Application Protocol

Culturing Human iPSC-derived Excitatory Neurons on Microelectrode Arrays:
Maestro Pro MEA
Introduction
Elixirgen Scientific’s proprietary transcription factor-based technology allows rapid and reproducible differentiation ofhuman iPS or ES cells into neurons without sacrificing the purity of the cells. Our excitatory neurons exhibit typicalneuronal morphology with outgrowing neurites and express markers characteristic of a variety of neuronal subtypes,
including the pan-neuronal marker tubulin beta 3 class III (TUBB3), the glutamatergic neuron marker vesicularglutamate transporter 1 (vGLUT1), and the cholinergic neuron marker choline acetyltransferase (ChAT).
In this Application Protocol, we describe how our human iPSC-derived excitatory neurons can be thawed, plated, andmaintained on the Axion Biosystems Maestro Pro microelectrode array (MEA) system for non-invasive, label-free 		measurement of neuronal activity. Our neurons fire synchronized bursts within 2 weeks after plating when co-cultured
with human primary astrocytes.

Required Equipment and Consumables
ItemVendor

Catalog Number

Equipment
Maestro Pro• AxIS Navigator softwareAxion Biosystems

Consumables
Cytoview MEA 48 Black or White

Axion Biosystems

M768-tMEA-48B or M768-tMEA-48W

4x Quick-Neuron™ Excitatory - Human iPSCderived Neurons. Each kit contains:• Frozen cells (>1.0 million viable cells, 0.5 ml)• Component N (2x 840 μl)• Component G2 (2x 16 μl)• Component P (14 μl)

Elixirgen Scientific

EX-SeV-CW*

DMEM/F12

ThermoFisher

21331020

Neurobasal Medium**

ThermoFisher

21103049

Glutamax (100x)

ThermoFisher

35050061

Penicillin-Streptomycin

ThermoFisher

15140-122

Poly(ethyleneimine) solution, PEI

Sigma-Aldrich

P3143

Pierce™ 20X Borate Buffer

ThermoFisher

28341

ThermoFisher

23017015

Phosphate-buffered saline (without Ca Mg )ThermoFisher

20012050

ROCK inhibitor Y27632

Selleckchem

S1049

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

ThermoFisher

A3653401

Laminin mouse protein, natural++++(multiple donor cell lines to choosefrom)

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

ThermoFisher

N7805200

Accutase

ThermoFisher

A1110501

Dimethyl sulfoxide (DMSO)

Sigma-Aldrich

D8418

Absolute Ethanol (≥99.5%)

Fisher ScientificBP2818100

Cell culture-grade water

ThermoFisher

15230162

Ascorbic acid

Sigma-Aldrich

A4544

Syringe filter (0.22 µm)

ThermoFisher

SLGP033RS* See https://elixirgenscientific.com/ipsc-derived-excitatory-neurons/ for our catalog of iPSC-derived excitatory neurons fromhealthy and affected donors.

** Please note that Neurobasal and Neurobasal Plus Medium are distinct and are used at different steps of this protocol.

Workflow
Astrocyte Preparation

Day -7...

Media Preparation
Plate Preparation

Harvest Astrocytes
Thaw Neurons
Plate Neurons/Astrocytes

Day -1

Day 0

Maintenance

...

Day 1
Medium iN(G2P)

Medium N(G2P)

...

Day 6

Day 28+

Neurobasal Plus

Day -7
Astrocyte Preparation
Our neurons fire synchronized bursts within 2 weeks after plating when co-cultured with human primary astrocytes astested with the Human Astrocytes Kit available through ThermoFisher (Cat No. N7805200). Astrocytes can be preparedby starting their culture one week in advance and harvesting them at Day 0 as instructed by the manufacturer. 1 vialof cryopreserved human primary astrocytes provides a sufficient number of cells (up to 1.5 x 106 cells) to plate theCytoview 48-well plate with our neurons. This protocol assumes that human primary astrocytes are cultured on one 100mm cell culture dish using manufacturer’s recommended protocol.

Day -1
Media Preparation
10 mM ROCK inhibitor Y27632 (iROCK)
1. Dissolve 10 mg ROCK inhibitor Y27632 in 3.12 ml DMSO.
2. Make aliquots of a convenient volume (e.g., 100 μl).
3. This solution is hereafter referred to as iROCK and can be stored at -20°C.
70% EtOH for MEA plate surface treatment
1. Mix Absolute Ethanol (more than 99.5% purity) with cell culture grade water at a ratio of 7 to 3 (e.g., 7 ml AbsoluteEthanol with 3 ml cell culture grade water).
1X Borate Buffer
1. Dilute 150 μl of 20X Borate Buffer with 2.85 ml of cell culture-grade water to make 1x Borate Buffer.
0.1% PEI solution
1. Add 5.4 μl PEI to 2.7 ml 1X Borate Buffer.
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1 mg/ml laminin stock solution (laminin)
1. Thaw Laminin Mouse Protein, Natural and chill PBS at 4°C or on ice.
2. Mix the Laminin Mouse Protein, Natural and PBS to make the 1 mg/ml stock solution (hereafter referred to aslaminin).
• Laminin concentration varies by lot, so use the number specified on the vial or CoA when making yourcalculations.
3. Make aliquots of a convenient volume (e.g., 15 μl) and store at -20°C.
200 mM ascorbic acid
1. Dissolve 352 mg ascorbic acid in 10 ml cell culture-grade water.
2. Sterilize using a 0.22 µm syringe filter.
3. Make aliquots of a convenient volume (e.g., 100 μl) and store at -20°C.
Medium N
1. Prepare Medium N using the reagents listed in the table below.
• Thaw 3 vials of Component N at room temperature for 20-30 minutes or at 4°C overnight.
• Warm/thaw all other reagents at room temperature for 20-30 minutes.
• Store Medium N for up to 2 weeks at 4°C. The leftover reagents can be discarded or saved for other uses.
Medium N Reagents

VolumeDMEM/F1231.2 ml

Neurobasal Medium

31.2 ml200 mM Glutamax (100x)

325 μlPenicillin-Streptomycin (10000 units/ml; 100x)

650 μlComponent N2 ml

Medium N(G2P)
1. Prepare Medium N(G2P) using the reagents listed in the table below.
• Thaw 3 vials of Component G2 on ice and 2 vials of Component P at room temperature for 20-30 minutes.
• Medium N(G2P) can be stored for up to 2 weeks when stored at 4°C.
• Store the rest of Medium N at 4°C for later use. The other leftover reagents can be discarded or saved forother uses.
Medium N(G2P) Reagents

VolumeMedium N44 ml

Component G2

44 μl

Component P

22 μl

Neurobasal Plus
1. Prepare the maturation medium (hereafter referred to as Neurobasal Plus) using the components listed in the tablebelow.
• Thaw the B-27™ Plus Supplement (50x) at room temperature for 1 hour or at 4°C overnight.
• Store Neurobasal Plus for up to 2 weeks at 4°C.
• IMPORTANT! 25 ml of Neurobasal Plus will be sufficient for about 3.5 full plate medium changes. We 		
recommend preparing 25 ml Neurobasal Plus fresh as needed.
Neurobasal Plus Reagents

VolumeNeurobasal Plus Medium24 ml

B-27™ Plus Supplement (50x)500 μl200 mM Glutamax (100x)

250 μlPenicillin-Streptomycin (10000 units/ml; 100x)

250 μl200 mM ascorbic acid (filter-sterilized)

25 μl

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3Plate Preparation
IMPORTANT! For all aspiration steps, use an aspirator with a P200 tip attached to avoid damaging the well bottom
center where the electrodes are located.
Surface treatment
1. Add 150 μl 70% EtOH for MEA plate surface treatment prepared above to each well.
• IMPORTANT! Do not use 70% EtOH for common lab use for this step.
2. Incubate the plate at room temperature for 10 minutes.
3. Remove 70% EtOH from all wells and add 150 μl cell-culture grade water to rinse wells.
4. Incubate the plate at room temperature for 10 minutes.
5. Remove the water and leave the plate to dry. Do not turn on UV at the biosafety cabinet.
PEI coating
1.
2.
3.
4.
5.
6.

Add 50 μl 0.1% PEI solution to cover electrodes in each well (or per 0.22 cm2).
Incubate the plate at 37°C, 5% CO2 for 1 hour.
Remove 0.1% PEI solution.
Add cell-culture grade water to each well and remove it from all wells.
Repeat Step 4 for 8 times.
Leave the plate inside a biosafety cabinet overnight without closing its lid.

Day 0
Medium iN(G2P)
1. Prepare Medium iN(G2P) by mixing together the following components in a 50 ml conical tube.
• Warm Medium N(G2P) and 10 mM ROCK inhibitor Y27632 at room temperature for at least 1 hour.
Medium iN(G2P) Reagents

VolumeMedium N(G2P)

20 mliROCK

20 μl

Harvesting Human Primary Astrocytes
1.
2.
3.
4.
5.

Remove culture medium from the dish with human primary astrocytes and add 10 ml PBS.
Remove PBS and add 3 ml Accutase to the dish.
Incubate the dish at 37°C, 5% CO2 for 5 minutes.
Add 3 ml astrocyte culture medium to the dish.
Disperse the medium quickly over the bottom surface of the well by pipetting 6-8 times to detach cells using aP1000 pipettor.
6. Collect 6 ml cell suspension in a 15 ml conical tube using the same P1000 pipettor.
7. Count the cells and determine quantity and viability. While counting the cells, the cell suspension should be kept onice.
8. Centrifuge the conical tube at 200 xg for 4 minutes.
9. Aspirate most of the supernatant from the conical tube but leave a small volume (<50 μl) to cover the pellet.
10. Gently tap the side of the conical tube to break the cell pellet.
11. Prepare 264 μl of a 4.0 x 106 live cells/ml cell suspension using Medium iN(G2P). If the total volume exceeds 264 μl
or cannot reach the cell density to 4.0 x 106 live cells/ml, centrifuge the excess volume at 200 xg for 4 minutes andresuspend the pellet to adjust the volume to 4.0 x 106 live cells/ml.
12. Gently tap the side of the conical tube and pipet up and down 2-3 times.
13. Keep the astrocytes on ice until ready for plating.

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Thawing neurons
1. Warm Medium N at room temperature for 20-30 minutes.
2. Take out the 4 cryovials of frozen human iPSC-derived excitatory neurons from the liquid nitrogen storage tank.
3. Incubate the cryovials in a water bath set at 37°C (do not submerge the cap) until most of the contents are thawed
but a small ice crystal remains (1.5~2 minutes).
4. Wipe the vials with a dry paper towel. Spray 70% ethanol on the vials and bring them inside a biosafety cabinet.
5. Using a P1000 pipettor, gently transfer the cell suspension from each vial to a 50 ml conical tube.
6. Take 1 ml Medium N and add it to each original cryovial. Pipet up and down 2-3 times and then transfer the medium
to the 50 ml conical tube with the cell suspension dropwise at 1 drop per 1-2 seconds.
7. Take 16 ml Medium N (4 ml per cryovial) and add it into the 50 ml conical tube dropwise at 1 drop/1-2 seconds whilegently shaking the tube.
8. Gently pipet the cell suspension up and down 2-3 times using a 5ml pipettor.
9. Count the cells and determine quantity and viability. While counting the cells, the cell suspension should be kept onice.
10. Centrifuge the conical tube at 200 xg for 4 minutes.
11. Aspirate most of the supernatant from the conical tube but leave a small volume (<50 μl) to cover the pellet.
12. Gently tap the side of the conical tube to break the cell pellet.
13. Prepare 264 μl of a 1.6 x 107 live cells/ml cell suspension using Medium iN(G2P). If the total volume exceeds 264 μl
or cannot reach the cell density to 1.6 x 107 live cells/ml, centrifuge the excess volume at 200 xg for 4 minutes andresuspend the pellet to adjust the volume to 1.6 x 107 live cells/ml.
14. Gently tap the side of the conical tube and pipet up and down 2-3 times.
Plating Neurons with Human Primary Astrocytes
1. Add 264 μl human primary astrocyte cell suspension to the human iPSC-derived neuron cell suspension.
2. Add 10.6 μl laminin into the cell suspension on ice to make a final laminin concentration 20 μg/ml. Mix well.
• IMPORTANT! Cells start aggregating immediately after adding laminin. Plate cell suspensions in all wells
within 10 minutes to prevent cells clumping together.
3. Plate 10 μl cell suspension to cover the electrodes in each well. Incubate the cultures at 37°C, 5% CO2 for at least 1
hour or up to 3 hours until cells settle down.
4. Add 300 μl Medium iN(G2P) to each well.
5. Add 5 ml PBS to the edge of the plate to keep from drying out.
6. Incubate the cultures at 37°C, 5% CO2 overnight.

Day 1
Maintenance
1. Warm Medium N(G2P) at room temperature for 20-30 minutes.
2. Pipet out the old medium from each well using a P1000 pipettor and add 500 μl PBS to wash away any cellulardebris.
3. Pipet out PBS from each well using a P1000 pipettor and add 300 μl Medium N(G2P).
4. Incubate the cultures at 37°C, 5% CO2 for 3 days.

Day 4
Maintenance
1. Warm Medium N(G2P) at room temperature for 20-30 minutes.
2. Pipet out 50% old medium (150 μl) from each well using a P1000 pipettor and add 150 μl Medium N(G2P).
3. Incubate the cultures at 37°C, 5% CO2 for 2 days.

Day 6+
Maintenance
1.
2.
3.
4.

Warm Neurobasal Plus at room temperature for 20-30 minutes.
Pipet out 50% old medium from each well using a P1000 pipettor and add 150 μl Neurobasal Plus to it.
Incubate the cultures at 37°C, 5% CO2 for 3-4 days.
Repeat Step 1 to 4 twice a week such as on Tuesday and Friday.

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