May 09, 2022

Public workspaceNPC to Astrocyte Differentiation 

DOI
dx.doi.org/10.17504/protocols.io.q26g781qqlwz/v1
  • 1Washington University in St Louis;
  • 2Washington University in Saint Louis - WUSTL (MO)
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DOI: dx.doi.org/10.17504/protocols.io.q26g781qqlwz/v1
Protocol Citation: Celeste M M. Karch, Jacob Marsh, Rj Martinez 2022. NPC to Astrocyte Differentiation . protocols.io https://dx.doi.org/10.17504/protocols.io.q26g781qqlwz/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: January 07, 2021
Last Modified: May 09, 2022
Protocol Integer ID: 46079
Abstract
Here, we provide a detailed protocol for differentiation of human induced pluripotent stem cell derived neural progenitor cells into astrocytes.
Preparation
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.
Note
Note: Fully prepared media should be used within two weeks when stored at Temperature4 °C , but can be aliquoted and stored at Temperature-20 °C for several months. Alternatively, aliquot the supplements and prepare smaller volumes of complete media, if desired.


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
NPC to Astrocyte Differentiation - Day 1
3m
3m
Prepare wells/plates/dishes to be used for the differentiation process with Matrigel
Note
For reference, we coat a single well of a six well plate with Amount1 mL of Matrigel


Remove media from NPCs and replace with Amount3 mL Accutase

Incubate at Temperature37 °C for Duration00:03:00

3m
Tap the plate to dissociate the cells and add Amount6 mL of room temperature DMEM/F12 and collect cells into a Amount15 mL tube

Count the number of cells and move the required volume to a new Amount15 mL tube. Use the table below as a guide for the number of cells to plate into specific vessels.

Plate Size# of CellsVolume of Media
6-well plate60,000/well2mL/well
24-well plate15,000/well500uL/well
10cm dish360,00010mL

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
NPC to Astrocyte Differentiation - Day 2
3m
3m
Remove NPC media and replace with equivalent volume of complete astrocyte medium
NPC to Astrocyte Differentiation - Day 4 and Onward
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.
Note
After full differentiation, cell confluency/number at passage is no longer important.

Astrocyte Freezing
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
Note
The slower spinning speed helps to reduce the percentage of cell death

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
Astrocyte Thawing
Prepare wells/plates/dishes to be used for the differentiation process with Matrigel
Note
For reference, we coat a single well of a six well plate with Amount1 mL of 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