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LUHMES

CRL-2927

LUHMES is a neuronal-like cell that was isolated from the mesencephalon of a patient. This cell line was deposited by J Lotharius in 1998 and can be used in neuroscience research.
Product category
Human cells
Organism
Homo sapiens, human
Morphology
neuronal
Tissue
Brain; Mesencephalon
Applications
3D cell culture
High-throughput screening
Neuroscience
Toxicology
Product format
Frozen
Storage conditions
Vapor phase of liquid nitrogen
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Documentation

Biosafety Icon Biosafety Level 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 v-myc retroviral vector

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

Characteristics

Growth properties
Adherent
Derivation
The Lund human mesencephalic (LUHMES) cell line is a subclone of the tetracycline-controlled, v-myc-overexpressing human mesencephalic-derived cell line MESC2.10. MESC2.10 was originally immortalized with a LINX v-myc retroviral vector.
Age
8 weeks gestation
Immortalization method
LINX v-myc retroviral transfection
Comments
LUHMES cells can be differentiated into morphologically and biochemically mature dopamine-like neurons following exposure to tetracycline, GDNF (glial cell line-derived neurotrophic factor), and db-cAMP. LUHMES cells exhibit the same dopaminergic and neuronal characteristics as MESC2.10 cells.

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 base medium for this cell line is ATCC-formulated DMEM:F12 Medium Catalog No. 30-2006. To make the complete growth medium, add the following components to the base medium:
  • 1% N2 supplement (Gibco-Invitrogen Cat No 17502-048)
  • 40 ng/ml b-FGF ( basic recombinant human Fibroblast Growth Factor; Gibco-Invitrogen Cat No 13256-029) added fresh at the last moment
Temperature
37°C
Atmosphere
95% Air, 5% CO2
Handling procedure

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

Note: Culture flasks should be pre-coated with 50µg/mL poly-L-ornithine and then with 1µg/mL human fibronectin. (See Subculturing Procedure)

 

  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 culture medium without bFGF and spin at approximately 125 x g for 5 to 10 minutes.
  4. Resuspend cell pellet with the recommended complete medium (see the specific batch information for the culture recommended dilution ratio) and dispense into a pre-coated vented 75 cm2 culture flask.  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).
  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.
Subculturing procedure

These cells should be recovered from cryopreservation and subcultured only on culture flasks that are sequentially pre-coated with 50 µg/mL poly-L-ornithine (Sigma, Cat. No. P-3655 or equivalent) and then with 1 µg/mL Human Fibronectin (Sigma, Cat. No. F-0895 or equivalent).

Note: Use flasks with vented caps for best results.

  1. Add 7.0 mL freshly diluted 50 µg/mL poly-L-ornithine to T-75 cm2 flask and allow flask to sit over night at room temperature.
  2. Remove and discard poly-L-ornithine solution. Rinse flask 3 times with culture grade water (Hyclone cat#SH30529.01). Discard last rinse and allow flask to air-dry uncapped and standing upright in a biological cabinet.
  3. Add 5.0 mL freshly diluted 1 µg/mL fibronectin and incubate 3 hours at 37°C.
  4. Remove and discard fibronectin solution. Rinse flask 3 times with culture grade water (Hyclone cat#SH30529.01). Discard last rinse and allow flask to air-dry uncapped and standing upright in a biological cabinet.
  5. Flask is ready for use when dry.

Volumes used in this protocol are for 75 cm2 flasks; proportionally reduce or increase amount of dissociation medium for culture vessels of other sizes.
Note: Subculture before cells reach 80% confluency. Warm aliquots of wash medium (growth medium without bFGF) used in Step 4, freshly made complete growth medium and freshly diluted tryspin solution to 37°C prior to use.
  1. Remove and discard culture medium.
  2. Briefly rinse the cell layer with 10.0 mL Ca++/Mg++ free Dulbecco's phosphate-buffered saline (D-PBS).
  3. Add 4.0 mL of freshly diluted, pre-warmed 0.025% Trypsin-0.1 g/L EDTA solution (see formula below) to flask and incubate for 3 minutes at 37°C. Knock the flask several times against the palm of your hand to dislodge cells. Observe flask under an inverted microscope to be sure cells have come off.
  4. Add 6.0 mL of pre-warmed wash medium (see Note above) and aspirate cells by gently pipetting.
  5. Transfer cell suspension to a centrifuge tube and spin at approximately 190 x g for 7 minutes. Discard supernatant and knock the tube against the palm of your hand to loosen the cell pellet. Using a 1 mL pipet, add 1.0 mL complete growth medium and pipet the pellet up and down to resuspend the cells (avoid creating foam or bubbles).
  6. Add an additional 2.0 mL of complete growth medium and dissociate cells further by pipetting up and down.
  7. Adjust cell concentration by adding the necessary volume of complete growth medium needed to seed new flasks. Pipet up and down to evenly resuspend cells.
  8. Add appropriate aliquots of the cell suspension to new pre-coated vented culture flasks.
  9. Incubate cultures in 5% CO2/95% air at 37°C.
Subcultivation ratio: A subcultivation ratio of 1:3 to 1:4 is recommended. Subculture approximately every 3 to 4 days.
Medium renewal: Every 2 to 3 days

2X Trypsin-EDTA Solution: 2X Trypsin-EDTA solution is 0.05% Trypsin-0.2g/L EDTA. Before use this must be diluted 1:1 in Ca++/Mg++ free Dulbucco's phosphate-buffered saline (D-PBS) to 0.025% Trypsin-0.1g/l EDTA
To make 1 liter of 2X Trypsin-EDTA solution:

  1. Add the following to 500 ml ddH2O:
    1. 8 g NaCl
    2. 0.4 g KCL
    3. 0.58 g NaHCO3
    4. 1 g Dextrose
    5. 0.2 g EDTA
    6. 0.5 g Trypsin (Sigma, Cat. No. T7409)
  2. Bring volume up to 1 liter with ddH2O, and pH to 7.4 with HCL.
  3. Incubate at 37°C for at least 1 hour to activate the trypsin.
  4. Sterile filter (0.2 µm) and make aliquots.
  5. Refrigerate at 4°C overnight and then store at -20°C.
  6. Before use, dilute trypsin 1:1 with Ca++/Mg++ free Dulbucco?s phosphate-buffered saline (D-PBS) and warm to 37°C.
Reagents for cryopreservation
Complete growth medium supplemented with 20% fetal bovine serum and 10% (v/v) DMSO (ATCC 4-x)

Quality control specifications

Mycoplasma contamination
Not detected
STR profiling
Amelogenin: X
CSF1PO: 13,14
D5S818: 11,13
D13S317: 9,11
D7S820: 11,13
D16S539: 11,12
vWA: 14,17
THO1: 7,9.3
TPOX: 8

History

Depositors
J Lotharius
Year of origin
1998

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

References

Curated Citations

Lotharius J, et al. Progressive degeneration of human mesencephalic neuron-derived cells triggered by dopamine-dependent oxidative stress is dependent on the mixed-lineage kinase pathway. J. Neurosci. 25 (27) : 6329-6342, 2005. PubMed: 16000623

Lotharius J, et al. Effect of mutant alpha-synuclein on dopamine homeostasis in a new human mesencephalic cell line. J. Bio.Chem. 277(41): 38884-388894, 2002. PubMed: 12145295

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