (no calcium, no magnesium).
Add 4 mL Accutase per 100 mm culture plate.
Incubate for 00:07:00 at 37 °C , 5 % CO2 incubator.
Add pre-warmed E8 medium up to 10 ml volume.
Centrifuge the cell suspension at 200 x g for 00:04:00 .
Resuspend the pellet with E8 medium and count cell number.
Centrifuge the cell suspension at 200 x g for 00:04:00 .
Resuspend the pellet with pre-warmed E8 medium supplemented with Y-27632 to obtain 3 million cells per 1 ml of medium.
Add 1 mL of this cell suspension to the previously prepared AggreWell plate, which contains 1 ml of E8 medium supplemented with Y-27632. Each well of AggreWell™800 plate contains 300 microwells, and one microwell will have 10,000 cells.
Centrifuge the AggreWell™800 plate at 100 x g for 00:03:00 to distribute the cells in the microwells.
Incubate for 24:00:00 at 37 °C , in a 5 % CO2 incubator.
Differentiation day 0: Dislodging and harvesting aggregated spheroids
Differentiation day 0: Dislodging and harvesting aggregated spheroids
Harvest the hiPSC-derived spheroids from the microwells by firmly pipetting medium in the well up and down with a 1 ml disposable tip that has been cut.
Place a 40 μm strainer on a 50 ml conical tube and pass the suspension of spheroids through the strainer.
Pipette 1 mL DMEM/F-12 medium across the entire surface of the well to dislodge any remaining spheroids. Collect washes and pass over the strainer until every spheroids are recovered by checking under a microscope.
Invert the strainer, and place over a new 50 ml conical tube. Collect the spheroids by washing with Essential 6™ (E6) medium for neural induction.
Observe the AggreWell™800 plate under the microscope to ensure that all aggregates have been removed from the wells. Repeat wash if necessary.
Neural Differentiation
Neural Differentiation
Harvested spheroids are placed in ultra-low attachment 100 mm plates in E6 medium supplemented with 2.5 micromolar (µM) Dorsomorphin (DM) and 10 micromolar (µM) SB-431542 (SB) .
Note
Optionally, 1.2 micromolar (µM) XAV 939 (XAV) can be added for the first five days.
Media changes are performed daily, except for day 1.
On day 6, E6 medium containing DM and SB is replaced with neural medium (NM) supplemented with EGF2 (20 ng/ml ) and FGF2 (20 ng/ml ) for the 19 days with daily medium change in the first 10 days, and every other day medium changes for the subsequent 9 days.
| Neural Medium (NM) Composition | Volume (∼ 500 ml) | |
| Neurobasal™-A Medium | 480 ml | |
| B-27™ Supplement (50X), minus vitamin A | 10 ml | |
| GlutaMAX™ Supplement (1:100) | 5 ml | |
| Penicillin-Streptomycin (10,000 U/mL) | 5 ml |
To promote differentiation of the neural progenitors into neurons, FGF2 and EGF are replaced with 20 ng/ml BDNF and 20 ng/ml NT-3 starting at day 25 (with media changes every other day).
From day 43 onwards only NM without growth factors is used for medium changes every four days or as needed.