Generating Mature Cerebellar Organoids From Induced Pluripotent Stem Cells Using Single-Use Bioreactors

Samenvatting

Source: Silva, T.P., et al. Scalable Generation of Mature Cerebellar Organoids from Human Pluripotent Stem Cells and Characterization by Immunostaining. J. Vis. Exp. (2020)

This video demonstrates a technique for generating mature cerebellar organoids from induced pluripotent stem cells (iPSCs) using a single-use 3D bioreactor. The bioreactor facilitates iPSC aggregate formation, which then undergoes treatment with a series of culture media enriched in growth factors and chemokines that induce the differentiation of the iPSC aggregates into mature cerebellar organoids. Subsequently, the organoids are harvested and cryopreserved for further analysis.

Protocol

1. Seeding of human iPSCs in the bioreactor

1. Incubate iPSCs grown as monolayers in mTeSR1 supplemented with 10 µM of ROCK inhibitor Y-27632 (ROCKi). Add 1 mL of supplemented medium to each well from a 6-well tissue culture plate and incubate for 1 h at 37 °C, 95% humidity, and 5% CO₂.
   NOTE: ROCKi is used to protect dissociated iPSCs from apoptosis.
2. After 1 h of incubation, aspirate the spent medium from each well and wash 1x with 1 mL of 1× PBS per well.
3. Add 1 mL of the cell detachment medium (see Table of Materials) to each well of a 6-well plate and inc....

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
BrainPhys Neuronal Medium N2-A & SM1 kit	5793 - 500mL	Stem cell tecnhnologies
Chemically defined lipid concentrate	11905031	ThermoFisher
Crystallization-purified BSA	5470	Sigma
DMEM-F12	32500-035	ThermoFisher
Fetal bovine serum	A3840001	ThermoFisher
Gelatin from bovine skin	G9391	Sigma
Glutamax I	10566-016	ThermoFisher
Dibutyryl cAMP	SC- 201567B -500mg	Frilabo
Ham's F12	21765029	ThermoFisher
Human Episomal iPSC Line	A18945	ThermoFisher	iPSC6.2
IMDM	12440046	ThermoFisher
Insulin	91077C	Sigma
iPS DF6-9-9T.B		WiCell
Iso-pentane	PHR1661-2ML	Sigma
L-Ascorbic acid	A-92902	Sigma
Matrigel	354230	Corning	basement membrane matrix
Monothioglycerol	M6154	Sigma
mTeSR1	85850 -500ml	Stem cell technologies
N2 supplement	17502048	ThermoFisher
Neurobasal	12348017	ThermoFisher
Paraformaldehyde	158127	Sigma
PBS-0.1 Single-Use Vessel	SKU: IA-0.1-D-001	PBS Biotech
PBS-MINI MagDrive Base Unit	SKU: IA-UNI-B-501	PBS Biotech
Recombinant human BDNF	450-02	Peprotech
Recombinant human bFGF/FGF2	100-18B	Peprotech
Recombinant human FGF19	100-32	Peprotech
Recombinant human GDNF	450-10	Peprotech
Recombinant human SDF1	300-28A	Peprotech
ROCK inhibitor	Y-27632 72302	Stem cell technologies
SB431542	S4317	Sigma
Sucrose	S7903	Sigma
Tissue-Tek O.C.T. Compound	25608-930	VWR