Application Protocol

Culturing Human iPSC-derived Mixed Neurons on Multielectrode Arrays:MaxTwo Multiwell MEAIntroduction(OL[LUJHQ6FLHQWLÀF·VSURSULHWDU\WUDQVFULSWLRQIDFWRUEDVHGWHFKQRORJ\DOORZVUDSLGDQGUHSURGXFLEOHGL੔HUHQWLDWLRQRIKXPDQL36&VLQWR
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express markers characteristic of a variety of neuronal subtypes, including the pan-neuronal marker tubulin beta 3 class III (TUBB3), thecholinergic neuron marker choline acetyltransferase (ChAT), the dopaminergic neuron marker tyrosine hydroxylase (TH), the GABAergic
neuron marker glutamate decarboxylase 1, 67 kDa isoform (GAD1/GAD67), the glutamatergic neuron marker vesicular glutamate transporter 1 (vGLUT1), and the serotonergic neuron marker tryptophan hydroxylase 2 (TPH2).
In this Application Protocol, we describe how our human iPSC-derived Mixed Neurons can be thawed, plated, and maintained on theMaxWell Biosystems MaxTwo Multiwell multielectrode array (MEA) system for non-invasive, label-free measurement of neuronal activity.
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Required Equipment and Consumables
Item

VendorCatalog Number

MaxTwo Multiwell MEA System- MaxLab Live Basic software, perpetual license

MaxWell Biosystems

MX2-SYS

MaxTwo 6-well Plates

MaxWell Biosystems

MX2-S-6W

Mixed Neurons from human iPSCs.* The kit contains:- Frozen cells (>1.0 million viable cells, 0.5 ml)&RPSRQHQW1 ǌO&RPSRQHQW3 ǌO(OL[LUJHQ6FLHQWLÀF

MN-SeV-C#(multiple donor cell lines to choose from)

DMEM/F12

ThermoFisher

21331-020

Neurobasal**

ThermoFisher

21103049

Glutamax (100x)

ThermoFisher

35050061

Penicillin-Streptomycin

ThermoFisher

15140-122

Poly-L-Ornithine Solution, 0.01%

Sigma-Aldrich

P4957-50ML

Laminin mouse protein, natural

ThermoFisher

23017015

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ThermoFisher

20012050ROCK inhibitor Y27632

Selleckchem

S1049

B-27™ Plus Neuronal Culture System. This kit contains:- Neurobasal Plus Medium**- B-27™ Plus Supplement (50x)

ThermoFisher

A3653401

Human Astrocytes Kit. This kit contains:- Human Astrocytes- Astrocyte Medium

ThermoFisher

N7805200

Trypan blue solution, 0.4%

ThermoFisher

15250061

Dimethyl sulfoxide (DMSO)

Sigma-Aldrich

D8418

Cell culture-grade water

ThermoFisher

15230162

Ascorbic acid

Sigma-Aldrich

A4544

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ThermoFisher

SLGP033RS++Mg )Formerly EXGS-QNMSVF-CW#++*See KWWSVHOL[LUJHQVFLHQWLÀFFRPLSVFGHULYHGPL[HGQHXURQV for a full list of mixed neuron products derived from human iPSCs.
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1

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IMPORTANT! Component N1 and B-27™ Plus Supplement (50x) should be thawed overnight at 4°C before being used to prepare
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Plus Supplement (50x) overnight 1-2 days before plating.

Astrocyte Preparation
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Media Preparation
Preparing the plating medium (Medium N1)
1.
2.
3.

Thaw Component N1 at 4°C overnight.
Prepare the plating medium (hereafter referred to as Medium N1) using the components listed in the table below.
Store Medium N1 for up to 2 weeks when stored at 4°C.
Medium N1 Components

VolumeDMEM/F1212 ml

Neurobasal

12 ml

N2 Supplement(100x)
200mM Glutamax(100x)
200 mM Glutamax (100x)
Penicillin-Streptomycin(1000 units/ml; 100x)

ǌO

Penicillin-Streptomycin(10000 units/ml; 100x)
Component
N1ǌO
250 µlǌO
750 µlComponent N1

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Preparing 10 mM ROCK inhibitor Y27632 (iROCK)
1.
2.
3.

Dissolve 10 mg ROCK inhibitor Y27632 in 3.1225 ml DMSO.
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Preparing 0.002% poly-L-ornithine solution (ornithine)
1.
2.

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Store the 0.002% poly-L-ornithine solution (hereafter referred to as ornithine) for up to 2 weeks at 4°C.

Preparing 1 mg/ml laminin stock solution (laminin)*
1.
2.

3.

2

Thaw Laminin Mouse Protein, Natural at 4°C or on ice and chill PBS at 4°C or on ice.
Take cold Laminin Mouse Protein, Natural and mix it with chilled PBS to make a 1 mg/ml stock solution hereafter referred to aslaminin.
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Calculate the volume needed to prepare 1 mg/ml stock solution accordingly.
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1.
2.
3.

Dissolve 352 mg ascorbic acid in 10 ml cell culture-grade water.
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Preparing the maturation medium (Neurobasal Plus)
1.
2.
3.

Thaw the B-27™ Plus Supplement (50x) at 4°C overnight.
Prepare the maturation medium (hereafter referred to as Neurobasal Plus) using the components listed in the table below.
Store Neurobasal Plus for up to 2 weeks at 4°C.
Neurobasal Plus Components

VolumeNeurobasal Plus Medium

48 ml

B-27™ Plus Supplement (50x)

1 ml

200 mM Glutamax (100x)

0.5 mlPenicillin-Streptomycin (10000 units/ml; 100x)

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Day 0
Sterilization of the MaxTwo plate
1.
2.
3.
4.
5.

Spray the MaxTwo plate and the lid with 70% EtOH and then put them inside the biosafety cabinet.
Fill all wells, areas outside the wells on the plate, and the lid with 70% EtOH all the way up to the edges and leave them for 30 minutes inside the biosafety cabinet.
• About 6-7 ml 70% EtOH per well (~50 ml total) is needed.
Remove 70% EtOH from the plate and the lid by decanting into a liquid waste beaker.
Rinse the wells with cell-culture grade water 3 times.
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IMPORTANT! For all aspiration steps, use an aspirator with a P200 tip attached to avoid damaging the well bottom center where theelectrodes are located. Do not scratch the electrodes.
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.

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Add 5 ml cell culture-grade water to the area outside the wells on the plate to keep humidity inside the wells.
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Aspirate water from each well completely and leave the plate in the biosafety cabinet for 30 minutes to allow air drying of the wells.
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Preparing Medium iN1(P)1.

Prepare Medium iN1(P) by mixing together the following components in a 15 mL conical tube.
• Warm Medium N1 and iROCK at room temperature for at least 1 hour and thaw Component P at room temperature for
20-30 min before combining.
• Store leftover Components P at 4°C for later use.
Medium iN1(P) Components

VolumeMedium N1

4.5 mliROCKǌO

Component PǌO

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Thawing mixed neurons
1.
2.

Take out the vial of Frozen Cells from the liquid nitrogen storage tank.
Incubate the cryovial in a water bath set at 37°C (do not submerge the cap) until most of the content is thawed but a small ice crystal
remains (~2 minutes).
3. Wipe the vial with dry paper towel. Spray 70% ethanol to the vial and bring it inside a biosafety cabinet.
4. Take 4.5 ml Medium N1 into a new 15 ml conical tube and store the rest of Medium N1 at 4°C for later use.
5. Using a P1000 pipettor, take 0.5 ml Medium N1 and add it into the cryovial dropwise at 1 drop/1-2 seconds.
• Use the same pipette tip for Steps 5-8 to minimize cell loss.
6. Gently pipet the cell suspension up and down once before transferring to the conical tube containing Medium N1.
7. Take 1 ml cell suspension from the conical tube, add it to the original cryovial, and pipet up and down 2-3 times before transferring
the entire volume back to the conical tube.
8. Mix the cell suspension by pipetting up and down 3 times.
9. Centrifuge the conical tube at 200 xg for 4 minutes.
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11. Gently tap the side of the conical tube to break the cell pellet.
12. Add 1 ml Medium iN1(P) to the conical tube using a P1000 pipettor and pipet up and down 2-3 times.
13. Count cells and determine cell viability by trypan blue staining.
Plating mixed neurons with human primary astrocytes
1.
2.
3.
4.

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Maintenance1.
2.

Warm Medium N1, Component P, and Neurobasal Plus at room temperature for 20-30 minutes.
Prepare 0.5x Neurobasal Plus using the components listed in the table below.
0.5x Neurobasal Plus Components3.
4.
5.

VolumeMedium N13 ml

Neurobasal Plus

3 ml

Component P

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1.
2.
3.
4.

Warm Neurobasal Plus and Component P at room temperature for 20-30 minutes.
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Pipet out the old medium from each well using a P1000 pipettor and add 1 ml Neurobasal Plus(P).
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1.
2.
3.

4

Warm Neurobasal Plus(P) at room temperature for 20-30 minutes.
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Day 7+Maintenance
1.
2.
3.
4.

Warm Neurobasal Plus (without Component P) at room temperature for 20-30 minutes.
Pipet out most of the old medium from each well using a P1000 pipettor and add 1 ml Neurobasal Plus.
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Replace 50% of the old medium with fresh Neurobasal Plus every 3-4 days.

Data Acquisition
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Spontaneous action potentials can be observed in some wells as early as Day 10 and synchronized network bursts by Day 14.

Representative Data

A

B

C

Electrophysiological activities of hiPSC-derived mixed neurons.0L[HGQHXURQV ï࢛FHOOV ZHUHFRFXOWXUHGZLWKDVWURF\WHV ï࢚ as described in this Application Protocol and their activity was measured using the MaxTwo Multiwell HD-MEA (MaxWell Biosystems).
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corresponding result of activity scan shows that active neurons are evenly distributed over the entire chip on Day 43. (C) By tracing thepropagation of axon potential in a single neuron, action potential velocity was analyzed. This graph indicates that the action potential
propagated rapidly along the long axon (~1.5 mm), demonstrating the maturity of neurons and their suitability for axonal conduction
studies.

Technical Support
For technical support, please contact us at FV#HOL[LUJHQVFLHQWLÀFFRP or call us at +1 (443) 869-5420 (M-F 9am-5 pm EST).
Last revised: May 13, 2020

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