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, forebrain, hypothalamus^7,8,9, and hippocampus¹⁰. There have been multiple examples of using organoids to study human nervous system diseases¹¹. Also, the organoids were implemented in drug discoveries¹² and used in studies of infectious diseases, including SARS-Cov-2^13,14.

The brain organoids can reach up to several millimeters in diameter. So, the inner zone of the organoid may suffer from hypoxia or malnutrition and eventually become necrotic. Therefore, many protocols include special bioreactors⁸, shakers, or microfluidic systems¹⁵. These devices may require large volumes of expensive cell culture media. Also, the cost of such equipment is usually high. Some bioreactors consist of many mechanical parts that make them difficult to sterilize for reuse.

Most protocols suffer from the "batch effect"¹⁶, which generates significant variability among organoids obtained from the identical iPSCs. This variability hinders drug testing or preclinical studies requiring uniformity. The high yield of organoids enough to select organoids of uniform size may partially solve this problem.

The time factor is also a significant problem. Matsui et al. (2018) showed that brain organoids require at least six months to reach maturity¹⁷. Trujillo et al. (2019) also demonstrated that electrophysiological activity occurred in organoids only after six months of cultivation¹⁸. Due to the long organoid maturation time, the researchers often launch new differentiation before completing the previous one. Multiple parallel processes of differentiation require additional expenses, equipment, and laboratory space.

We have recently developed a mini bioreactor that mainly solves the problems mentioned above¹⁹. This home-made bioreactor consists of an ultra-low adhesion or untreated Petri dish with a plastic knob in the center. This plastic knob prevents crowding of organoids and their conglutination in the center of the Petri dish, which is caused by the rotation of the shaker. This paper describes how this inexpensive and simple home-made mini bioreactor allows generating high-quality brain organoids in large quantities.

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 chloroform overnight to prepare liquid plastic.
   ​CAUTION: Work in a fume hood.
3. Check that the resulting liquid plastic is viscous enough for pipetting; its drop retains a spherical form and does not spread on the surface. If it is very liquid, add more plastic crumbs. If it is thick, then add chloroform.
4. Make a plastic knob in the center of a sterile ultra-low adhesion 6-cm Petri dish. There are two equally suitable ways, as detailed below.
   1. Put the autoclaved plastic ring on the center and drop the liquid plastic to the inside of the ring.
   2. Without any plastic ring, drop the liquid plastic on the center of the Petri dish.
5. Leave the dishes open for 2-3 h in a laminar flow hood until dried complete. Treat the dried dishes with ultraviolet radiation for 15-20 min.

2. Induction of neuronal differentiation of iPSCs

1. Cultivate iPSCs in the medium for pluripotent stem cells up to 75-90% confluence in 35 mm Petri dishes precoated with a matrix consisting of extracellular proteins.
2. Prepare medium A-SR. See Table 1 for details.
3. Aspirate the cultivation medium and add 2 mL of A-SR medium at Day 0 of differentiation.
4. Prepare medium A (see Table 2).
5. Cultivate cells in medium A for two weeks from Days 2-14, refreshing medium in Petri dishes every other day.

3. The formation of spheroids from neuroepithelial precursor cells at Day 14

1. At Day 14, make spheroids from neuroepithelial precursor cells using a special 24-well culture plate containing approximately 1,200 microwells in each well (Figure 2C). Follow the procedure given below.
   NOTE: At this differentiation stage, a 35 mm Petri dish usually contains 3 - 3.5 x 10⁶ neuroepithelial precursor cells. Thus, one 35 mm Petri dish with neuroepithelial precursor cells is sufficient for 3 - 4 wells of 24-well culture plate with microwells.
2. Prepare a 24-well culture plate with microwells: To each well, add 1 mL of medium A. Centrifuge briefly at 1,300 x g for 5 min in swinging bucket rotor fitted with plate holder. Control under the microscope that there are no bubbles in microwells.
3. Prepare the medium B (Table 3).
4. Remove the medium from the Petri dish with neuroepithelial precursor cells; wash the cells with 2 mL of DMEM/F12. For the cell detachment, treat the cells with 1.5 mL of 0.48 mM EDTA solution prepared in PBS. Control the cell detachment under the microscope.
5. Harvest the cells into a 15 mL tube. Add 5 mL of DMEM/F12 in the tube to wash the cells. Centrifuge at 200 x g for 5 min. Remove the supernatant and resuspend cells in 2 mL of medium B.
6. Check the cell concentration and viability by Trypan Blue staining and a hemocytometer. Calculate the volume of suspension needed to contain 1 x 10⁶ viable cells in total.
7. Transfer the cell suspension containing 1 x 10⁶ cells into each well of a 24-well plate with microwells. Add medium B up to 2 mL into each well, gently pipette cells up and down several times, and centrifuge briefly 100 x g for 1 min to capture cells in the microwells.
   NOTE: The number of cells per well should not exceed 1 x 10⁶. Otherwise, spheroids from neighboring microwells fuse.
8. Check under the microscope that cells are evenly distributed in microwells. Repeat pipetting and centrifugation if cells are distributed unevenly.
9. Incubate the plate overnight to let cells aggregate in spheroids.

4. Obtaining and cultivation of organoids

1. The next morning (Day 15), check the quality of spheroids under the microscope. Ensure that they are transparent and smooth, if healthy (Figure 2A,B). Carefully collect the spheroids from each well into a 15 mL tube, leave the spheroids to precipitate by gravity for 2-3 min, and then remove the supernatant.
2. Add to the spheroids 2 mL of the matrix thawed during the same time on ice. Mix gently by pipetting and incubate at room temperature for 30 min.
3. To wash excess of the matrix, add to the tube 8 mL of medium B. Pipette gently, then centrifuge the tube for 1 min at 100 x g.
   NOTE: Do not exceed the time and the speed of centrifugation to avoid the irreversible aggregation of spheroids.
4. Remove the supernatant. Add to the tube 2 mL of medium B, pipette gently. Split the spheroid suspension between two mini bioreactors, each containing 4 mL of medium B. Place the mini bioreactors into a 15 cm Petri dish to prevent the evaporation of water and to avoid contamination.
5. Put the Petri dish with mini bioreactors on an orbital shaker. Cultivate the organoids at a rotation rate of 70-75 rpm.
6. On Day 16, prepare medium C (Table 4).
7. Transfer the organoids into 15 mL tube. For 5 min, let them fall to the bottom, aspirate the supernatant, add 5 mL of medium C. Return the organoids into the mini bioreactors.
   NOTE: Be careful not to lose the spheroids, which are transparent and barely visible.
8. Cultivate spheroids in medium C for two weeks, refreshing the medium every two days. At the end of these two weeks, leave about 100 spheroids per mini bioreactor for the following cultivation. Freeze excessive spheroids in a freezing medium in liquid nitrogen.
9. On Day 30, prepare medium D (Table 5).
10. Change the cultivation medium to medium D, which is a maturation medium. Refresh cultivation medium every 2-3 days for three weeks, then use medium D without BDNF and GDNF.

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...

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

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