Complete Growth Medium
Complete growth media for Neural Progenitor Cells (NPCs) includes DMEM: F12 (ATCC 30-2006) supplemented with the Growth Kit for Neural Progenitor Cell Expansion (ATCC ACS-3003). To make complete NPC medium add the following components of the kit to 464 mL DMEM: F12:
- 5 mL L-Alanyl-L-Glutamine
- 5 mL Non-Essential Amino Acids
- 10 mL NPC Growth Kit Component A
- 5 mL NPC Growth Kit Component B
- 1 mL NPC Growth Kit Component C
- 10 mL NPC Growth Kit Component D
Complete dopaminergic differentiation medium for NPCs is DMEM: F12 (ATCC 30-2006); supplemented with the Neural Progenitor Cell Dopaminergic Differentiation Kit (ATCC ACS-3004). To make complete NPC dopaminergic differentiation medium add the following components of this kit to 237 mL DMEM: F12:
Also add CHIR-99021 (from Stemgent, catalog number 3003) to a final concentration of 5 µM (625 µl to 250 ml of media)
- 2.5 mL L-Alanyl-L-Glutamine
- 2.5 mL Non-Essential Amino Acids + 0.5 mL Ascrobic Acid
- 5 mL Dopaminergic Differentiation Kit Component A
- 2.5 mL Dopaminergic Differentiation Kit Component B
- 0.5 mL Dopaminergic Differentiation Kit Component C
Post-thaw: 40,000 viable cells/cm2 on CellMatrix-coated dishes/plates
Subculture: 40,000 viable cells/cm2 on CellMatrix-coated dishes/plates
Post thaw day 1, perform a 100% medium change and remove all cells that did not attach. Perform a 100% medium change every other day thereafter. Passage the cells cells with diluted Accutase (50% Accutase and 50% DPBS) when they reach ~95% confluence and reseed the NPCs at 40,000 viable cells/cm2 on CellMatrix-coated dishes/plates.
Dopaminergic Differentiation: Seed NPCs at 10,000 cells/cm2 in a 12-well plate pre-coated with CellMatrix. Culture overnight in complete NPC expansion medium. The next day, aspirate the medium and add 1.5 ml of pre-warmed complete Dopaminergic Differentiation Medium containing CHIR. Change the Dopaminergic media every other day (e.g., Monday, Wednesday, and Friday) for 3 weeks as the following:
- For each media change in the process, gently remove approximately 85% of the medium using a 5 ml serological pipette from each well and discard.
During the first week of the differentiation, slowly add 1.5 ml of fresh Dopaminergic Differentiation Medium to each well along the wall of the well on Monday and Wednesday; add 2 ml of of fresh Dopaminergic Differentiation Medium on Friday.
During the second and third week of the differentiation, slowly add 2 ml of fresh Dopaminergic Differentiation Medium to each well along the wall of the well on Mondays and Wednesdays; add 2.5 ml of of fresh Dopaminergic Differentiation Medium on Fridays.
Monitor NPC differentiation.
Coat plates with CellMatrix (ATCC ACS-3035) and culture the NPCs with NPC Growth Medium (ATCC ACS-3003) to provide a surface for the attachment of NPCs.
- Thaw CellMatrix Gel on ice and swirl gently to mix. Important: CellMatrix Gel will solidify in 15 to 30 minutes above 15°C. Keep CellMatrix Gel, vials and pipette tips on ice at all times to prevent CellMatrix Gel from solidifying. If air bubbles form, they may be eliminated by centrifuging CellMatrix Gel at 300 x g for 10 minutes at 2°C to 8°C.
- Determine the appropriate volume per aliquot based on concentration and usage. For seeding, plate cells at 40,000 viable cells/cm2 (1.50 x 106 cells/ well of 12-well plate).
Dilute CellMatrix in DMEM:F12 to a working concentration of 150 μg/mL. Add 0.5 ml diluted CellMatrix gel per well of 12 well plate.
- Cell culture dishes coated with CellMatrix Basement Membrane Gel should be incubated at 37°C for one hour. Aspirate coating solution and immediately plate the cells. It is critical that the coating does not dry out.