NPC to Astrocyte Differentiation
Celeste M M. Karch, Jacob Marsh, Rj Martinez
Abstract
Here, we provide a detailed protocol for differentiation of human induced pluripotent stem cell derived neural progenitor cells into astrocytes.
Steps
Preparation
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.
Prior to starting, NPCs are cultured in one well of a six well plate until they reach 70% confluency
NPC to Astrocyte Differentiation - Day 1
Prepare wells/plates/dishes to be used for the differentiation process with Matrigel
Remove media from NPCs and replace with 3mL Accutase
Incubate at 37°C for 0h 3m 0s
Tap the plate to dissociate the cells and add 6mL of room temperature DMEM/F12 and collect cells into a 15mL tube
Count the number of cells and move the required volume to a new 15mL tube. Use the table below as a guide for the number of cells to plate into specific vessels.
| A | B | C |
|---|---|---|
| 6-well plate | 60,000/well | 2mL/well |
| 24-well plate | 15,000/well | 500uL/well |
| 10cm dish | 360,000 | 10mL |
Centrifuge the required volume at 750-800 rpm for 5 minutes
Aspirate the supernatant and resuspend the cell pellet in NPC medium
NPC to Astrocyte Differentiation - Day 2
Remove NPC media and replace with equivalent volume of complete astrocyte medium
NPC to Astrocyte Differentiation - Day 4 and Onward
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
Within the first 30 days, keep the number of cells per well low to aid in differentiation
After 30 days, cells should be >98% positive for S100b. Some lines may express high levels of GFAP, others will not.
Astrocyte Freezing
Dissociate cells with Accutase and collect in complete astrocyte medium in a 15mL conical tube
Spin cells at 750-800 rpm for 10 minutes
Aspirate media from pellet and resuspend in freezing media (50% complete astrocyte media, 40% FBS, 10% DMSO)
Transfer cell suspension to cryovials and place in -80C for 24-48 hours before transferring to liquid nitrogen for long-term storage
Astrocyte Thawing
Prepare wells/plates/dishes to be used for the differentiation process with Matrigel
Add 9mL of complete astrocyte medium to a 15mL conical tube
Thaw frozen vial by swirling in water-bath at 37C for 60-90 seconds
Add thawed cells to 15mL conical tube containing astrocyte medium
Spin conical tube at 750-800 rpm for 10 minutes to pellet cells
Aspirate media from cell pellet and replace with 2mL of complete astrocyte medium
Transfer cell suspension to Matrigel coated plate and incubate at 37C
