Immortalized Human Dopaminergic Neuronal Precursor Cells (LUHMES)

The Immortalized Human Dopaminergic Neuronal Precursor Cells, also known as the Lund Human Mesencephalic (LUHMES) cell line, is a subclone of the tetracycline-controlled, v-myc-overexpressing human mesencephalic-derived cell line MESC2.10. This cell line is unique in that it can be differentiated to acquire a dopaminergic neuron-like phenotype under appropriate growth conditions. LUHMES expresses functional dopamine transporter (DAT), vesicular monoamine transporter (VMAT-2), tyrosine hydroxylase (TH) and the neuronal form of β-III tubulin after differentiation. In addition to retaining dopaminergic neuronal-specific markers, LUHMES also exhibit electrophysiological properties, thus making this cell line a valuable neuronal model for neurodevelopmental studies, disease modelling and neuropharmacology.

Immortalization Method: Conditional immortalization by tetracyclin-controlled transduction with retrovirus carrying v-myc genes

BioSafety Level: II

Species: Human

Source Organ: 8-week-old fetal human ventral mesencephalon

Growth Properties: Adherent

Morphology: Flattened|Dendritic processes

Population Doubling: 30 - 40 hours

Seeding Density: 50,000 - 100,000 cells/cm²; cells will have viability of around 40-50%. Post thawing, it will need 4-5 days for cells to recover in culture; Recommended split ratio is 1:4 to 1:5

Markers: DAT, VMAT-2, TH, α-SYN, β-III tubulin

Donor Age: 8 week

Donor Gender: undetermined

Donor Ethnicity: N/A

Propagation:Grow the cells in culture vessel pre-coated with 50 μg/ml poly-L-ornithine (PLO) (TM062) and 1 μg/ml fibronectin (EMD Millipore; Cat. FC010) in H2O for at least 3 hours at 37°C.
The base medium for this cell line is Advanced DMEM/F12 (Gibco; 12634010). To make the complete growth medium, add the following components to the base medium: 1x N2 supplement (ThermoFisher Scientific), 2 mM L-glutamine (G275) and 40 ng/ml recombinant bFGF (Z101455).
Carbon dioxide (CO₂): 5%, Temperature: +37.0°C.
Change the complete media every 2-3 days. Do not let media colour change to orange-yellow. The cells will form round clumps instead of a monolayer when stressed.
It is recommended to subculture when cells are grown to a cell density of 50-60%. To subculture the cells, use 0.25% Trypsin-EDTA (TM051). After adding trypsin, incubate the cells at room temperature for 2-3 minutes and agitate the culture vessel until 90% of the cells have detached. Immediately neutralize the trypsin using Trypsin Neutralizing Solution (Lonza, Cat. CC-5002). Add Trypsin Neutralizing Solution twice the volume of trypsin used. Centrifuge at 200x g for 2-3 minutes. Aspirate supernatant and resuspend cells in complete media. Plate cells into pre-warmed PLO-fibronectin-coated vessels at 37^oC. Avoid subculturing if the cells appear stressed.
Note: If leaving the cells over the weekend (or more than 2 days), make sure to do a high split ratio (1:4 to 1:5).
To differentiate the cells into neurons, change the medium to differentiation medium after the cells have grown to a density of 40-50%. To make complete differentiation medium, add the following components to the basal is Advanced DMEM/F12 (Gibco;12634010): 1x N2 supplement, 2 mM L-glutamine, 1mM dbcAMP, 1 μg/ml tetracycline, and 2 ng/ml recombinant human GDNF (Z101055). Allow the cells in differentiation medium for 4-6 days before testing for neuron specific markers.

Quality Control: mRNA and protein expression levels of various markers pre and post differentiation are verified by RT-PCR and western blotting.

Shipping Condition: Dry Ice

Storage Condition: liquid nitrogen or -180°C