LUHMES (lund human mesencephalic) culturing and differentiation protocol

Jason Waligorski, William J Buchser, Mallory Wright, Colin Kremitzki, Serena Elia, Graham Bachman, emanuel gerbi

Published: 2024-02-15 DOI: 10.17504/protocols.io.kxygx36ykg8j/v3

Dopaminergic Neuron Differentiation

AI 解读

Abstract

Steps

LUHMES coating protocol

0.1mg/mL
50µg/µL Thaw an aliquot of Poly-L-Ornithine solution at room temperature.

Dilute Poly-L-Ornithine solution to 50ug/mL in Nuclease-free water

Add 500µL of PLO for every 500µL 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)

Storage: –20 °C in aliquots

1mg/mL
2µg/µL 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 2µL to 998µL

Place fibronectin coated flask in incubator for 3 hours

Rinse 3 times with HBSS.

10.

Air dry for 15 minutes and add fresh LUHMES growth media.

LUHMES growth media

11.

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.

12.

Add recombinant human FGF to media after seeding cells See material section for media concentration information

LUHMES Passaging (~ every 2-3 days)

13.

Remove media and rinse with DPBS

14.

Add fresh culture media to newly coated flask for at least 15 minutes before seeding cells to

allow media to reach normal pH

15.

Add 4mL of the pre-warmed .025% Trypsin/EDTA and place in the incubator for 3 minutes.

16.

Neutralize trypsin with 6mL of pre-warmed DMEM/F12

17.

Transfer cells to 15mL tube and centrifuge for 5 minutes at 1200 RPM

18.

Discard the supernatant and resuspend in 1mL LUHMES growth media

19.

use a 5mL pipette to Triturate cells only 1 or 2 times before seeding.

Cryopreservation

20.

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

21.

Day 0, when the LUHMES are 80% confluent, add differentiation media and incubate overnight.

22.

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.

23.

Replace LUHMES differentiation media every day while differentiating. LUHMES become mature dopamine-like neuron in 7-days.

See material section for media concentration information