LUHMES (ATCC® CRL-2927)

Organism: Homo sapiens, human  /  Tissue: mesencephalon  / 

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Organism Homo sapiens, human
Tissue mesencephalon
Product Format frozen
Morphology neuronal
Culture Properties adherent
Biosafety Level 2  [Cells contain v-myc retroviral vector]
Age fetus, 8 weeks gestation
Storage Conditions liquid nitrogen vapor phase
Images
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.
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.
Complete Growth 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
Subculturing

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 sterile double distilled water. 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 sterile water. 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.
Cryopreservation
Freeze medium: complete growth medium supplemented with 20% fetal bovine serum and 10% (v/v) DMSO
Storage temperature: liquid nitrogen vapor phase
Culture Conditions
Temperature: 37°C
Atmosphere: air, 95%; carbon dioxide (CO2), 5%
STR Profile
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
Name of Depositor J Lotharius
Year of Origin 1998
References

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

Notice: Necessary PermitsPermits

These permits may be required for shipping this product:

  • Customers located in the state of Hawaii will need to contact the Hawaii Department of Agriculture to determine if an Import Permit is required. A copy of the permit or documentation that a permit is not required must be sent to ATCC in advance of shipment.
Basic Documentation
Other Documentation
References

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