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How to Culture Human Pluripotent Stem Cell (hPSC)-Derived Induced Forebrain Neurons for MEA Analysis Using the Maestro MEA™ System

How to Culture Human Pluripotent Stem Cell (hPSC)-Derived Induced Forebrain Neurons for MEA Analysis Using the Maestro MEA™ System

Immunofluorescence image of neurons differentiated from H9 hPSCs using STEMdiff™-TF Forebrain Induced Neuron Differentiation Kit.
4 Parts 16 Materials Print

Multi-electrode array (MEA) systems provide a powerful way to record and quantify activity of cell populations simultaneously. They capture voltage fluctuations in the extracellular space around electrically active cells, such as neurons, which are cultured on specialized cell culture plates containing a grid of electrodes within each well. Because they can monitor signals from multiple electrodes at once, MEAs enable researchers to analyze the dynamics of neuronal networks. Importantly, this method is non-invasive and does not require cell labeling, making it suitable for long-term tracking of electrophysiological development. As a result, MEA platforms are highly valuable for both disease modeling and drug discovery studies.

Neuronal cultures generated with STEMdiff™-TF Forebrain Induced Neuron Differentiation Kit consist of a highly pure excitatory glutamatergic neuron population that recapitulates key features of forebrain neuron identity and function. Differentiation is driven by transcription factor (TF) overexpression, achieved through lipid nanoparticle (LNP)-mediated delivery of non-integrating Neurogenin-2 (NGN2) mRNA. This forward programming approach generates forebrain-type neurons within just five days. The resulting neurons are functionally mature, electrically active, and can be maintained in culture for more than 50 days. The protocol yields uniform, adherent monolayers well-suited for downstream MEA analysis. These cultures develop robust synaptic connectivity and exhibit spontaneous and evoked activity that can be reliably measured and modulated over time.

To ensure reproducible electrophysiological outcomes, we present a detailed step-by-step protocol that standardizes every aspect of MEA experimentation—from seeding density to media changes and recording timelines. By supporting consistent functional readouts of forebrain neurons, this streamlined workflow provides a robust platform for investigating disease mechanisms, evaluating neurotoxicity, and screening compounds.

Read on for a step-by-step guide for culturing human pluripotent stem cell (hPSC)-derived forebrain neurons on CytoView MEA™ plates to produce mature, consistent, and stable neuronal activity using Maestro MEA™ systems.

Materials and Reagents

*The MEA plate can be coated with either Poly-D-Lysine and Laminin-521 or Poly-D-Lysine and Matrigel®.

Protocol

Part I. Reagent Preparation

A. Coating Cultureware with Poly-D-Lysine and Laminin-521 or Poly-D-Lysine and Corning® Matrigel® hESC-Qualified Matrix

Use sterile technique to coat tissue culture-treated cultureware with both Poly-D-Lysine and CellAdhere™ Laminin-521 Matrix or Poly-D-Lysine and Corning® Matrigel®.

Poly-D-Lysine

  1. Use Poly-D-Lysine at 100 µg/mL. If needed, dilute Poly-D-Lysine solution in phosphate-buffered saline (PBS) to reach a final concentration of 100 µg/mL.
  2. Gently mix Poly-D-Lysine solution by inverting the bottle several times to ensure homogeneity. Avoid vigorous shaking to prevent foaming.
  3. Add 200 µL of Poly-D-Lysine solution per well into the MEA plate to cover the entire growth surface.
  4. Distribute the solution evenly and incubate at 37°C and 5% CO2 for 3 hours.
  5. Wash the Poly-D-Lysine-coated cultureware three times by adding D-PBS (Without Ca++ and Mg++) gently and removing the solution by tilting the cultureware, putting the pipette tip toward the corner of the cultureware to avoid damaging the Poly-D-Lysine coating.
  6. Allow the Poly-D-Lysine-coated cultureware to dry out completely by incubating at room temperature for 30 minutes with the lid open in a biosafety cabinet.

CellAdhere™ Laminin-521

  1. Thaw CellAdhere™ Laminin-521 at 2 - 8°C before use.
    Note: If not used immediately, store at 2 - 8°C for up to 3 months.
  2. Dilute CellAdhere™ Laminin-521 in D-PBS with Ca++ and Mg++ to a final concentration of 10 µg/mL.
  3. Gently mix the diluted CellAdhere™ Laminin-521. Do not vortex.
  4. Immediately add 0.2mL of diluted CellAdhere™ Laminin-521 per well into the MEA plate to cover the entire growth surface.
  5. Gently rock the cultureware back and forth to spread the CellAdhere™ Laminin-521 solution evenly across the entire surface.
  6. Seal the cultureware to prevent evaporation of the CellAdhere™ Laminin-521 solution (e.g. with Parafilm®). Incubate at 2 - 8°C overnight.
  7. Aspirate CellAdhere™ Laminin-521 when cells are ready to be plated.
    Note: The coating does not require washing before use.

Corning® Matrigel® hESC-Qualified Matrix

Note: Matrigel® should be aliquoted and frozen. Consult the Matrigel® Certificate of Analysis for the recommended aliquot size (“Dilution Factor”) to prepare 24 mL of diluted matrix. Ensure Matrigel® is always kept on ice when thawing and handling to prevent it from gelling.
  1. Thaw one aliquot of Corning® Matrigel® on ice.
  2. Dispense 24 mL of cold DMEM/F-12 with 15 mM HEPES into a 50 mL conical tube and keep on ice.
  3. Add thawed Matrigel® to the cold DMEM/F-12 with 15 mM HEPES (in the 50 mL tube) and mix well. The vial may be washed with cold medium if desired.
  4. Immediately add 0.2 mL of diluted Matrigel® solution per well into the MEA plate to cover the entire growth surface.
  5. Swirl the cultureware to spread the solution evenly across the surface. If the surface of the cultureware is not fully coated by the Matrigel® solution, it should not be used.
  6. Incubate at room temperature (15 - 25°C) for 1 hour or incubate at 2 - 8°C overnight. Do not let the Matrigel® solution evaporate.
    Note: Using freshly coated cultureware is recommended. However, if not used immediately, the coated cultureware must be sealed (e.g. with Parafilm®) and can be stored at 2 - 8°C for up to 24 hours. Allow stored coated cultureware to come to room temperature before proceeding to the next step.
  7. Aspirate Matrigel® solution immediately prior to seeding cells. Ensure that the coated surface is not scratched. Do not let the surface dry.
    Note: It is not necessary to wash cultureware after removing the Matrigel® solution.

B. Preparing Complete STEMdiff™-TF Forebrain Induced Neuron Differentiation Medium B (Complete Medium B)

Use sterile technique when preparing complete STEMdiff™-TF Forebrain Induced Neuron Differentiation Medium B (complete medium B; STEMdiff™-TF Forebrain Induced Neuron Basal Medium + STEMdiff™-TF Forebrain Induced Neuron Supplement B [50X] ). For complete instructions on preparing seeding medium, refer to the Product Information Sheet.

C. Preparing Complete STEMdiff™-TF Forebrain Induced Neuron Differentiation Medium AB (Complete Medium AB)

Use sterile technique to prepare complete STEMdiff™-TF Forebrain Induced Neuron Differentiation Medium AB (complete Medium AB; complete medium B + STEMdiff™-TF Forebrain Induced Neuron Supplement A).

This protocol is for preparing complete medium AB for a single well of a 48-well CytoView MEA™ Plate. If using a higher number of wells or other cultureware, adjust volumes accordingly.

Note: Complete medium B and AB are stable at 2 - 8°C for up to 2 weeks. Warm complete medium B and AB to room temperature (15 - 25°C) before use. Mix thoroughly by pipetting up and down. Do not store. Do not filter.
  1. Thaw STEMdiff™-TF Forebrain Induced Neuron Supplement A overnight at 2 - 8°C. Mix thoroughly by gentle pipetting. Do not vortex.
    Notes:
    • If not used immediately, store at 2 - 8°C for up to 3 weeks. For long-term storage, aliquot Supplement A into tubes, place tubes in a pre-cooled slow freezing chamber (e.g. Mr Frosty™) and store the chamber at -80°C. The next day, transfer the frozen tubes to long-term storage in a -80°C freezer.
    • After thawing the aliquots, use immediately or store at 2 - 8°C for up to 3 weeks. Do not re-freeze. Do not exceed the shelf life of the supplement.
  2. Add 1 µL of STEMdiff™-TF Forebrain Induced Neuron Supplement A to 0.2 mL of complete medium B for each well of a 48-well CytoView MEA™ Plate that will be cultured. Mix thoroughly by pipetting up and down. Do not vortex.
    Note: Complete medium AB is stable at 2 - 8°C for up to 2 weeks. Warm complete medium AB to room temperature (15 - 25°C) before use. Mix thoroughly by pipetting up and down. Do not store. Do not freeze.

D. Preparing STEMdiff™ Forebrain Neuron Maturation Medium

Use sterile technique to prepare STEMdiff™ Forebrain Neuron Maturation Medium. For complete instructions on preparation, refer to the Product Information Sheet.

E. Supplementing STEMdiff™ Forebrain Neuron Maturation Medium with Uridine and 5-Fluoro-2′-deoxyuridine

Use sterile technique to supplement the STEMdiff™ Forebrain Neuron Maturation Medium with 3µM Uridine and 3µM 5-Fluoro-2′-deoxyuridine (Fdu/U).

Notes:
  • By Day 5 of culture, approximately 5 - 10% of proliferating cells may persist. To maintain culture purity beyond this point, supplement the STEMdiff™ Forebrain Neuron Maturation Medium with 3µM Uridine and 3µM 5-Fluoro-2′-deoxyuridine (Fdu/U), beginning 48 hours after the transition to maturation medium (i.e. starting on Day 7). Continue supplementation with Fdu/U for at least two additional feedings every 48 hours (e.g. on Days 9 and 11). If undifferentiated or proliferative cells remain after this period, continue Fdu/U treatment as needed.
  • For co-culture applications, discontinue Fdu/U supplementation after the third feeding and introduce the co-culture cell line at that point.

Part II. Plating and Culturing Forebrain Neurons on MEA Plates

Please read the entire protocol before proceeding. Use sterile technique when performing the following protocols:

A. Harvesting and Seeding hPSCs As Single Cells (Day -1)
B. Differentiation of hPSCs to Neurons (Day 0 - 5)
C. Neuron Maturation (Day 5 Onward)

Schematic for Culturing STEMdiff™-TF Forebrain Induced Neurons for MEA Analysis Using the Maestro MEA™ System

Figure 1. Schematic for Culturing STEMdiff™-TF Forebrain Induced Neurons for MEA Analysis Using the Maestro MEA™ System

Excitatory glutamatergic forebrain neurons are generated from hPSCs within five days using STEMdiff™-TF Forebrain Induced Neuron Differentiation Kit and maintained in STEMdiff™ Forebrain Neuron Maturation Kit. These neurons are plated on a 48-well CytoView MEA™ Plate for differentiation, allowing MEA recording using Maestro Pro™. MEA = micro-electrode array; hPSCs = human pluripotent stem cells

A. Harvesting and Seeding hPSCs As Single Cells (Day -1)

This protocol is for harvesting hPSCs from a single well of a 6-well plate and seeding them into a 48-well CytoView MEA™ Plate format for differentiation. If using other cultureware, adjust volumes accordingly. Warm cultureware, media, and reagents to room temperature (15 - 25°C) before use. Cells can be previously maintained in mTeSR™ Plus (Catalog #100-0276) or eTeSR™ (Catalog #100-1215). For further information on maintaining high-quality hPSCs, refer to the Technical Manuals for mTeSR™ Plus or eTeSR™.

Note: The whole-well method was used for seeding the plate during this protocol. This approach involves coating and seeding cells over the entire growth area of each well.
  1. Coat cultureware with Poly-D-Lysine and Laminin-521 or Poly-D-Lysine and Matrigel® (see Part I. Reagent Preparation, Section A). Warm cultureware at room temperature if it was prepared the day before.
  2. Aliquot sufficient volumes of seeding medium (mTeSR™ Plus Basal Medium + mTeSR™ Plus 5X Supplement), Y-27632 (Dihydrochloride), and your preferred cell dissociation reagent (TrypLE™ Express Enzyme (1X), phenol red or ACCUTASE®). Warm to room temperature before use.
    Note: Before starting, ensure you have the seeding medium supplemented with 10 µM Y-27632 (Dihydrochloride) ready. Prepare the appropriate volume of medium by adding Y-27632 (Dihydrochloride) to the seeding medium to obtain a final concentration of 10 µM. Mix thoroughly.
  3. Use a microscope to visually identify regions of differentiation in the hPSC culture. Mark these using a felt tip or lens marker on the bottom of the plate. Remove regions of differentiation by scraping with a pipette tip or by aspiration. Avoid having the culture plate out of the incubator for more than 15 minutes at a time.
  4. Wash the well with 2 mL of room temperature D-PBS (Without Ca++ and Mg++). Discard the wash.
  5. Add 0.5 - 1 mL of TrypLE™ Express Enzyme (1X), phenol red or ACCUTASE®.
  6. Incubate at 37°C and 5% CO2 for 4 - 8 minutes.
    Note: The incubation time may vary when using different cell lines and matrices. For cultures maintained on Corning® Matrigel®, 4 - 5 minutes is typically optimal for dissociation.
  7. Add 1 mL of seeding medium + 10 µM Y-27632 (Dihydrochloride) to the well.
  8. Harvest the cells by carefully tilting the plate and gently pipetting up and down with a 1 mL pipette to detach the cells. Transfer the single-cell suspension to a 15 mL conical tube.
    Note: Avoid excessive or harsh trituration, as this may adversely impact cell viability. If the cells do not readily detach, a longer incubation time may be required.
  9. Centrifuge the cell suspension at 300 x g for 5 minutes.
  10. Carefully aspirate the supernatant and gently flick the tube 3 - 5 times to resuspend the cell pellet.
  11. Add 1 - 2 mL of seeding medium + 10 µM Y-27632 (Dihydrochloride) to the cell pellet. Mix gently by pipetting.
  12. Count cells using Trypan Blue and a hemocytometer (e.g. Catalog #100-1181) or an automated cell counting method.
  13. Adjust the cell suspension using additional seeding medium + 10 µM Y-27632 to an appropriate concentration to allow for the desired cell number to be seeded in 0.2 mL of suspension per well. Suggested cell numbers range from 6.5 × 10⁴ to 7.5 × 10⁴ per well in a 48-well CytoView MEA™ Plate.
  14. Add 0.2 mL of the cell suspension onto the entire growth area of each coated MEA plate well.
  15. Add sterile distilled water to reservoirs around the edge of the plate to prevent evaporation.
  16. Move the plate in several quick, short, back-and-forth and side-to-side motions to distribute the cells across the surface of the wells.
  17. Incubate at 37°C and 5% CO2. Do not disturb the plate for 24 hours.
Note: Cell density is a critical factor for the success of this kit. For optimal performance, we recommend seeding 6.5 × 10⁴ to 7.5 × 10⁴ cells per well in a 48-well CytoView MEA™ Plate. However, culture appearance may vary between users. To evaluate your culture density, refer to the Day 0 reference images (captured 24 hours after seeding). If your culture appears noticeably denser or sparser than the reference, do not proceed with transfection. Instead, discard the culture, adjust the seeding density accordingly, and repeat the procedure. Maintaining appropriate cell density is essential for consistent and reproducible results. To determine the optimal seeding density for your specific conditions, we recommend testing 5.0 × 10⁴, 6.5 × 10⁴, 7.0 × 10⁴, 7.5 × 10⁴, and 8.0 × 10⁴ cells per well in a 48-well CytoView MEA™ Plate. Compare your Day 0 cultures to the reference images to identify the most suitable density for your experiments.

B. Differentiation of hPSCs to Neurons (Day 0 - 5)

Figure 2. Day 0 STEMdiff™-TF Forebrain Induced Neurons Cultured on a 48-well CytoView MEA™ Plate

Figure 2. Day 0 STEMdiff™-TF Forebrain Induced Neurons Cultured on a 48-well CytoView MEA™ Plate

Representative images illustrating the recommended cell density on Day 0 for differentiation of hPSCs into neurons using the STEMdiff™-TF Forebrain Induced Neuron Differentiation Kit on MEA plates. If cultures appear substantially denser or sparser than those shown, discard the culture, adjust the seeding density, and restart the differentiation. Maintaining the appropriate cell density is essential to ensure consistent and reliable results with this kit. hPSCs = human pluripotent stem cells; MEA = multi-electrode array

Day 0 - 2

Prepare complete medium B (See Part I. Reagent Preparation, Section B) and complete medium AB (See Part I. Reagent Preparation, Section C). Complete medium AB is required for Day 0 - 2 of induction and complete medium B is required for Day 3 - 5 of induction.

Notes:
  • Complete medium B can be stored at 2 - 8°C for up to 2 weeks. Warm the complete medium B to room temperature before use. Do not freeze. Do not filter.
  • Complete medium AB is stable at 2 - 8°C for up to 2 weeks. Warm complete medium AB to room temperature (15 - 25°C) before use. Mix thoroughly by pipetting up and down. Do not freeze. Do not filter.
  • Add 1 µL of STEMdiff™-TF Forebrain Induced Neuron Supplement A to 0.2 mL of complete medium B for each well of a 48-well plate that will be cultured. Adjust the volumes accordingly to the total number of wells being cultured.
  1. On Day 0, use a 1 mL pipette tip to gently remove and discard the medium from each well. If using an aspirator, remove the solutions by tilting the cultureware and put the tip toward the corner of the cultureware to avoid any contact with the cells and coating matrix.
  2. Add 0.2 mL of complete medium AB per well using a serological pipette or 1 mL pipette tip. Avoid disturbing the cells and coating matrix.
  3. Incubate at 37°C and 5% CO2 for 24 hours.
  4. Repeat steps 1 - 3 again on Day 1 and 2.
Note: On Day 0, completely remove the seeding medium from each well and replace it with complete medium AB. On Days 1 and 2, avoid removing all the medium; instead, leave a small volume to prevent cells from being exposed to air, as they are more sensitive at this stage.

Day 3

Warm an appropriate volume of complete medium B to room temperature. Add PluriSin-1 to complete medium B at a final concentration of 20 µM. Mix thoroughly. Do not filter.

Note: To prepare 10 mM stock solution of PluriSin-1, resuspend 1 mg in 469 µL of fresh DMSO. Aliquot and store at -20°C to prevent repeated freeze-thaw cycles and protect from light exposure to maintain stability.
  1. Using a 1 mL pipette tip, gently remove and discard the medium from each well. If using an aspirator, tilt the cultureware and position the tip at the corner of the cultureware to prevent contact with the cells and coating matrices. Be sure to leave a small volume of medium in the we
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