Feb 15, 2024
Version 3
LUHMES (lund human mesencephalic) culturing and differentiation protocol V.3
- Mallory Wright1,
- William J Buchser2,
- Colin Kremitzki2,
- Jason Waligorski2,
- Graham Bachman2,
- Serena Elia2,
- emanuel gerbi2
- 1Washington University, Saint Louis. McDonnell Genome Institute (MGI);
- 2Washington University in St. Louis
Protocol Citation: Mallory Wright, William J Buchser, Colin Kremitzki, Jason Waligorski, Graham Bachman, Serena Elia, emanuel gerbi 2024. LUHMES (lund human mesencephalic) culturing and differentiation protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.kxygx36ykg8j/v3Version created by Mallory Wright
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: Working
We use this protocol and it's working
Created: February 15, 2024
Last Modified: March 09, 2024
Protocol Integer ID: 95286
Keywords: LUHMES differentiation, Neuronal differentiation, Dopaminergic neurons, Human Midbrain, Dopaminergic Neuron Differentiation
Abstract
Materials
| Reagents | Stock Concentration | Final Concentration | Final Solution Volume= 20mL | |
| DMEM/F12 | x | x | 19,792uL | |
| B27 | 50X | 1X | 200ul | |
| Recombinant Human FGF basic (100ug) store at -20C | 100ug/mL Reconstitute: add 1000ul PBS to 100ug vial of fgf | 40ng/mL | 8uL | |
| Penicillin Streptomycin (10,000 U/mL) | X | X | 20uL |
LUHMES Growth Media
| Reagents | Note | Stock Concentration | Working Concentration | Final 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°C | 0.1mg/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/mL | 1ug/mL | 2uL | |
| TGF β-III (10ug vial) | Storage Conditions 4° C | 0.25 mg/mL | 20ng/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
Human Recombinant BDNFSTEMCELL Technologies Inc.Catalog #78005
Materials
Human Recombinant LIFSTEMCELL Technologies Inc.Catalog #78055
Materials
Penicillin Streptomycin (10,000 U/mL)Gibco - Thermo FischerCatalog #15140122
Step 12
B-27 Supplement (50X)Thermo Fisher ScientificCatalog #17504044
Step 12
Recombinant Human FGF basic/FGF2/bFGF (145 aa) Protein, CFR&D SystemsCatalog #3718-FB
Step 12
Tetracyline HydrochlorideThermo ScientificCatalog #A39246
Materials
Human Recombinant GDNFSTEMCELL Technologies Inc.Catalog #78058
Materials
DMEM/F12Thermo Fisher ScientificCatalog #11320033
Step 12
Fibronectin human plasma,liquid, 0.1% (Solution),Merck MilliporeSigma (Sigma-Aldrich)Catalog #F0895-1MG
Step 6
Tgf beta 3 (human) Recombinant ProteinInvitrogen - Thermo FisherCatalog #RP8600
Materials
Dibutyryl-cAMPSTEMCELL Technologies Inc.Catalog #73884
Materials
Absorbic AcidSTEMCELL Technologies Inc.Catalog #72132
Materials
LUHMES coating protocol
LUHMES coating protocol
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 Nuclease-free water
Add 500 µL of PLO for every 500 µL Nuclease-free water
Add 7mL of the 50ug/mL PLO to a T-75 overnight at RT.
Rinse flask 3 times with Nuclease-free water.
Allow the flask to air dry for 15 minutes uncapped and standing upright in the hood. (turn on UV)
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 an aliquot of Fibronectin solution at 5°C .
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 (HBSS).
Add 2 µL Fibronectin to 998 µL HBSS
Place fibronectin coated flask in incubator for 3 hours
Rinse 3 times with HBSS.
Air dry for 15 minutes and add fresh LUHMES growth media.
LUHMES growth media
LUHMES growth media
Change (pre-warmed) media every 1-2 days
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
Penicillin Streptomycin (10,000 U/mL)Gibco - Thermo FischerCatalog #15140122 B-27 Supplement (50X)Thermo Fisher ScientificCatalog #17504044 Recombinant Human FGF basic/FGF2/bFGF (145 aa) Protein, CFR&D SystemsCatalog #3718-FB
- Add recombinant human FGF to media after seeding cells
**See material section for media concentration information**
LUHMES Passaging (~ every 2-3 days)
LUHMES Passaging (~ every 2-3 days)
Remove media and rinse with DPBS
Add fresh culture media to newly coated flask for at least 15 minutes before seeding cells to
allow media to reach normal pH
Add 4mL of the pre-warmed .025% Trypsin/EDTA and place in the incubator for 3 minutes.Trypsin/edta Solution (TE)Thermo ScientificCatalog #R001100
Neutralize trypsin with 6mL of pre-warmed DMEM/F12
Transfer cells to 15mL tube and centrifuge for 5 minutes at 1200 RPM
Discard the supernatant and resuspend in 1mL LUHMES growth media
use a 5mL pipette to Triturate cells only 1 or 2 times before seeding.
Cryopreservation
Cryopreservation
Label 2mL cryovials with the date, name, FIV#, Qbench number, passage number and cell type.
Add about 1.5 million cells per vial with 1mL freezing media.
Freezing Media:
- 7mL LUHMES Media
- 4uL B-fgf (40ng)
- 2mL FBS (20%)
- 1mL DMSO (1%)
LUHMES Differentiation
LUHMES 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