Luminescence-Based Assay to Assess Neurotoxicity of Test Compounds on Neurons

Overview

In this video, we describe a luminescence-based method to assess the neurotoxicity of neurite outgrowth-promoting small molecule compounds on the viability of hNPC-derived neurons. The assay measures intracellular ATP level, an indicator of cell viability, to evaluate neurotoxicity following test compound treatment.

Protocol

All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human welfare and have been reviewed by the local institutional review board

1. Isolation and culture of human neural progenitor cells (hNPCs)

1. Place the brain tissue in a 100 mm Petri dish and carefully remove the meninges using forceps.
2. Transfer the brain tissue to a 50 mL conical tube and wash it twice with 20 mL of PBS by gently inverting the tube.
3. Incubate the brain tissue in a new 50 mL conical tube by submerging the tissue in cell dissociation solution (see Table of Materials) and DNase I (10 U/mL) for 10 min at 37 °C.
4. Add 5 mL of neuronal cell culture medium (see Table of Materials) to the brain tissue-containing tube and mechanically dissociate the neurospheres by triturating 20 to 30 times through a 1000 µL pipet tip to make a single-cell suspension.
5. Filter the cell suspension through a 70 µm cell strainer to remove cell clusters.
6. Seed the single-cell suspension in a vented T-25 flask with 5-10 mL of neuronal cell culture medium supplemented with components detailed in Table 1.
   1. Sterilize the heparin solution by filtration through a 0.2 µm filter. The B-27 supplement without Vitamin A is a serum-free supplement for the cultivation of neural progenitors and stem cells without inducing differentiation.
      NOTE: Human neural progenitor cells (hNPCs) are isolated from a human fetal brain collected from an aborted fetus. Following 7–10 days in culture, neural stem cells (NSCs) form free-floating neurosphere colonies, whereas other cell types remain in suspension as single cells or attach to the bottom of the flask. The isolated hNPCs can be cultured as neurospheres in suspension for several months.

2. Passaging the hNPCs

1. Collect the media containing the floating spheres, big and small neurospheres, and transfer them to a 50 mL conical tube.
   NOTE: Due to unknown reasons, the timing for splitting the neurospheres is variable from 7 days up to 30 days. However, generally, neurospheres need to be passaged when they reach a diameter greater than 700-900 µm. This is when the center of the neurosphere starts to darken, which is considered as a sign of a high rate of cell death.
2. Spin the neurospheres down by centrifugation at 300-400 x g for 3 min.
3. Carefully aspirate the supernatant and then submerge the spheres in 500 µL of defrosted cell dissociation reagent (see Table of Materials).
   1. Make aliquots of cell dissociation reagent by adding 500 µL per 1.5 mL microtubes and store at -20 °C. To avoid losing enzyme activity, thaw the cell dissociation reagent by holding it at RT or warm for 5 min in a 37 °C water/bead bath.
4. Depending upon the density and size of the spheres, incubate the submerged spheres at 37 °C for 5-15 min.
5. Add 5-10 mL of pre-warmed culture media to the neurospheres containing 50 mL conical tube and centrifuge at 300-400 x g for 5 min to sediment the neurospheres.
6. Aspirate the supernatant and gently pipette up and down, using a 1000 µL pipette, in 2 mL of culture media until all the neurospheres are in a single-cell suspension.
   NOTE: The dissociation will become visible to the naked eye. Before dissociation, the neurospheres are in the form of spheres. After submerging in dissociation reagent and by pipetting up and down, they will become single cells.
   1. Count and plate the single cells, 2 to 3 million cells per T-25 flask, in 10 mL of culture media.
7. Feed the cells every 3 days by replacing half the culture media.
   1. Settle neurospheres by leaning the flask so that it is on its bottom corner. Hold the flask in the position for about 1-2 min until the neurospheres sediment. Then aspirate half the media gently by inserting the serological pipette in the media above the settled neurospheres. Dissociated cells can aggregate to form spheres after 2 to 3 days in culture.

3. Freezing the hNPCs

1. Prepare the cell freezing medium by adding DMSO to the culture media to a final concentration of 10% (v/v) or use a commercially available cryopreservation medium for sensitive cell types (see Table of Materials).
2. Sort out big spheres by transferring the media into a 50 mL conical tube and letting the spheres settle by gravity. Then remove and transfer the big neurospheres into a new 50 mL conical tube for passaging using a 200 or 1000 µL pipet tip.
   NOTE: Neurospheres with a diameter greater than 900 µm are considered big, and the ones with a diameter smaller than 500 µm are considered small.
3. Spin the remaining neurospheres down by centrifugation at 300-400 x g for 3 min. Carefully remove the supernatant.
4. Resuspend up to 100 spheres in 1 mL of cryopreservation reagent and transfer it to a cryotube.
5. Store overnight at -80 °C in a cell freezing container (see Table of Materials) and move it to liquid nitrogen the next day for long-term storage.
   NOTE: It is preferable to freeze small to medium-sized neurospheres (lower than 900 µm in diameter) and avoid freezing big-sized neurospheres (greater than 900 µm in diameter) or single cells. In order to reduce cell damage while thawing the sample, keep the cells dense by seeding the thawed neurospheres into a small T25 flask.

4. Neurotoxicity assessment

NOTE: Cytotoxicity of test compounds is evaluated in 384-well plates (see Table of Materials) using a luminescent cell viability assay (see Table of Materials). The hNPCs are prepared following the same method, except for slight modifications, described in the “Differentiation and Treatment of hNPCs” section. Subsequently, a luminescent signal generated by luminescent cell viability assay is measured utilizing a microplate reader. The luminescent signal is proportional to the cellular ATP concentration, which itself is directly proportional to the number of viable cells present in each well.

1. Coating
   1. Add 30 µL of poly-L-lysine (PLL) per well of a 384-well plate.
   2. Incubate for 1 h at RT.
   3. Wash 2x with PBS.
   4. Let it dry at RT (for about 30 min).
   5. Add 30 µL of laminin (50 µg/mL) per well of a 384-well plate.
   6. Incubate for 2 h at 37 °C.
   7. Wash 2x with PBS.
      NOTE: Coated 384-well plates can be stored at 4 °C for 1 month.
2. Plating the cells
   1. Count and plate 20,000 single-cell neurospheres per well of a 384-well plate in 25 µL of differentiation media.
   2. Incubate for 5 days at 37 °C.
   3. After 5 days, treat the cells for 24 h by test compounds prepared at 6x the final desired concentration in 5 µL volume (to make the final volume of 30 µL per well).
3. Cell viability assay
   1. Add 30 µL of luminescent cell viability assay reagent per well of a 384-well plate.
      NOTE: Add a volume of luminescent cell viability assay reagent equal to the volume present in each well. Thaw the luminescent cell viability assay reagent and equilibrate it to RT prior to use.
   2. Shake on a plate shaker for 2 minutes (to mix and induce cell lysis).
   3. Spin down the mixture by centrifugation at 300-400 x g for 30 s.
   4. Incubate the 384-well plate for 10 min at RT in a place protected from light (to stabilize the luminescent signal).
   5. Record luminescence with a microplate reader.
      NOTE: Use appropriate controls for the viability assay, including Velcade (at a final concentration of 10 µM) as positive and HBSS containing DMSO (with a final concentration of 0.1% or 0.2%) as the negative control.

Table 1. Components required for making 100 mL of culture media

Amount	Component
100 µL	EGF (20 ng/mL)
100 µL	FGF (10 ng/mL)
2 mL	B-27, Minus vitamin A (50X)
1 mL	L-alanyl-L-glutamine (100X) (see table of materials)
4 µL	Heparin (2 μg/mL)
96.8 mL	Neuronal cell culture medium (see table of materials)

Materials

Name	Company	Catalog Number	Comments
Antibiotic-Antimycotic	Gibco	15240062
B27	Thermo	17504001
B27 - minus vitamin A	Thermo	12587010
BDNF	PeproTech	450-02
BSA	Sigma	A8531
CellTiter-Glo	Promega	G7572
CoolCell	Corning	432000	Cell freezing containers ensuring standardized controlled-rate -1°C/minute cell freezing in a -80°C freezer
CryoStor CS10	StemCell Technologies	7930	Cryopreservation medium containing 10% DMSO
DMEM/F12	Gibco	11320033
DMSO	Sigma	34869-100ML
EGF	Gibco	PHG0311
FGF	Gibco	PHG6015
GDNF	PeproTech	450-10
Glutamax	Gibco	35050061	L-alanyl-L-glutamine supplement
Heparin	Calbiochem	375095
Laminin	Sigma	L2020-1MG
L-Ascorbic Acid	Sigma	A92902-25G
mFreSR	StemCell Technologies	5854	Serum-free cryopreservation medium designed for the cryopreservation of human embryonic and induced pluripotent stem cells
N2	Gibco	17502048
Nunc 384-Well Polystyrene White Microplates	Thermo	164610
PBS	Thermo	10010-049
Poly-L-lysine	Sigma	P5899-5MG
StemPro Accutase	Gibco	A1110501	Cell dissociation reagent containing proteolytic and collagenolytic enzymes
Neurobasal Medium	Gibco	21103049