May 19, 2026

LUHMES (lund human mesencephalic) culturing and differentiation protocol

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This  protocol  is a draft, published without a DOI.
LUHMES (lund human mesencephalic) culturing and differentiation protocol
  • 1Washington University in St. Louis;
  • 2Washington University: MGI
  • WashU FIVE @ MGI
Serena Elia
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Protocol Citation: William Buchser, Colin Kremit, Jason Waligorski, Graha Bachman, Serena Elia, Emanuel Gerbi 2026. LUHMES (lund human mesencephalic) culturing and differentiation protocol. protocols.io https://dx.doi.org/
Manuscript citation:
License: This is an open access  protocol  distributed under the terms of the  Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: In development
We are still developing and optimizing this protocol
Created: May 13, 2025
Last Modified: May 19, 2026
Protocol  Integer ID: 218215
Keywords: LUHMES differentiation, Neuronal differentiation, Dopaminergic neurons, Human Midbrain, Dopaminergic Neuron Differentiation, human mesencephalic, lund human mesencephalic, luhme, differentiation protocol
Abstract




Materials
ReagentsStock ConcentrationFinal ConcentrationFinal Solution Volume= 20mL
DMEM/F12 x x 19,792uL
LUHMES Growth Media
ReagentsNoteStock ConcentrationWorking ConcentrationFinal Solution Volume = 20mL
DMEM/F12 X X 19,568uL
B27-supplement 100X 1X 200uL
DibutyrylcAMP MW: 491.4 g/mol Mass: 100mg Reconstitue: Add 1.56mL of PBS to 100mg Vial 100mM 1mM 200uL
Ascorbic Acid (500mg Vial)Reconstitute: Add 14.195mL PBS (pH 7.2) to 500mg of absorbic acid 200mM 0.2mM 20uL
Human Recombinant LIF (50ug vial)Reconstitute: Add 500ul of nuclease free water to 50ug vial. *2 weeks or at -20°C to -80°C for up to 3 months 0.1ug/mL 10ng/mL 2uL
Human Recombinant BDNF (10ug vial)Reconstitute: Add 100ul to vial. centrifuged before opening, Do Not Vortex 0.1 mg/mL 20ng/mL 4uL
Human Recombinant GDNF (10ug vial)Reconstitute: Add 100ul of nuclease free water to 10ug vial of GDNF. Make 5ul aliquots Store aliquots at -20°C0.1mg/mL 20ng/mL 4uL
Tetracycline MW: 480.91 g/mol Mass: 500mg Reconstitue: Add 50mL of bio-grade water to 500mg of tetracycline. Store in -20 10mg/mL1ug/mL  2uL
TGF β-III (10ug vial)Storage Conditions 4° C 0.25 mg/mL20ng/mL 0.25 mg/mL
LUHMES Differentiation Media
Tgf beta 3 (human) Recombinant ProteinInvitrogen - Thermo FisherCatalog #RP8600 Human Recombinant LIFSTEMCELL Technologies Inc.Catalog #78055
Dibutyryl-cAMPSTEMCELL Technologies Inc.Catalog #73884
Tetracyline HydrochlorideThermo ScientificCatalog #A39246
Human Recombinant GDNFSTEMCELL Technologies Inc.Catalog #78058 Human Recombinant BDNFSTEMCELL Technologies Inc.Catalog #78005
Absorbic AcidSTEMCELL Technologies Inc.Catalog #72132

Protocol materials
N2 supplementGibco - Thermo Fisher ScientificCatalog #17502048
Coating
Poly-L- OrnithineMerck MilliporeSigma (Sigma-Aldrich)Catalog #A-004-C
  • 0.1 mg/mL Stock concentration
  • 50 µg/µL Working concentration
Thaw an aliquot of Poly-L-Ornithine solution at room temperature.
Dilute Poly-L-Ornithine solution to 50ug/mL in molecular biology grade water

Add 500 µL of PLO for every 500 µL molecular biology grade water

Add 1 mL/well to a 6-well plate or 3 mL to a T25 flask.

Incubate closed overnight at room temperature in a sterile hood.
Rinse vessel 3 times with molecular biology grade water.
Allow to air dry open under UV light (in hood)
Fibronectin human plasma,liquid, 0.1% (Solution),Merck MilliporeSigma (Sigma-Aldrich)Catalog #F0895-1MG
Storage:  –20 °C in aliquots
  • 1 mg/mL Stock Concentration
  • 2 µg/µL Working concentration
Thaw aliquots of fibronectin solution at room temperature.
Note
Do not vortex or shake vigorously to resuspend the fibronectin. This will cause the fibronectin to “crash” out of solution, which is irreversible

Dilute the fibronectin in sterile Hank’s Balanced Salt Solution without CaCl2 or MgCl2 (HBSS -/-).

Add 2 µL Fibronectin to 998 µL HBSS -/-
After PLO has completely dried, add 1 mL/well of fibronectin to a 6-well plate or 3 mL to a T25.
Rinse 3 times with molecular biology grade water.
Air dry open at room temperature in sterile hood.
Culture Media
Change media every 2-3 days. Rinse with DPBS first.
Note
LUHMES are sensitive to changes in the media pH and oxidative stress. Always use fresh DMEM/F12 because the HEPES buffer in DMEM is subject to photooxidation upon exposure to light and produces hydrogen peroxide.

DMEM/F12Thermo Fisher ScientificCatalog #11320033
N2 supplementGibco - Thermo Fisher ScientificCatalog #17502048
Recombinant Human FGF basic/FGF2/bFGF (145 aa) Protein, CFR&D SystemsCatalog #3718-FB
**See material section for media concentration information**
Thawing
Make 50 mL of media without b-FGF and use that to make media with b-FGF.
Add 2 mL/well of media with b-FGF into a 6-well plate and allow to warm in the incubator.

Note
Each cryovial will be thawed into a single well so you only need to fill as many wells as you have cryovials. However, it is recommended to fill empty wells to allow for even evaporation and potential use later, but this media does not have to contain b-FGF


For each cryovial, prepare a 15 mL conical tube with 9 mL of culture media without b-FGF.
Remove cryovials from LN2 freezer and thaw in the water bath for 1-2 minutes.
Holding the cap of the cryovial, spray bottom with ethanol and bring into hood
Using a 5 mL serological pipette, aspirate as much of the cell suspension as possible from the cryovial and dispense into one of the 15 mL conical tubes.
Centrifuge for 7 minutes at 1200 RPM.

Aspirate the supernatant fluid.
Gently flick the cell pellet to break it up.
Note
With larger cell pellets, you will be able to see it slosh against the sides of the tube which is good

Using a 5 mL serological pipette, resuspend in 1 mL of media with b-FGF and dispense dropwise into 1 well of warmed media.
Passaging
Allow TE buffer (0.025% Trypsin/EDTA) to come to room temperature.Trypsin/edta Solution (TE)Thermo ScientificCatalog #R001100

Fill destination vessel with appropriate volume of media: 2 mL/well for a 6-well plate or 3 mL for a T25.
Note
These volumes are important in ensuring LUHMES cells do not grow upward and spread evenly into a monolayer.

Aspirate old media from plate or flask.
Rinse with DPBS.
Add 1 mL/well to a 6-well plate or 3 mL to a T25 flask of warmed TE buffer and incubate for 3 minutes.Trypsin/edta Solution (TE)Thermo ScientificCatalog #R001100

Remove from incubator and knock gently on the side of the vessel to loosen the cells.
Neutralize the dissociation reagent by adding at least the same amount of media.
Note
LUHMES cells do not grow well with residual TE so feel free to add an extra mL of media when neutralizing.

Using a 5 mL serological pipette, rinse the well or flask by pipetting the neutralized TE/media solution up and down around the vessel to remove as many cells as you can and transfer to a 15 mL conical tube.
Note
Sometimes LUHMES cells can be difficult to remove so it may help to dispense the solution directly onto the areas they can be seen when rinsing, however, this step must be done gently as to not mechanically disturb them too much.

If a large portion of the cells are still adhered, transfer the current TE/media/cell solution to a 15 mL conical tube and repeat steps 28-31, ensuring to combine cells from the same original vessels.
Centrifuge for 7 minutes at 1200 RPM.
Aspirate supernatant fluid.
Gently flick the cell pellet.
Cells should be resuspended using a 5 mL serological pipette and passaged at a 1:4 ratio.
Cryopreservation
Freezing Media:
  • 45% LUHMES media without b-FGF
  • 45% Gibco FBS
  • 10% DMSO
Allow TE buffer to come to room temperature.
Follow Passaging steps 26-34.

Resuspend in an appropriate volume of media without b-FGF based on the size of the cell pellet.
Note
Be sure the suspension is not too dilute to get an accurate cell count.

Load both sides of a cell counter slide and find the average to determine the count per mL.

If this is not working (i.e. the counter is failing, the two counts are very far apart, etc.), load a hemacytometer and obtain the count by hand.
Calculate the volume needed to obtain aliquots of 1-2 million cells.
Move the appropriate cell volume to a new 15 mL conical tube if you will not need the entire suspension.
Centrifuge for 7 minutes at 1200 RPM.
Depending on the number of aliquots to be frozen, prepare the appropriate number of cryovials and mL of freezing media using the recipe above.
Aspirate the supernatant fluid and gently flick the cell pellet.
Resuspend in the freezing media and gently triturate as few times as possible in order to break up the pellet.
Dispense 1 mL/cryovial.
Cap each vial and move directly to the -80 degree freezer.
Differentiation
Day 0, when the LUHMES are 80% confluent, add differentiation media and incubate overnight.
Day 1, replate cells onto a new poly-l-ornithine and fibronectin-coated plate. Replate cells at a density of 1 million cells per T-75 flask or ~ 400,000 per well of a 6-well.
Replace LUHMES differentiation media every day while differentiating. LUHMES become mature dopamine-like neuron in 7-days.

LUHMES Differentiation Timeline
**See material section for media concentration information**

Protocol references
ATCC LUHMES CRL-2927