LUHMES細胞

ATCC Number： CRL-2927?

數量： 大量

年限： fetus, 8 weeks gestation

是否是腫瘤細胞： 0

物種來源： 人

組織來源： mesencephalon

運輸方式： 凍存運輸

生長狀態： 貼壁生長

細胞形態： 神經元

Designations: LUHMES

Depositors: J Lotharius

Biosafety Level: 2

Shipped: frozen

LUHMES細胞Medium & Serum: See Propagation

Growth Properties: adherent

Organism: Homo sapiens

Morphology: neuronal

Source: Tissue: mesencephalon

Permits/Forms: In addition to the MTA mentioned above, other ATCC and/or regulatory permits may be required for the transfer of this ATCC material. Anyone purchasing ATCC material is ultimately responsible for obtaining the permits. Please click here for information regarding the specific requirements for shipment to your location.

Isolation: Isolation date: 1998

DNA Profile (STR): 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

Age: fetus, 8 weeks gestation

Comments: The Lund human mesencephalic (LUHMES) cell line is a subclone of the tetracycline-controlled, v-myc-overexpressing human mesencephalic-derived cell line MESC2.10. LUHMES細胞MESC2.10 was originally immortalized with a LINX v-myc retroviral vector. [16173252] [16173253]

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. [16173253]

Propagation: ATCC 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

Temperature: 37.0°C

Atmosphere: air, 95%; carbon dioxide (CO2), 5%

Subculturing: Flask Coating Procedure:

Culture flasks should be sequentially pre-coated with 50μg/ml poly-L-ornithine (Sigma, Cat. No. P-3655 or equivalent) and then with 1μg/ml Human Fibronectin (Sigma, Cat. No. F-0895 or equivalent).

Note: Use flasks with vented caps for best results.

LUHMES細胞Add 7.0 ml freshly diluted 50μg/ml poly-L-ornithine to T-75 sq cm flask and allow flask to sit over night at room temperature.

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.

Add 5.0 ml freshly diluted 1μg/ml fibronectin and incubate 3 hours at 37°C.

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.

Flask is ready for use when dry.

Protocol:

Subculture before cells reach 80% confluency.

Volumes used in this protocol are for 75 sq cm flasks; proportionally reduce or increase amount of dissociation medium for culture vessels of other sizes.

Note: 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.

Remove and discard culture medium.

Briefly rinse the cell layer with 10.0 ml Ca++/Mg++ free Dulbecco's phosphate-buffered saline (D-PBS).

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.

Add 6.0 ml of pre-warmed wash medium (see Note above) and aspirate cells by gently pipetting.

Transfer cell suspension to a centrifuge tube and spin at approximately 190 xg 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).

Add an additional 2.0 ml of complete growth medium and dissociate cells further by pipetting up and down.

LUHMES細胞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.

Add appropriate aliquots of the cell suspension to new pre-coated vented culture flasks.

Incubate cultures in 5% CO2/95% air at 37°C.

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:

Add the following to 500 ml ddH2O:

8 g NaCl

0.4 g KCL

0.58 g NaHCO3

1 g Dextrose

0.2 g EDTA

0.5 g Trypsin (Sigma, Cat. No. T7409)

Bring volume up to 1 liter with ddH2O, and pH to 7.4 with HCL.

Incubate at 37°C for at least 1 hour to activate the trypsin.

Sterile filter (0.2 μm) and make aliquots.

Refrigerate at 4°C overnight and then store at -20°C.

Before use, dilute trypsin 1:1 with Ca++/Mg++ free Dulbucco?s phosphate-buffered saline (D-PBS) and warm to 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

Preservation: Freeze medium: complete growth medium supplemented with 20% fetal bovine serum and 10% (v/v) DMSO

Storage temperature: liquid nitrogen vapor phase

Related Products: Recommended medium LUHMES細胞(without the additional serum described under ATCC Medium): ATCC 30-2006

Cell culture tested DMSO: ATCC 4-X

Recommended serum: ATCC 30-2020

Phosphate-buffered saline: ATCC 30-2200

References: 16173252: 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

16173253: 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