Cerebral and ChP organoid culture conditions

Choroid plexus organoids - Lancaster Lab

Materials

• STEMdiff^TM Cerebral Organoid Kit: Stem Cell Technology cat#08570 #08571
• StemFlex^TM Medium: Thermo Fisher cat#A3349401
• EDTA: Sigma Aldrich cat#E6758
• DMEM/F12: Invitrogen cat#11330-032
• GlutaMAX: Invitrogen cat#35050-038
• P/S: Penicillin/Streptomycin: Sigma cat#P0781
• MEM-NEAA: MEM-Non-essential amino acids: Sigma cat#M7145
• 2-Mercaptoethanol: 50 mM solution of 2ME: Life Technologies #31350-010
• Accutase: A6964 SIGMA
• Rock inhibitor Y27632, 5mg: VWR cat# 688000-5
  • Working solution prepared by reconstituting 5mg in 2.96 ml water
• N2 supplement: Invitrogen cat# 17502048
  • Aliquot and store at -20°C for up to 1 year
• B27+vit. A supplement: Invitrogen cat# 17504044
  • Aliquot and store at -20°C for up to 1 year
• Neurobasal medium: Invitrogen cat# 21103049
• Insulin solution: Sigma cat# I9278-5ML
• Vitamin C solution: Dissolve 352 mg Ascorbic Acid in 50 ml DMEM/F12, Store in dark at 4°C
• Low attachment 96-well plates: CLS7007 SIGMA
• 5cm tissue culture dishes 
• Regular Matrigel: Corning cat#354234
• Parafilm
• Sterile forceps and scissors
• CHIR:  Tocris cat#4423 - Prepare 3mM stock (1000x) in DMSO
• BMP4: R&D cat#314-BP – Prepare 10μg/ml stock in sterile 4mM HCl with 0.1% BSA

Media

From kit:

EB media 

NI media

Expansion media 

Maturation media

Improved Differentiation Media (IDM+A) (500ml) – store at 4°C, up to 1 month

• 250 ml DMEM/F12
• 250 ml Neurobasal
• 2.5 ml N2 supplement
• 10 ml B27+ vitamin A supplement
• 125 μl insulin
• 500 μl of 50mM 2-ME solution
• 5 ml Glutamax supplement
• 2.5 ml MEM-NEAA
• 5 ml P/S
• 1.49g HEPES
• 5 ml Vitamin C solution (40mM stock)

Improved Differentiation Media + MG (50 ml) – Make fresh

• 50 ml Improved Differentiation Media +A– Keep cold!
• 1 ml Matrigel – Slowly thaw on ice and add slowly to cold media to dissolve

Procedure

Making embryoid bodies (1-2h)

1. Grow hESC or iPSC colonies (we have successfully tested H9, H1 and iPS IMR90-4 cells) in one well of a 6-well plate (coated with Matrigel) until 70-80% confluent. All or part of the well can be used to make EBs. One confluent well can yield an entire 96-well plate. Feeder-free cells PSCs are regularly maintained and fed with 2ml of StemFlex Media every other day.
   Important: The morphology of the colonies is important for the generation of cerebral tissue. The colonies should display clear sign of pluripotency and have clear, defined borders. 
2. Wash colonies twice with 600μL of EDTA, then leave 600μL EDTA on 4min at 37°C to facilitate colonies detachment. 
3. Aspirate EDTA solution and use StemFlex to spray off quantity needed for subsequent splitting or directly proceed with adding 500μL Accutase and incubate 4min at 37°C. 
4. Tap the plate to detach cells and add 500μL StemFlex to inactivate Accutase. Pipette up and down 4-5 times to generate a single cell suspension. Transfer the 1ml solution into a 15ml conical tube and take 5μL to mix with 5μL of Trypan blue for counting cells. Repeat this twice to get average cell count. 
5. Spin conical tube with cells at 200g for 4min. Count cells while tube is spinning. 
6. Remove supernatant and resuspend cell pellet with 1ml of EB media with 1:100 Rock Inhibitor. 
7. Prepare a tube with 150μL of EB media per well (number of desired wells of 96well-plate) and add 1:100 Rock Inhibitor to the solution. 
8. Transfer appropriate volume of cells to EB media in order to plate 4000 cells per well. 
9. Using multi-channel pipette, transfer 150μL of EB media+RI+cells solution to each well of a low adhesion 96-well plate. 
10. Change medium with EB media (without RI) on day 3.

Making primitive neuroepithelia

1. On day 5, when EBs begin to brighten and display smooth edges, replace EB media with NI media, in the same 96-well plate. 

Optional: wash one time with 100μL NI media and then add 150μL of NI per well. 

Making cerebral tissue (1-2h)

1. On day 7, EBs should become brighter around the edges and should display a visible neuroepithelia, forming a defined line around the EBs. Healthy cell aggregates should have smooth edges. 

2. When neuroepithelia are evident, EBs can be embedded and transferred to Matrigel droplets:

   a. Using a cut P200 tip, transfer EBs one by one to dimpled Parafilm (cover a tip holder with a square of Parafilm and slightly push parafilm to create a 4x4 grid of dimples) placed inside a 5cm dish using sterile forceps. 

   b. Remove excess media from EBs on parafilm and add 20-30μL drops of Matrigel (keep on ice). Position each aggregate to the centre of the droplet using a pipette tip (this must be done immediately as Matrigel rapidly solidify at room temperature). 

   c. Move the 5cm dish with the Parafilm sheet in the incubator at 37°C for 20min to allow Matrigel to polymerize. 

   d. Transfer the Parafilm sheet to a 1 well of a 6well plate and spray EBs off with Expansion media (2ml) until they fall out of the sheet (16 EBs per 1 well). 

   e. Continue culturing the tissue droplets in Expansion media until day 10 in the incubator.

Caution: Parafilm cannot be properly sterilized, as it cannot be autoclaved. Keep Parafilm in a clean environment and spray your gloved with 70% (v/v) ethanol before preparing dimples. 

Choroid plexus induction (4-7 days)

1. By day 10, tissues should begin forming rounded buds of neuroepithelium containing fluid-filled cavities. 
2. At day 10, move tissue using a cut P1000 tip to a 5cm dish (5-6 organoids per dish) and replace the Expansion media with Maturation media with 3μM CHIR and 20ng/ml of BMP4. 
3. Replace the media with Maturation media with 3μM CHIR and 20ng/ml of BMP4 after 4 days. 

Notes: After 2 days of treatment with BMP4 and CHIR the neuroepithelial buds start to elongate. Treatment with BMP4 and CHIR can be left for 4 to 7 days in Maturation media. 

Organoid maturation 

1. At day 14 transfer organoids to orbital shaker (47 rpm) at 37°C and feed every 3-4 days with Maturation media. 
2. At day 30 start feeding with Improved Differentiation Media (IDM+A) + Matrigel and notice appearance and initial expansion of fluid-filled compartments surrounded by a thin epithelial tissue. 

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