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

ACS-5001

Product category
Human cells
Product type
Cell model
Organism
Homo sapiens
Cell type
neural progenitor cell
Morphology
short spindle shape
Tissue
Skin; Dermis
Disease
Parkinsons disease
Applications
Drug development
High-throughput screening
Neuroscience
Stem cell research
Toxicology
Product format
Frozen
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Price: $1,030.00 EA
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Documentation

Biosafety Icon BSL 2

ATCC determines the biosafety level of a material based on our risk assessment as guided by the current edition of Biosafety in Microbiological and Biomedical Laboratories (BMBL), U.S. Department of Health and Human Services. It is your responsibility to understand the hazards associated with the material per your organization’s policies and procedures as well as any other applicable regulations as enforced by your local or national agencies.

Cells contain Sendai virus (SeV) DNA sequences

ATCC highly recommends that appropriate personal protective equipment is always used when handling vials. For cultures that require storage in liquid nitrogen, it is important to note that some vials may leak when submersed in liquid nitrogen and will slowly fill with liquid nitrogen. Upon thawing, the conversion of the liquid nitrogen back to its gas phase may result in the vial exploding or blowing off its cap with dangerous force creating flying debris. Unless necessary, ATCC recommends that these cultures be stored in the vapor phase of liquid nitrogen rather than submersed in liquid nitrogen.

Required Products

These products are vital for the proper use of this item and have been confirmed as effective in supporting functionality. If you use alternative products, the quality and effectiveness of the item may be affected.

Growth Kit for Neural Progenitor Cell Expansion

ACS-3003

Price: $329.00 ea

Stem Cell Freezing Media

ACS-3020

Price: $55.00 ea

CellMatrix Basement Membrane Gel

ACS-3035

Price: $221.00 ea

Stem Cell Dissociation Reagent

ACS-3010

Price: $98.00 ea

DMEM: F-12 Medium

30-2006

Price: $24.00 ea

Detailed product information

General

Specific applications
Neuronal differentiation; drug and toxicological screening

Characteristics

Cells per vial
1.0 x 106
Volume
1.0 mL
Growth properties
Adherent
Derivation
Parkinson's disease NPCs (ATCC® ACS-5001™) are derived from ATCC-DYS0530 Parkinson's disease Human [Caucasian Male] Induced Pluripotent Stem Cells (ATCC® ACS-1013™)

Age
63 years
Ethnicity
White
Gender
Male
Antigen expression
Nestin, Pax-6
Comments
Parkinson's disease research; astrocyte, oligodendrocyte, and neuron differentiation; drug screening, toxicology

Handling information

Unpacking and storage instructions
  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.
Complete 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:

  • 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
Also add CHIR-99021 (from Stemgent, catalog number 3003) to a final concentration of 5 µM (625 µl to 250 ml of media)
Seeding density
Post-thaw: 4.0 x 104 viable cells/cm2 on CellMatrix-coated dishes/
Subculture: 4.0 x 104 viable cells/cm2 on CellMatrix-coated dishes/plates
Handling procedure

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.
Coating Procedure:

  1. 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.
  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 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. 
  4. 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.
To insure the highest level of viability, thaw the vial and initiate the culture as soon as possible upon receipt. If, upon arrival, continued storage of the frozen culture is necessary, it should be stored in liquid nitrogen vapor phase and not at –80°C. Storage at –80°C will result in loss of viability. Preparation of CellMatrix Basement Membrane Gel (ATCC ACS-3035) coated plates:
  1. Thaw CellMatrix Basement Membrane Gel on ice or at 4°C
  2. Prepare a 150 µg/ mL working concentration of CellMatrix in cold DMEM: F-12 medium
  3. Add enough CellMatrix solution to cover the surface of the plate (e.g. 1 mL diluted CellMatrix/well of a 12-well plate)
  4. Incubate for 1 hour at 37°C prior to use
Initiation of Cultures
  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 CellMatrix Basement Membrane. Aspirate the CellMatrix 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 CellMatrix-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
Note: Don’t passage NPCs when the cells are <85% confluency.
Subculturing procedure

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:

  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.

Quality control specifications

Bacterial and fungal testing
Not detected
Mycoplasma contamination
Not detected
Virus testing
Cytomegalovirus (CMV): Not detected
Human papillomavirus (HPV): Not detected
Human Immunodeficiency virus (HIV): Not detected
Epstein-Barr virus (EBV): Not detected
Hepatitis B virus (HBV): Not detected
Functional tests
Exhibit differentiation potential of NPCs into Tuj1+ early neurons and TH+ dopaminergic neurons using NPC Expansion Medium (ACS-3003) and Dopaminergic Differentiation Kit (ACS-3004)
Population doubling time
Approximately 42 hrs
STR profiling
Amelogenin: X,Y
CSF1PO: 10,12
D13S17: 11
D16S539: 10,11
D5S818: 11,12
D7S820: 10,12
TH01: 7
TPOX: 8,11
vWA: 17,18
Viability
≥ 80%

History

Depositors
ATCC
Year of origin
2016

Legal disclaimers

Intended use
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.

Astrocyte, oligodendrocyte, and neuron differentiation; drug screening

Warranty

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.

Disclaimers

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.

Permits & Restrictions

Import Permit for the State of Hawaii

If shipping to the U.S. state of Hawaii, you must provide either an import permit or documentation stating that an import permit is not required. We cannot ship this item until we receive this documentation. Contact the Hawaii Department of Agriculture (HDOA), Plant Industry Division, Plant Quarantine Branch to determine if an import permit is required.

This material is subject to the following restrictions in addition to those outlined in the ATCC Material Transfer Agreement:

  1. iPS Label License 
  2. This product was generated using Sendai virus vectors from ID Pharma

For information on obtaining additional rights, please contact:

ATCC Licensing
Email: [email protected]

MORE INFORMATION ABOUT PERMITS AND RESTRICTIONS

References

Curated Citations

Malik N, et al. Compounds with species and cell type specific toxicity identified in a 2000 compound drug screen of neural stem cells and rat mixed cortical neurons. Neurotoxicology 45: 192-200, 2014. PubMed: 25454721

Frequently Asked Questions

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