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hTERT NF1 ipNF05.5 (Mixed clones)

CRL-3387

Product category
Human cells
Product type
hTERT-immortalized cell
Organism
Homo sapiens, human
Cell type
Schwann Cell
Morphology
fibroblast/spindle-like
Tissue
Neural; Hand
Disease
Plexiform Neurofibroma; Tumor not malignant
Applications
3D cell culture
Drug development
High-throughput screening
Neuroscience
Product format
Frozen
Storage conditions
Vapor phase of liquid nitrogen
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Documentation

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.

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.

Detailed product information

General

Specific applications

Through generous support from the Neurofibromatosis Therapeutic Acceleration Program (NTAP), purchasers of these cells are eligible to receive up to a 100% reimbursement from NTAP.  For additional information and to apply for reimbursement after a purchase, please contact NTAP at [email protected] or visit the NTAP Website.

For study of Schwann cell biology, diseases and tumors. These cells are invaluable tools for dissecting the processes contributing to the formation of neurofibromas and possibly other diseases, and the development of disease models and therapeutics.

The collection contains “normal” (wild-type) NF1(+/+),  NF1(+/-) (heterozygous), and NF1(-/-) homozygous (neurofibroma derived) human Schwann cell lines.  (NOTE: Cell-lines generated under auspices of the lab of Dr. Peggy Wallace, University of Florida, with funding support by the Neurofibromatosis Therapeutic Acceleration Program (NTAP) at Johns Hopkins University)


Characteristics

Cells per vial
Approximately 2.0 to 3.0 x 106
Growth properties
Adherent
Age
22 years
Ethnicity
White
Gender
Male
Karyotype
46,XY,t(2;3)(q23;p26) (p32)
Comments

A team of researchers from the University of Florida, the National Center for Advancing Translational Sciences (NCATS), and Sage Bionetworks with support from the Neurofibromatosis Therapeutic Acceleration Program (NTAP) at Johns Hopkins, have developed and characterized a set of immortalized patient derived plexiform neurofibroma Schwann cells in an initial effort to create a series of cells that represent the genetic diversity of human plexiform tumors.1 

Through generous support from the Neurofibromatosis Therapeutic Acceleration Program (NTAP), purchasers of these cells are eligible to receive up to a 100% reimbursement from NTAP.  For additional information and to apply for reimbursement after a purchase, please contact NTAP at [email protected] or visit the NTAP Website.

Through exogenous expression of human telomerase reverse transcriptase (hTERT) and murine cyclin-dependent kinase (mCdk4) using retroviral (and subsequently lentiviral) vectors carrying the hTERT and mCdk4 genes, normal (NF1 wild-type, +/+), neurofibroma-derived Schwann cells that were heterozygous (+/-) for NF1 mutation, and homozygous (-/-) for NF1 mutation, were immortalized. 1

This product, and other immortalized cells from the collection, have undergone in-depth characterization and authentication.

Real time PCR for hTERT and mCdk4 Proliferation rate
NF1 mutational analysis Doubling time
Karyotyping Apoptosis
Cell authentication Microsatellite instability
Contact inhibition Tumorigenicity

 

To learn more and view characterization data, go to: https://www.synapse.org/#!Synapse:syn7239479/wiki/405899

Gosline, SJC Synapse http://doi.org/10.7303/syn4939906 (2017).
Gosline, SJC Synapse http://doi.org/10.7303/syn4940963 (2017).
Gosline, SJC Synapse http://doi.org/10.7303/syn4939874 (2017).
NCBI Sequence Read Archive SRP125359 (2018)
  • Mixture of 6 single-cell clones derived from P16 immortalized primary culture of plexiform neurofibroma cells (ipNF05.5)
  • Tumor: homozygous disease (NF1-/-), plexiform neurofibroma
  • Oncogenes: transduced with mCdk4 cDNA and hTERT
  • Neurofibroma Schwann cell culture transduced with human telomerase (TERT) cDNA and mouse Cdk4 cDNA in lentivirus vectors (no selection), to immortalize the cells.  
Proliferation rate: 82% BrdU positive at 75% confluency

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
The basal medium for this cell line is Dulbecco's Modified Eagle's Medium (DMEM; ATCC 30-2002). To make the complete medium add the following components to 500 mL DMEM:

Handling procedure

To ensure 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 -70°C.  Storage at -70°C will result in loss of viability.  

  1. Thaw the vial by gentle agitation in a 37°C water bath. To reduce the possibility of contamination, keep the O-ring and cap out of the water.  Thawing should be rapid (approximately 2 minutes).
  2. Remove the vial from the water bath as soon as the contents are thawed, and decontaminate by dipping in or spraying with 70% ethanol. All of the operations from this point on should be carried out under strict aseptic conditions.
  3. Transfer the vial contents to a centrifuge tube containing  9.0 mL complete culture medium. and spin at approximately 125 x g for 5 to 7 minutes.
  4. Resuspend cell pellet with the recommended complete medium (see the specific batch information for the culture recommended dilution ratio). It is important to avoid excessive alkalinity of the medium during recovery of the cells.  It is suggested that, prior to the addition of the vial contents, the culture vessel containing the complete growth medium be placed into the incubator for at least 15 minutes to allow the medium to reach its normal pH (7.0 to 7.6). pH (7.0 to 7.6).
  5. Incubate the culture at 37°C in a suitable incubator.  A 5% CO2 in air atmosphere is recommended if using the medium described on this product sheet.

         

Subculturing procedure
Volumes used in this protocol are for 75 cm2 flask; proportionally reduce or increase amount of dissociation medium for culture vessels of other sizes. Corning® T-75 flasks (catalog #430641) are recommended for subculturing this product.
  1. Remove and discard culture medium.
  2. Briefly rinse the cell layer with 0.25% (w/v) Trypsin- 0.53 mM EDTA solution to remove all traces of serum that contains trypsin inhibitor.
  3. Add 2.0 to 3.0 mL of Trypsin-EDTA solution to flask and observe cells under an inverted microscope until cell layer is dispersed (usually within 5 to 15 minutes).
    Note: To avoid clumping do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach. Cells that are difficult to detach may be placed at 37°C to facilitate dispersal.
  4. Add 6.0 to 8.0 mL of complete growth medium and aspirate cells by gently pipetting.
  5. Add appropriate aliquots of the cell suspension to new culture vessels.
    Cultures can be established between 2 x 104 and 4 x 104 viable cells/cm2.
  6. Incubate cultures at 37°C.
Interval: Maintain cultures at a cell concentration between 1.0 X 104 and 2.4 X 105 cell/cm2.
Subcultivation Ratio: A subcultivation ratio of 1:3 to 1:8 is recommended
Medium Renewal: 2 to 3 times per week
Reagents for cryopreservation
Complete growth medium supplemented with 5% (v/v) DMSO (ATCC 4-X)

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
Population doubling time
Approximately 59 hrs
STR profiling
Amelogenin: X,Y
CSF1PO: 10,11
D13S317: 11
D16S539: 13,14
D5S818: 11
D7S820: 10,12
THO1: 8,9
TPOX: 8,11
vWA: 16
Viability
≥ 50%

History

Depositors
M Wallace, University of Florida, College of Medicine, Gainesville, FL
Year of origin
2008

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.
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.

MORE INFORMATION ABOUT PERMITS AND RESTRICTIONS

Frequently Asked Questions

References

Curated Citations

Li H, et al. Immortalization of Human Normal and NF1 Neurofibroma Schwann Cells. Lab Invest 96(10):1105-15, 2016. PubMed: 27617404

Ryman DA, et al. Pharmacological and genomic profiling of neurofibromatosis type 1 plexiform neurofibroma-derived schwann cells. Sci Data 5:180106, 2018. PubMed: 29893754

Kraniak JM, et al. Development of 3D culture models of plexiform neurofibroma and initial application for phenotypic characterization and drug screening. Exp Neurol 299(Pt B):289-298, 2018. PubMed: 29055717

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