Neural Progenitor Cells Derived from ATCC-DYS0530 Parkinson's Disease

1. Check all containers for leakage or breakage.
2. Remove the frozen cells from the dry ice packaging and immediately place the cells at a temperature below ­-130°C, preferably in liquid nitrogen vapor, until ready for use.

• 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:

• 2.5 mL L-Alanyl-L-Glutamine
• 2.5 mL Non-Essential Amino Acids + 0.5 mL Ascorbic Acid
• 5 mL Dopaminergic Differentiation Kit Component A
• 2.5 mL Dopaminergic Differentiation Kit Component B
• 0.5 mL  Dopaminergic Differentiation Kit Component C

Coat plates with Cell Basement Membrane Gel (ATCC ACS-3035) and culture the NPCs with NPC Growth Medium (ATCC ACS-3003) to provide a surface for the attachment of NPCs.
Coating Procedure:

1. Thaw Cell Basement Membrane Gel on ice and swirl gently to mix. Important: Cell Basement Membrane Gel will solidify in 15 to 30 minutes above 15°C. Keep Cell Basement Membrane Gel, vials and pipette tips on ice at all times to prevent Cell Basement Membrane Gel from solidifying. If air bubbles form, they may be eliminated by centrifuging Cell Basement Membrane Gel at 300 x g for 10 minutes at 2°C to 8°C.
2. 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).
3. Dilute Cell Basement Membrane in DMEM:F12 to a working concentration of 150 μg/mL. Add 0.5 ml diluted Cell Basement Membrane gel per well of 12 well plate. 
4. Cell culture dishes coated with Cell Basement Membrane 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.

1. Thaw Cell Basement Membrane Basement Membrane Gel on ice or at 4°C
2. Prepare a 150 µg/ mL working concentration of Cell Basement Membrane in cold DMEM: F-12 medium
3. Add enough Cell Basement Membrane solution to cover the surface of the plate (e.g. 1 mL diluted Cell Basement Membrane/well of a 12-well plate)
4. Incubate for 1 hour at 37°C prior to use

1. Prepare complete NPC growth medium (ATCC ACS-3003) following the instructions in the package and pre-warm that medium as well as DMEM:F12 in a 37°C water bath for 15-30 min. If using a small volume of medium (50 mL or less), warm only the volume needed in a sterile conical tube. Avoid warming complete medium multiple times.
2. Obtain a 12-well plate with Cell Basement Membrane Basement Membrane. Aspirate the Cell Basement Membrane medium and directly add 1.5 mL of the complete NPC Growth Medium per well. Place the plate in the incubator for 15 minutes to allow the medium to reach its normal pH (7.0-7.6). Four to five wells of a 6-well plate may be needed for each vial of cells thawed.
3. Transfer 9 mL of pre-warmed DMEM:F12 into a 15 mL conical tube for recovery of the NPCs from the frozen stock.
4. Remove cryovial of frozen cells from liquid nitrogen storage.
5. Thaw the cells by gently swirling in a 37°C water bath. To reduce the possibility of contamination, keep the cap out of the water. Thawing should be rapid (approximately 1 to 2 minutes). Remove the cryovial from water bath when only a few ice crystals are remaining.
6. Sterilize the cryovial with 70% ethanol. All of the operations from this point on should be carried out under strict aseptic conditions.
7. Remove cells from the vial using a P1000 micropipette and transfer cells drop-wise into the 15 mL conical tube containing 9 mL DMEM:F12.
8. Centrifuge cells at 270 x g for 5 minutes at room temperature.
9. Aspirate the supernatant and discard. Gently tap the bottom of the tube to loosen the cell pellet.
10. Add 4 mL of the complete NPC Growth Medium to the tube. Gently resuspend the pellet by pipetting up and down 3 or 4 times to make a single-cell suspension.
11. Perform cell count by a Vi-Cell Analyzer or hemocytometer. Note: Don’t perform cell count by a Vi-Cell Analyzer without removal of serum-free freezing medium.
12. Seed NPCs at a seeding density of 80,000 viable cells/cm² (e.g. 0.30 x10⁶/well of a 12-well plate) onto a Cell Basement Membrane-coated plate described above.
13. Incubate the plate at 37°C with 5% CO₂ overnight.
14. Change medium at 100% media change rate (1 mL media/well) next day and every other day thereafter.
15. Monitor cell growth and passage the cells when they reach ~95% confluency

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 Cell Basement Membrane-coated dishes/plates.

Dopaminergic Differentiation: Seed NPCs at 10,000 cells/cm² in a 12-well plate pre-coated with Cell Basement Membrane. 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:

1. 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.
2. 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.
3. 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.
4. Monitor NPC differentiation.

Astrocyte, oligodendrocyte, and neuron differentiation; drug screening

The product is provided 'AS IS' and the viability of ATCC^® products is warranted for 30 days from the date of shipment, provided that the customer has stored and handled the product according to the information included on the product information sheet, website, and Certificate of Analysis. For living cultures, ATCC lists the media formulation and reagents that have been found to be effective for the product. While other unspecified media and reagents may also produce satisfactory results, a change in the ATCC and/or depositor-recommended protocols may affect the recovery, growth, and/or function of the product. If an alternative medium formulation or reagent is used, the ATCC warranty for viability is no longer valid.  Except as expressly set forth herein, no other warranties of any kind are provided, express or implied, including, but not limited to, any implied warranties of merchantability, fitness for a particular purpose, manufacture according to cGMP standards, typicality, safety, accuracy, and/or noninfringement.

This product is intended for laboratory research use only. It is not intended for any animal or human therapeutic use, any human or animal consumption, or any diagnostic use. Any proposed commercial use is prohibited without a license from ATCC.

While ATCC uses reasonable efforts to include accurate and up-to-date information on this product sheet, ATCC makes no warranties or representations as to its accuracy. Citations from scientific literature and patents are provided for informational purposes only. ATCC does not warrant that such information has been confirmed to be accurate or complete and the customer bears the sole responsibility of confirming the accuracy and completeness of any such information.

This product is sent on the condition that the customer is responsible for and assumes all risk and responsibility in connection with the receipt, handling, storage, disposal, and use of the ATCC product including without limitation taking all appropriate safety and handling precautions to minimize health or environmental risk. As a condition of receiving the material, the customer agrees that any activity undertaken with the ATCC product and any progeny or modifications will be conducted in compliance with all applicable laws, regulations, and guidelines. This product is provided 'AS IS' with no representations or warranties whatsoever except as expressly set forth herein and in no event shall ATCC, its parents, subsidiaries, directors, officers, agents, employees, assigns, successors, and affiliates be liable for indirect, special, incidental, or consequential damages of any kind in connection with or arising out of the customer's use of the product. While reasonable effort is made to ensure authenticity and reliability of materials on deposit, ATCC is not liable for damages arising from the misidentification or misrepresentation of such materials.

Please see the material transfer agreement (MTA) for further details regarding the use of this product. The MTA is available at www.atcc.org.