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: In development
We are still developing and optimizing this protocol
Created: May 08, 2024
Last Modified: May 17, 2024
Protocol Integer ID: 99474
Abstract
Protocol to differentiate neural progenitor cells to mature neurons and astrocytes in 2D culture.
d1 full media change to start differentiation to neurons/astrocytes
d7 passage (without ROCKi)
d14 passage (without ROCKi)
d42 Mitomycin C treatment (for neurons only)
~d60-100 replate neurons/astrocytes for experiments
Note
If more cells are needed, the NPCs can be maintained additional passages in +EGF+FGF before differentiation, but can't be refrozen and thawed again. If frozen/thawed again, they don't proliferate or differentiate well.
Media Recipe
Media Recipe
NPC Base Media (500mL):
240mL 50% DMEM/F12 (Hyclone VWR Cat # SH30023.02)
240mL 50% Neurobasal (FisherSci Cat # 21103-049)
2.5mL 0.5X N2 (100X; Invitrogen Cat # 17502048)
5mL 0.5X B27 without VA (50X; Invitrogen, 12587010)
5mL 1X Glutamax (100x Invitrogen Cat # 35050061)
5mL 1X NEAA (Invitrogen Cat # 11140050)
*250 uL 5 ug/mL Human Insulin Solution (250mg per 500mL) (Sigma Cat # I9278-5ML)
To keep coating consistent across lots, I check the concentration and make aliquots. However, if you don't have the lot number information, 0.5mg/mL is close to a 1:20 dilution.
To coat plates:
Thaw an aliquot of matrigel at4 °CorOn ice
Dilute with the appropriate amount of ice cold DMEM/F12
6.5 mLfor 6.5-wells = 3.25 mg matrigel
Aliquot 1 mL per well in a cold 6-well plate
Shake/tilt the plate until the matrigel evenly coats the bottom
Incubate 37 °Cfor 00:30:00
After incubating, aspirate the matrigel
Add 2 mL per wellDMEM/F12
Place the plate in the incubator at 37 °C
Note
Plates can be stored in the incubator with DMEM/F12 for up to 1 week before use
30m
Pre-treat the cells with 10 micromolar (µM) ROCK inhibitor (ROCKi)
Pick up the media in the well to a 15mL conical tube.
Add 10 micromolar (µM) ROCKi (2 µL per well) and mix well.
Gently return the media to the dish.
Incubate the cells with ROCKi for00:30:00 - 01:00:00at 37 °C
1h 30m
Lift the cells with Accutase.
Aspirate the media.
Wash with 2mL/well PBS (without Ca/Mg).
Aspirate the PBS.
Add 1mL/well Accutase.
Incubate 5 minutes at 37C.
Pick up the cells in accutase with a glass pipette and blow the cells off the dish.
Transfer the cells to a 15mL conical tube.
Add 6mL NPC base media to the dish and blow the remaining cells off the dish.
Transfer the remaining cells to the 15mL conical tube.
Pipette up/down 3-5x in the conical tube to break apart clumps. To break apart clumps, pipette the cells up, and then press the pipette against the side of the tube and blow out quickly.
Pick up the entire cell mixture and filter through a 100um cell strainer over a 50mL conical tube to remove any remaining large clumps.
Transfer the cell mixture back to the 15mL conical tube.
Centrifuge at 180g for 5 minutes room temperature.
Aspirate the media.
Close the conical tube lid and tap the pellet to loosen the cell pellet.
Resuspend in 2-3mL of NPC base media.
Pipette up/down 3-5x or until the cell solution is cloudy.
Count the cells and aliquot to new plates.
Count 10uL cells + 10uL trypan blue with a hemocytometer.
Determine the #uL for 1 million cells.
Add 1 million cells/well in 2 mL per wellNPC+EGF+FGF+ROCKi in new 0.5 mg/mL matrigel coated plates.
NPC+EGF+FGF+ROCKI (10mL):
10mL NPC base media
1uL 20ng/mL EGF (stock 200ug/mL)
2uL 20ng/mL FGF (stock 100ug/mL)
10uL 10uM ROCKi (stock 10mM)
Gently place the plate in the incubator and shake evenly (4x forward/back, 4x side-to-side, 4x forward/back).
Note
If the cells were confluent, 1 million cells/well is typically around a 1:2-1:4 passage dilution.
Expansion of NPCs
Expansion of NPCs
d1 after passaging NPCs (see STEP 3)
Perform a full media change and replace with NPC maintenance media without ROCKi (NPC base +EGF+FGF)
NPC+EGF+FGF (10mL):
10mL NPC base media
1uL 20ng/mL EGF (stock 200ug/mL)
2uL 20ng/mL FGF (stock 100ug/mL)
d3
Perform a half media change. Remove 900uL/well, and add a fresh 1mL/well NPC base +EGF+FGF.
d5
Perform a half media change. Remove 900uL/well, and add a fresh 1mL/well NPC base +EGF+FGF.
d6-7
Passage to new dishes coated with 0.5mg/mL standard matrigel in NPC base +EGF+FGF.
for 6w plates -> 1 million cells/well in 2mL NPC base +EGF+FGF
for 5cm cellBIND dishes -> 2.4 million cells/dish in 6mL NPC base +EGF+FGF
Note
The high plating density is important, the cells need to be very dense to maintain progenitor identity.
I've stains the cells after maintaining in +EGF+FGF at high density up to 5x passages after thawing and > 90% of the cells remain PAX6+SOX2+NESTIN+. If passaged at lower density, the cells will stop dividing and/or differentiate.
Differentiation of NPCs to Neurons
Differentiation of NPCs to Neurons
d1 after passaging NPCs (see STEP 3)
Perform a full media change and replace with neuron differentiation media (NPC base +BDNF+GDNF+cAMP)
NPC+BDNF+GDNF+cAMP (10mL):
10mL NPC base media
1uL 20 ng/mL BDNF (stock 200 ug/mL)
2uL 20 ng/mL GDNF (stock 100 ug/mL)
1uL 1 uM cAMP (stock 10mM)
d3
Perform a half media change. Remove 900uL/well, add a fresh 1mL/well NPC base +BDNF+GDNF+cAMP.
d5
Perform a half media change. Remove 900uL/well, add a fresh 1mL/well NPC base +BDNF+GDNF+cAMP.
d7 [differentiation passage 1 - no ROCKi during this step]
Passage to new dishes coated with 0.5mg/mL standard matrigel in NPC base +BDNF+GDNF+cAMP.
for 6w plates -> 1 million cells/well in 2mL media
for 5cm cellBIND -> 2.4 million cells/dish in 6mL media
for 10cm cellBIND dishes -> 5.8 million cells/dish in 12mL media
d9
Perform a full media change and replace with neuron differentiation media (NPC base +BDNF+GDNF+cAMP) to remove any dead cells.
d11
Perform a half media change. Remove 900uL/well, add a fresh 1mL/well NPC base +BDNF+GDNF+cAMP.
d13 [differentiation passage 2 - no ROCKi during this step]
Passage to new dishes coated with 0.5mg/mL standard matrigel in NPC base +BDNF+GDNF+cAMP.
for 6w plates -> 400K cells/well in 2mL media
for 5cm cellBIND -> 1 million cells/dish in 6mL media
for 10cm cellBIND dishes -> 2.5 million cells/dish in 12mL media
d16+
Perform a half media change every 3-4 days until 90-95% confluent. When the cells are 90-95% confluent, passage at the same density as d13 and continue with half media changes every 3-4 days until 90-95% confluent again.
for 5cm cellBIND -> remove ~2-2.5mL (depending how much media evaporated, leave enough media
to cover the bottom), and add a fresh 3mL/well NPC base +BDNF+GDNF+cAMP.
for 10cm cellBIND -> remove ~4.5-5mL (depending how much media evaporated, leave enough media
to cover the bottom), and add a fresh 6mL/well NPC base +BDNF+GDNF+cAMP.
Note
Seeding at the lower density and feeding the cells less often (every 4 days) results in more progenitor differentiation, plan to have around the number of dishes you want to end with after the 2nd differentiation passage.
~d42
To enrich for neurons, around d42 (or close to this date when the dishes are 95% confluent again), treat the cells with 5ug/mL Mitomycin C for 1hour at 37C.
After 1hr, perform 3x washes with 6mL PBS, and then replace with 6mL (5cm dishes) or 12mL (10cm dish) of fresh NPC base +BDNF+GDNF+cAMP.
Note
If the cells were ~95% confluent before Mitomycin C treatment, 1x 10cm typically yields around 3-6 million cells.
Continue performing half media changes until used for experiments. After Mitomycin C treatment, the cells should not expand further, so they likely won't need to be passaged again until plating for experiments.
I've been using these for experiments ~d60-100.
Differentiation of NPCs to Astrocytes
Differentiation of NPCs to Astrocytes
d1 after passaging NPCs (see STEP 3)
Perform a full media change and replace with astrocyte differentiation media #1 (NPC base +hLIF+EGF)
NPC+hLIF+EGF (10mL):
10mL NPC base media
1uL 10 ng/mL hLIF (stock 100 ug/mL)
0.5uL 10 ng/mL EGF (stock 200 ug/mL)
d3
Perform a half media change. Remove 900uL/well, and add a fresh 1mL/well NPC base +hLIF+EGF.
d5
Perform a half media change. Remove 900uL/well, and add a fresh 1mL/well NPC base +hLIF+EGF.
d6-7 [differentiation passage 1 - no ROCKi during this step]
Passage to new dishes coated with 0.5mg/mL standard matrigel in NPC base +hLIF+EGF.
for 6w plates -> 1 million cells/well in 2mL media
for 5cm cellBIND -> 2.4 million cells/dish in 6mL media
for 10cm cellBIND dishes -> 5.8 million cells/dish in 12mL media
~d9
Perform a full media change and replace with neuron differentiation media (NPC base +hLIF+EGF) to remove any dead cells.
~d11
Perform a half media change. Remove 900uL/well, and add a fresh 1mL/well NPC base +hLIF+EGF.
~d13-14 [differentiation passage 2 - no ROCKi during this step]
Passage to new dishes coated with 0.5mg/mL standard matrigel in NPC base +hLIF+CNTF.
for 6w plates -> 400K cells/well in 2mL media
for 5cm cellBIND -> 1 million cells/dish in 6mL media
for 10cm cellBIND dishes -> 2.5 million cells/dish in 12mL media
NPC+hLIF+CNTF (10mL):
10mL NPC base media
1uL 10 ng/mL hLIF (stock 100 ug/mL)
1uL 10 ng/mL CNTF (stock 100 ug/mL)
~d16+
Perform a half media change every 3-4 days with NPC base +hLIF+CNTF until 95-100% confluent.
When the cells are 95-100% confluent, passage at the same density as ~d13 and continue with half media changes with NPC base +hLIF+CNTF every 3-4 days until 95-100% confluent again.
for 5cm cellBIND -> remove ~2-2.5mL (depending how much media evaporated, leave enough media
to cover the bottom), and add a fresh 3mL/well NPC base +hLIF+CNTF.
for 10cm cellBIND -> remove ~4.5-5mL (depending how much media evaporated, leave enough media
to cover the bottom), and add a fresh 6mL/well NPC base +hLIF+CNTF.
Note
If the cells were ~95% confluent before passaging, 1x 10cm typically yields around 9-14 million cells.
The cells will expand slower with each passage, avoid passaging until 95-100% confluent. Continue performing half media changes until used for experiments, around d50-60 astrocytes with more complex branching should appear and increase in number.
I've been using these for experiments ~d60-100.
After replating for experiments or if maintained past d100, I change the media to NPC +BDNF+GDNF+cAMP to prevent the astrocytes from overgrowing and detaching from the plate.