TITLE: NPC to Astrocyte Differentiation 
AUTHORS: Celeste M M. Karch, Jacob  Marsh, Rj  Martinez

[DESCRIPTION]
Here, we provide a detailed protocol for differentiation of human induced pluripotent stem cell derived neural progenitor cells into astrocytes. 

[STEPS]
SECTION: Preparation
1. Prepare astrocyte medium (ScienCell Astrocyte Medium #1801) by adding FBS, antibiotic, and astrocyte growth supplement to basal media. All components are provided in ScienCell Astrocyte Medium #1801 kit. 
 

SECTION: Preparation
2. Prior to starting, NPCs are cultured in one well of a six well plate until they reach 70% confluency 
SECTION: NPC to Astrocyte Differentiation - Day 1
3. Prepare wells/plates/dishes to be used for the differentiation process with Matrigel
 

SECTION: NPC to Astrocyte Differentiation - Day 1
4. Remove media from NPCs and replace with 3 mL Accutase 

SECTION: NPC to Astrocyte Differentiation - Day 1
5. Incubate at 37 °C for 3 min 

SECTION: NPC to Astrocyte Differentiation - Day 1
6. Tap the plate to dissociate the cells and add 6 mL of room temperature DMEM/F12 and collect cells into a 15 mL tube

SECTION: NPC to Astrocyte Differentiation - Day 1
7. Count the number of cells and move the required volume to a new 15 mL tube. Use the table below as a guide for the number of cells to plate into specific vessels.

 

SECTION: NPC to Astrocyte Differentiation - Day 1
8. Centrifuge the required volume at 750-800 rpm for 5 minutes 
SECTION: NPC to Astrocyte Differentiation - Day 1
9. Aspirate the supernatant and resuspend the cell pellet in NPC medium
SECTION: NPC to Astrocyte Differentiation - Day 2
10. Remove NPC media and replace with equivalent volume of complete astrocyte medium
SECTION: NPC to Astrocyte Differentiation - Day 4 and Onward
11. Change media every 2-3 days for 30 days and passage with Accutase, centrifuge at 750-800 rpm for 10 minutes and re-plate on a Matrigel coated plate whenever culture becomes 100% confluent 
SECTION: NPC to Astrocyte Differentiation - Day 4 and Onward
12. Within the first 30 days, keep the number of cells per well low to aid in differentiation 
SECTION: NPC to Astrocyte Differentiation - Day 4 and Onward
13. After 30 days, cells should be >98% positive for S100b. Some lines may express high levels of GFAP, others will not. 
 

SECTION: Astrocyte Freezing
14. Dissociate cells with Accutase and collect in complete astrocyte medium in a 15mL conical tube
SECTION: Astrocyte Freezing
15. Spin cells at 750-800 rpm for 10 minutes
 

SECTION: Astrocyte Freezing
16. Aspirate media from pellet and resuspend in freezing media (50% complete astrocyte media, 40% FBS, 10% DMSO)
SECTION: Astrocyte Freezing
17. Transfer cell suspension to cryovials and place in -80C for 24-48 hours before transferring to liquid nitrogen for long-term storage 
SECTION: Astrocyte Thawing
18. Prepare wells/plates/dishes to be used for the differentiation process with Matrigel
 

SECTION: Astrocyte Thawing
19. Add 9mL of complete astrocyte medium to a 15mL conical tube
SECTION: Astrocyte Thawing
20. Thaw frozen vial by swirling in water-bath at 37C for 60-90 seconds
SECTION: Astrocyte Thawing
21. Add thawed cells to 15mL conical tube containing astrocyte medium 
SECTION: Astrocyte Thawing
22. Spin conical tube at 750-800 rpm for 10 minutes to pellet cells
SECTION: Astrocyte Thawing
23. Aspirate media from cell pellet and replace with 2mL of complete astrocyte medium 
SECTION: Astrocyte Thawing
24. Transfer cell suspension to Matrigel coated plate and incubate at 37C