TITLE: Neural differentiation on EM grids - iNeurons sample preparation for cryo-ET and CLEM 
AUTHORS: Cristina   Capitanio, Victoria Trinkaus, Melissa Hoyer

[DESCRIPTION]
This is a protocol for differentiating AAVS1-TRE3G-NGN2 iPSCs and hESCs to iNeurons directly on EM grids for cryo-ET and cryo-CLEM. The protocol comprises the following steps:
EM grids coating with Matrigel or Poly-Ornithine and Laminin; 
differentiation and seeding of iNeurons on EM grids; 
lentiviral transduction for transient expression of fluorescently-labelled proteins; 
plunge freezing. 


[STEPS]
SECTION: Starting the neural differentiation (Day 0-Day6)
2. We based our neural differentiation protocol, for both iPSCs and hESCs AAVS1-TRE3G-NGN2 cells, on: 
 
We also refer to that protocol for a complete list of media and reagents. 

Follow the neural differentiation protocol until cell splitting on Day 6;
One well of a six well plate is enough to seed iNeurons on 10-20 EM grids at Day 6, depending on the desired cell density.
SECTION: EM grid preparation for neuronal culture seeding (Day 5)
3. Choosing grid type:
Use gold (Au) grids only for mammalian cell culture; copper (Cu) is cytotoxic;  
iNeurons grow well on films with 1 μm and 2 μm holes. All following grid geometries worked in our hands: R 1/4, R2/4, R2/2, R2/1. Films with 2 μm holes may be preferred for direct TEM data acquisition on the processes. 
Both carbon and silicon dioxide (SiO2) films work. SiO2  is preferred when preparing samples for FIB-milling. In our hands, iNeurons did not grow on UltraAUfoil gold film grids.

Start preparing EM grids one day before seeding the iNeurons. 
SECTION: Maintenance of iPSCs and hESC cell lines
1. For maintenance of both induced pluripotent stem cells (iPSCs) and human embryonic stem cells (hESCs) as well as for a complete list of media and reagents, we refer to this protocol:
 

SECTION: EM grid preparation for neuronal culture seeding (Day 5)
4. Plasma cleaning
Carefully place EM grids on a glass slide and plasma clean the grids for 30 seconds. 
SECTION: EM grid preparation for neuronal culture seeding (Day 5)
6. UV-treating
Treat the grids with UV-light for 20 min. 
SECTION: EM grid preparation for neuronal culture seeding (Day 5)
7. Coating grids 
We suggest two methods, coating grids with matrigel or poly-Ornithin + Laminin (PO+L). Both methods worked well for hESC-derived iNeurons, while iPSC-derived iNeurons seemed to grow better on POL+L coated grids.  

 

SECTION: EM grid preparation for neuronal culture seeding (Day 5)
7.1. Method 1: Coating grids with Matrigel 
thaw the Matrigel in ice and dissolve it in cold DMEM/F12 medium; 
dispense 100 μl on top of each grid to form a a small dome;
incubate overnight at 37 °C; 
the grids are ready for use the next day. 
SECTION: EM grid preparation for neuronal culture seeding (Day 5)
7.2. Method 2: Coating grids with PO+L
dispense 100 μl of poly-ornithine solution (0.01%) on top of every grid;
incubate at RT for 4 hours in a sterile container;
remove the PO solution and add 100 μl of Laminin (dissolved in PBS at a final concentration of 0.2 μg/ml);
incubate overnight at RT;
the next day wash 3 times with PBS and store with 100 μl ND2 or DMEM/F12 on top till cell seeding;
SECTION: EM grid preparation for neuronal culture seeding (Day 5)
5. Prepare 3.5 mm dishes with four inner rings (see materials). This kind of dish allow to use smaller quantity of medium and cells for seeding. 
Use tweezers to transfer each grid inside of one inner ring of the culture dish. 
SECTION: Seeding iNeurons on the grids (Day 6)
8. iPSC and hESC-derived iNeurons can be successfully seeded on grid on any day between 5 and 7. 

Detach the cells and count:
wash the cells with PBS;
add Accutase (1 ml for one well) and incubate for 5 minutes at 37 °C;
add 5 ml of ND2 medium;
use a 1 ml pipette to pipette the cells up and down to obtain single cells;
OPTIONAL: pass the cells through a cell strainer;
transfer to 15 ml tube and count the cells.
SECTION: Seeding iNeurons on the grids (Day 6)
9. After the cells attached, carefully add 2 ml of ND2 medium. 
 

SECTION: Medium exchange and Doxycycline withdrawal (Day 7-15)
10. Exchange 50% of the medium every other day, as explained in the neural differentiation protocol; 
Withdraw Doxycycline on Day 10 for hESC-derived iNeurons, or around Day 15 for iPSC-derived iNeurons. 
SECTION: Seeding iNeurons on the grids (Day 6)
8.1. Dilute to desired density and seed: 
Prepare cell dilution in ND2 medium: seed 1500 cells in a volume of 150 μl on top of each grid (See note: How many cells should I seed on the grid? );
Leave the cells in the incubator for 1 or 2 hours while they attach on the grid. 
 

SECTION: Lentiviral transduction for fluorescence marker expression on EM grids (Day 9-14)
11. In order to express fluorescently-labelled proteins for CLEM experiments, we use transient expression via lentiviral transduction. Before transducing iNeurons on EM grids, determine the correct quantity of virus to add by titrating it on control cell culture dishes.

On the day of viral transduction:
Exchange half of the medium as usual;
If co-transducing with different viruses, pre-mix the different viruses in 200 μl of medium;
Dilute the virus in 200 μl of culture medium;
Apply the virus solution dropwise on top of the dish;
Incubate the cells and do not exchange medium the following day.

Two days after viral transduction: 
Start exchanging half of the medium with fresh ND2. 
                                                                                                                                                                                                                                                                                                                                                                                                                                                        
 

 

SECTION: Plunge-freezing of iNeurons on EM grids (Day 20-24)
12. If possible, store cells in an incubator close to the plunge-freezing set up during plunging day;  
Be very careful when moving and transporting grids and dishes, shake the dishes as little as you can and minimize their time outside of the incubator. iNeurons are very fragile and have a tendency to detach from the grid when shaken. 

We propose two methods for two commonly used plunge-freezing instruments, Vitrobot Mark IV and Leica EM GP2. 

 

 

SECTION: Plunge-freezing of iNeurons on EM grids (Day 20-24)
12.2. Method 1: Plunging with Vitrobot Mark IV
apply 4 μl;
Settings:
Blot force: 8;  Blot time: 10;  Chamber humidity: 70%.
Blot from the back: one teflon sheet + blotting paper, teflon only on front pad. 
SECTION: Plunge-freezing of iNeurons on EM grids (Day 20-24)
12.3. Method 2: Plunging with Leica EM GP2
apply 4 μl;
Settings:
Blot time: 7.0 sec; Horizontal blot position: 43.2 mm /43.0 mm; Vertical blot position: 2.7 mm; Chamber humidity: 95%.

SECTION: Plunge-freezing of iNeurons on EM grids (Day 20-24)
12.1. When preparing sample for correlative FIB-milling, apply 4 μl of medium containing 1 μm autofluorescent beads (Dynabeads, diluted 1:20) before blotting. If not, just apply 4 μl of culture medium.
Plunge in ethane-propane or ethane. 
SECTION: Plunge-freezing of iNeurons on EM grids (Day 20-24)
12.4. Store your grids in LN2. The iNeurons samples are ready to be directly imaged by Cryo-ET or imaged by cryo-FLM and subsequently FIB-milled.