Brain Organoid Generation from Induced Pluripotent Stem Cells in Home-Made Mini Bioreactors

Summary

Here we describe a protocol for generating brain organoids from human induced pluripotent stem cells (iPSCs). To obtain brain organoids in large quantities and of high quality, we use home-made mini bioreactors.

Abstract

The iPSC-derived brain organoid is a promising technology for in vitro modeling the pathologies of the nervous system and drug screening. This technology has emerged recently. It is still in its infancy and has some limitations unsolved yet. The current protocols do not allow obtaining organoids to be consistent enough for drug discovery and preclinical studies. The maturation of organoids can take up to a year, pushing the researchers to launch multiple differentiation processes simultaneously. It imposes additional costs for the laboratory in terms of space and equipment. In addition, brain organoids often have a necrotic zone in the center, which suffers from nutrient and oxygen deficiency. Hence, most current protocols use a circulating system for culture medium to improve nutrition.

Meanwhile, there are no inexpensive dynamic systems or bioreactors for organoid cultivation. This paper describes a protocol for producing brain organoids in compact and inexpensive home-made mini bioreactors. This protocol allows obtaining high quality organoids in large quantities.

Introduction

Human iPSC-derived models are widely used in the studies of neurodevelopmental and neurodegenerative disorders¹. Over the past decade, 3D brain tissue models, so-called brain organoids, essentially complemented traditional 2D neuronal cultures². The organoids recapitulate to some extent the 3D architecture of the embryonic brain and allow more precise modeling. Many protocols are published for the generation of organoids representing different brain regions: cerebral cortex^3,4,5, cerebellum⁶, midbrain, fo....

Protocol

NOTE : Use sterile technique throughout the protocol, excluding steps 1.2 and 1.3. Warm all culture media and solutions to 37 °C before applying to cells or organoids. Cultivate cells in a CO₂ incubator at 37 °C in 5% CO₂ upon 80% humidity. The protocol scheme is shown in Figure 1.

1. Transforming Petri dishes into mini bioreactors

1. Cut sterile 15 mL centrifuge tubes in rings of 7-8 mm in height; autoclave the rings.
2. Break low-adhesion, untreated or microbiological Petri dishes into crumbs. Dissolve about 1 g of plastic crumbs in 10 mL of chl....

Results

The protocol scheme is shown in Figure 1. The protocol included five media in which iPSCs differentiated into brain organoids during at least one month. The differentiation was started then iPSCs reached the 75-90% confluence (Figure 2A,B). The first signs of differentiation towards neurons were observed on days 10-11 of iPSC cultivation in medium A when cells began to cluster into "rosettes" (Figure 2C). At days 14.......

Discussion

The described protocol has two crucial steps allowing the generation of high-quality organoids of uniform size. First, the organoids grow from spheroids which are near identical in cell number and cell maturity. Second, the home-made bioreactors provide each organoid a uniform environment, where organoids do not crowd or stick together.

The cell quality and state of cell maturation are essential to perform the protocol. It is critical to start neuronal differentiation at 75-90% confluence of i.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Advanced DMEM/F-12	Gibco	12634010	DMEM/F-12
AggreWell400	STEMCELL Technologies Inc	34425	24-well culture plate with microwells
B-27 Supplement	Gibco	17504044	Neuronal supplement B
GlutaMAX Supplement	Gibco	35050061	200 mM L-alanyl-L-glutamine
Human BDNF	Miltenyi Biotec	130-096-285
Human FGF-2	Miltenyi Biotec	130-093-839
Human GDNF	Miltenyi Biotec	130-096-290
KnockOut Serum Replacement	Gibco	10828028	Serum replacement
mTESR1	STEMCELL Technologies Inc	85850	Pliripotent stem cell medium
N2 Supplement	Gibco	17502001
Neurobasal Medium	Gibco	21103049	Basal medium for neuronal cell maintenance
Penicillin-Streptomycin Solution	Gibco	15140130
Plasmocin	InvivoGen	ant-mpt-1	Antimicrobials
Purmorphamine	EMD Millipore	540220
StemMACS Y27632	Miltenyi Biotec	130-106-538	Y27632
StemMACS Dorsomorphin	Miltenyi Biotec	130-104-466	Dorsomorphin
StemMACS LDN-193189	Miltenyi Biotec	130-106-540	LDN-193189
StemMACS SB431542	Miltenyi Biotec	130-106-543	SB431542
Trypan Blue Solution	Gibco	15250061
Versen solution	Gibco	15040066	0.48 mM EDTA in PBS
β-mercaptoethanol	Gibco	31350010