# Differentiation of cerebral orgnaoid

## Reagent
- Cell
- Knockout Serum Replacement (KOSR)
- DMEM /F12
- Neurobasal medium
- Penicillin/Streptomycin (P/S)
- N2 supplement  
- B27 (+ Vitamin A) plus supplement
- Human insulin
- Ascorbic acid
- GlutaMAX
- dibutyryl-cAMP  
- NEAA
- LDN-193189
- SB431542
- XAV939
- BDNF

---
## Preparation

### Neural induction medim

| Reagent            | Volume |
|:-------------------|-------:|
| Knock out DMEM/F12 |  75 ml |
| KSOR               |  15 ml |
| GlutaMAX           |    1 % |
| NEAA               |    1 % |
| LDN-193189         | 100 nM |
| SB431542           |  10 μM |
| XAV939             |  10 μM |
|                    |        |
| Total              | 100 ml |

### Differentiaion medium

| Reagent                           |           Volume |
|:----------------------------------|-----------------:|
| Knock out DMEM/F12                |            50 ml |
| Neurobasal medium                 |            50 ml |
| N2 supplement                     |            0.5 % |
| B27 (+ Vitamin A) plus supplement |              1 % |
| GlutaMAX                          |              1 % |
| MEM-NEAA                          |              1 % |
| Human insulin                     | 0.25 mg/ml (v/v) |
|                                   |                  |
| Total                             |           100 ml |

### Maturation medium

| Reagent                            |           Volume |
|:-----------------------------------|-----------------:|
| Neurobasal medium                  |           100 ml |
| N2 supplement                      |              1 % |
| B27 (+ Vitamin A)  plus supplement |              2 % |
| GlutaMAX                           |              1 % |
| MEM-NEAA                           |              1 % |
| Human insulin                      | 0.25 mg/ml (v/v) |
| Ascorbic acid                      |           200 mM |
| BDNF                               |         20 ng/ul |
| dibutyryl-cAMP                     |             1 mM |
|                                    |                  |
| Total                              |           100 ml |

### Maintanance medium

| Reagent                            |   Volume |
|:-----------------------------------|---------:|
| Brainphys                          |   100 ml |
| B27 (+ Vitamin A)  plus supplement |       2% |
| GlutaMAX                           |      1 % |
| P/S                                |      1 % |
| BDNF                               | 20 ng/ml |
| GDNF                               | 20 ng/ml |
|                                    |          |
| Total                              |   100 ml |

### Ca<sup>2+</sup> imaging recording medium

| Reagent                               |   Volume |
|:--------------------------------------|---------:|
| Brainphys Imaging optimization medium |   100 ml |
| B27 (+ Vitamin A)  plus supplement    |       2% |
| GlutaMAX                              |      1 % |
| P/S                                   |      1 % |
| BDNF                                  | 20 ng/ml |
| GDNF                                  | 20 ng/ml |
|                                       |          |
| Total                                 |   100 ml |

## Protocols

1. iPS cells on 6 well plate were dissociated into single cells with TrypLE express.
2. Plated at 30,000 cell per each well of U-bottom ultra-low attachment 96 well plate (Corning) with mTeSR plus supplemented with 20 μM of Y-23632. 
3. After 24 h, culture medium was replaced with neural induction medium and changed every other day.
4. After 10 days of culture, the culture medium was replaced with neuronal differentiation medium and the medium was changed every 2 days until 18 days.
5. After 18 days of culture, organoid were transferred to flat bottom ultra-low attachment 24 well plate (Corning) on the rocking shaker (120 rpm) with switching to maturation medium.
6. After 30 days from the differentiation, the culture medium was changed two-time per week with maintanance medium until imaging session.