Mar 15, 2024
Non-guided neural organoids differentiation
- 1Laboratory of Molecular Neurogenetics, Department of Experimental Medical Science, Wallenberg Neuroscience Center and Lund Stem Cell Center, BMC A11, Lund University, 221 84 Lund, Sweden.
Protocol Citation: anita.adami 2024. Non-guided neural organoids differentiation. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvwjo27lmk/v1
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: February 04, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 76386
Keywords: ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson’s through the Michael J. Fox Foundation for Parkinson’s Research
Grant ID: ASAP-000520
Swedish Research Council
Grant ID: 2018-02694
Swedish Brain Foundation
Grant ID: FO2019-0098
Cancerfonden
Grant ID: 190326
Barncancerfonden
Grant ID: PR2017-0053
NIHR Cambridge Biomedical Research Centre
Grant ID: NIHR203312
Swedish Society for Medical Research
Grant ID: S19-0100
National Institutes of Health
Grant ID: HG002385
Swedish Research Council
Grant ID: 2021-03494
Swedish Research Council
Grant ID: 2020-01660
Abstract
This protocol describes how to perform non-guided neural organoid differentiation
hiPSCs lines (LINC01876 KD)
hiPSCs lines (LINC01876 KD)
To generate the human cerebral-like organoids we followed the protocol detailed in Johansson et al., 2022, based on the protocol published by Lancaster et al., 2013.
We used three hIPSC6-derived lines obtained by transduction and FACS sorting as described in detail in the protocol CRISPR inhibition of LINC01876 in hiPSCs and fbNPCs.
The cell lines included one control line (with a gRNA against LacZ) and two LINC01876 CRISPRi KD lines (guide 2 and guide 3).
Embroid body formation
Embroid body formation
8000 cells/well were plated in a 96-wells plate (Costar, Ultra Low Attachment, round bottom; REF 7007) with 250 μL of mTeSR1 (StemCell Technologies, Inc.) and RY27632 10 μM. This is considered day −5 of the differentiation of the iPSCs-derived non-guided neural organoids.
On days −3 and −1 the medium was changed (150 μL and 200 μL of mTeSR1, respectively).
Neural induction
Neural induction
At day 0 the cells are fed with Neural Induction Medium (NIM; DMEM/F12 media, N2 Supplement (1:100), L-Glutamine (2mM), Penicillin/Streptomycin (1:500), Non-Essential Amino acids (1:100) and Heparin (2ug/ml).) enriched with 3% KSR.
At day 2, 4, and 6, the organoids were fed with NIM with no added KSR.
Embedding
Embedding
On day 8 the organoids were embedded in 30-50 μL of Matrigel (Corning) and incubated at 37°C for 25 minutes to allow the Matrigel to solidify.
Differentiation
Differentiation
The organoids were then transferred in Corning REF 3471 6-wells plates with flat bottom containing 4ml/well of Cortical Differentiation Medium (CDM; F12 Media (-Glut) (48.5%), Neurobasal (48.5%), N2 Supplement (1:200), B27 Supplement (-Vit.A, 1:100), L-Glutamine (2mM), Penicillin/Streptomycin (1:500), Non-Essential Amino acids (1:200), Beta MercaptoEtOH (50uM) and Insulin (2.5 ug/mL)).
On day 10 and 12 of the differentiation, the medium was changed exchanging 3 ml/well for 3 mL of fresh CDM.
On day 15, 17, 19, 21 and 23, ∼4 mL the medium was replaced with 4 mL of Improved Differentiation Medium + A (IDM, F12 Media (-Glut) (48.5%), Neurobasal (48.5%), N2 Supplement (1:200), B27 Supplement (+Vit.A, 1:50), L-Glutamine (2mM), Penicillin/Streptomycin (1:500), Non-Essential Amino acids (1:200), Beta MercaptoEtOH (50uM), Insulin (2.5 ug/mL) and Ascorbic Acid (400uM)).
From day 25, the medium was changed every 3 days with 3-4 mL of Cortical Terminal Differentiation Medium (CTDM, F12 Media (-Glut) (48.5%), Neurobasal (48.5%), N2 Supplement (1:200), B27 Supplement (+Vit.A) – (1:50) 800uL, L-Glutamine (2mM), Penicillin/Streptomycin (1:500), Non-Essential Amino acids (1:200), Beta MercaptoEtOH (50uM), Insulin (2.5 ug/mL) and Ascorbic Acid (400uM), BDNF (10ng/uL), cAMP (200uM), GDNF (10ng/uL)).
Organoids' size measurements
Organoids' size measurements
All the diameter measurements of the organoids were taken with the Measure tool from the Image J software. The chosen measuring unit was mm.