TITLE: Formation of ventral midbrain Assembloid/Organoids
AUTHORS: Regine Tipon, Gist Croft

[DESCRIPTION]
Brain organoids are three-dimensional (3D) structures derived from human pluripotent stem cells (hPSCs) that reflect early brain organization. Compared with traditional cell cultures, brain organoids offer a more accurate representation of human brain development and function, rendering them suitable models for neurodevelopmental diseases. In this early pilot to form brain organoids using iPSCs, we focused on verifying a successful approach to form organoids using our vmDA generated from our protocol, optimizing conditions to maintain the organoids (media, feeding schedule) as well as trying to understand the different ways to analyze these formed organoids. This approach also tested the possibility of producing a tri-culture system by adding iPSC-derived microglia into the organoids (composed of neurons and glia), and investigating the interactions between the distinct cell types. 

[STEPS]
SECTION: Formation of ventral midbrain Assembloid/Organoids
1. Following Croft Lab edited vmDA protocol (dx.doi.org/10.17504/protocols.io.yxmvmebdog3p/v1, ventral midbrain organoids are dissociated at day 33 using the Worthington Kit protocol (dx.doi.org/10.17504/protocols.io.j8nlk8dbxl5r/v1) into single cell suspension 
SECTION: Formation of ventral midbrain Assembloid/Organoids
2. During the final spin (300 XG for 5 mins) , count cells 
SECTION: Formation of ventral midbrain Assembloid/Organoids
3. Resuspend cells in ventral midbrain maturation media

 

SECTION: Formation of ventral midbrain Assembloid/Organoids
4. 1X10^3 mDA neurons were added to one well of a 96wp V bottom plate 
SECTION: Formation of ventral midbrain Assembloid/Organoids
5. For the neuron/microglia cocultures, 1-2K iPSC-derived microglia were seeded into each well (washer protocol) 
SECTION: Formation of ventral midbrain Assembloid/Organoids
6. Centrifuge V bottom plate 300 X g for 5 mins to allow for cells to pellet and aggregate 
SECTION: Formation of ventral midbrain Assembloid/Organoids
7. Plate placed in 37C for ~week to allow the assembloids to form, within 1 day post seeding there should be visible aggregates 
SECTION: Formation of ventral midbrain Assembloid/Organoids
8. For maintenance: 
Day 33-100 Feed organoids weekly with ½ media change weekly
Day 100-430 Feed organoids with ½ media change every other week
SECTION: Notes:
9. Additional methods to analyze/assess organoid formation/composition and triculture interactions 
-Lentiviral transduciton and imaging
-iPSC reporter lines and imaging 

SECTION: Important:
10. Use ROCk inhibitor or CEPT for aggregation day 33.  
Dissociated cryopreserved midbrain organoids cells day 16, day25, day 33, or day 60, can be used to reconstitute organoids, chimeroids, or assembloids 
Organoids can be maintained intact (without dissociation and reconstitution) at day 33.  Unreconstituted assembloids can be seeded with microglia progenitors, which will attach to and enter organoids.  Unreconstituted organoids are more heterogenous in size and cell composition on a per organoid basis than those reassembled from a large population  of normally heterogenous organoids.  We have not observed extremely strong differences between individual organoids before day 33, a tight normal distribution. Organoids fusion is the key driver of size heterogeneity but does not affect cell fate/composition, patterning or, viability, 
 Assembly.  Organoids can be reassembled, or seeded with microglia, mature astrocytes, MSNs, or Rapid Synucleinopathy iNeurons, or vascular endothelial cells.