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How to Culture Human Pluripotent Stem Cell (hPSC)-Derived Forebrain Neurons for MEA Analysis Using the Maestro MEA™ System
How to Culture Human Pluripotent Stem Cell (hPSC)-Derived Forebrain Neurons for MEA Analysis Using the Maestro MEA™ System
Micro-electrode array (MEA) systems are powerful tools to measure activity from a population of cells simultaneously. They detect activity-associated voltage changes in the extracellular space around electrically active cells, such as neurons, that are grown on specialized cell culture plates containing a grid of electrodes in each well. By measuring activity from several electrodes simultaneously, researchers can characterize the activity of neuronal networks. Using MEAs to measure cellular activity is non-invasive and does not require cell labeling, allowing for long-term monitoring of electrophysiological maturation. This makes MEA systems ideal for both disease modeling and drug studies.
Because cells must cover the electrodes uniformly and consistently to produce high-fidelity data, culture substrates, and seeding densities must be optimized for each cell type. 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.
Workflow Specifications
Obtaining hPSC-Derived Forebrain Neuron Precursor Cells
Important Note: For this protocol, cells must be seeded on the MEA plate at the “forebrain neuron precursor” stage. This stage corresponds to the time point after the differentiation stage and before the maturation stage of the STEMdiff™ Forebrain Neuron workflow (see below). Forebrain neuron precursor cells cannot be further dissociated or replated once seeded on MEA plates in STEMdiff™ Forebrain Neuron Maturation Medium.
There are multiple options for generating or sourcing forebrain neuron precursor cells:
A. Starting from hPSCs
B. Starting from Neural Progenitor Cells (NPCs)
C. Starting from Forebrain Neuron Precursor Cells
This protocol can also be used with hPSC-derived midbrain neurons instead of forebrain neurons. If working with midbrain neurons, cells should be seeded onto MEA plates at the “midbrain neuron precursor” stage using STEMdiff™ Midbrain Neuron Maturation Kit (Catalog #100-0041). Refer to the Product Information Sheet for STEMdiff™ Midbrain Neuron Kits for detailed instructions and additional materials required to generate midbrain neuron precursor cells. All other protocol details outlined below remain unchanged (e.g. coating procedures, recommended seeding density ranges, etc).
Choosing an MEA Plate Format
This protocol is optimized for culturing cells on polyethyleneimine (PEI)-coated 24-, 48-, and 96-well CytoView MEA™ plates. Refer to Figure 1 and Table 1 below for a schematic diagram showing the bottom surfaces of the wells for each of these plate formats, as well as plate specifications.
We recommended seeding at least 3 - 8 replicate wells per condition to calculate sample-to-sample variability alongside well-to-well variability.
Figure 1. CytoView MEA™ Plate Electrode Layouts.
Schematic diagrams of the bottom surfaces of different CytoView MEA™ Plate formats depicting recording electrodes (blue), grounds (orange), and on-plate spotting guides (gray), and where present, a large dedicated stimulation electrode (blue; 24-well format).
Table 1. CytoView MEA™ Plate Specifications and Compatibility
Selecting a Coating/Plating Method
Two different coating/plating methods are outlined throughout this protocol. Refer to the guidance below to determine which method is best suited for your application and experimental design.
Whole-Well Method
- This approach involves coating and seeding cells over the entire growth area of each well.
- This option is well-suited for those requiring higher throughput, as it tends to be easier from a technical standpoint and is more amenable to the use of multichannel pipettes or automated platforms.
Spot Method
- This approach involves coating and seeding cells using a small droplet placed only over the recording electrodes within each well.
- This option is advantageous for researchers using high plating densities, as it concentrates the cells over the recording area and, therefore, reduces the total cell number required for an assay compared to that for the whole-well method. However, the spot method tends to be more technically difficult and less amenable to the use of multichannel pipettes or automated platforms.
Materials and Reagents
- STEMdiff™ Forebrain Neuron Maturation Kit (Catalog #08605)
- BrainPhys™ Neuronal Medium (Catalog #05797)
- STEMdiff™ Forebrain Neuron Maturation Supplement (Catalog #08602)
- D-PBS (Without Ca++ and Mg++) (Catalog #37350)
- 50% (w/v) Poly(ethyleneimine) solution (Sigma P3143)
- Boric acid (Sigma B6768)
- Sodium tetraborate (Sigma 221732)
- D-(+)-glucose (Sigma G8270 or G8644)
- Trypan Blue (Catalog #07050)
- Hemocytometer and cover slip (e.g. Hausser Scientific™ Bright-Line Hemocytometer; Catalog #100-1181)
Protocol
Part I. Reagent Preparation
Borate Buffer, pH 8.4
- Add 100 mL of distilled water to a glass beaker and heat to 50°C.
- Stir at 300 rpm and add the following to the beaker of water:
- 310 mg of boric acid
- 475 mg of sodium tetraborate
- Continue to stir at 300 rpm at 50°C for 30 minutes.
- Adjust pH to pH 8.4 with 12 N HCl.
Note: Adjust pH while the chemicals are dissolving. pH adjustment can accelerate dissolution.
- Stir until all chemicals are dissolved and the solution becomes clear.
- Measure pH and adjust to pH 8.4 with HCl or NaOH accordingly.
- Remove the beaker from the hot plate and allow the solution to equilibrate to room temperature (15 - 25°C).
- Filter sterilize and store at room temperature until use.
5% Polyethyleneimine (PEI) Intermediate Stock Solution in Borate Buffer
- Pour approximately 2 mL of 50% (w/v) PEI stock solution into a tared 50 mL tube.
- Centrifuge at 1000 rpm for 5 minutes.
- Re-weigh the tube to determine the weight (in grams) of 50% PEI solution added to the tared tube.
- Add borate buffer (pH 8.4) to dilute the 50% PEI solution to a final concentration of 5% (w/v). Vortex to mix.
- E.g. 2 g of 50% (w/v) PEI solution is diluted with 18 mL of borate buffer to reach a final volume of 20 mL, resulting in a 5% PEI solution.
- Store the 5% PEI solution at 2 - 8°C for up to 6 months.
1 M Glucose Stock Solution
- The following instructions are for preparing 10 mL of 1 M D-(+)-glucose stock solution from D-(+)-glucose powder. If preparing other volumes, adjust accordingly. Alternatively, a sterile 10% w/v D-(+)-glucose solution can be used by diluting to a 1 M solution with distilled water.
- Use sterile technique to prepare 1 M glucose stock solution.
- Dispense 7 mL distilled water into a 50 mL tube.
- Weigh out 1.8 g of D-(+)-glucose powder and add to the tube containing 7 mL water.
- Mix to dissolve.
- Check the volume of the solution and add distilled water to bring the total volume up to 10 mL.
- Filter sterilize the glucose solution and store at 4°C.
Glucose-Supplemented STEMdiff™ Forebrain Neuron Maturation Medium
- BrainPhys™ contains glucose at a physiological level of 2.5 mM. However, in applications with high cellular metabolic demands, this glucose level may deplete rapidly. To ensure cell survival and support neuronal activity, supplementing the cultures with additional glucose is recommended. For MEA cultures of hPSC-derived neurons, add 7.5 mM glucose to achieve a final concentration of 10 mM. This process is described below.
- Use sterile technique to prepare STEMdiff™ Forebrain Neuron Maturation Medium. The following example is for preparing 125 mL of medium. If preparing other volumes, adjust accordingly.
- Thaw STEMdiff™ Forebrain Neuron Maturation Supplement at room temperature (15 - 25°C) or at 2 - 8°C overnight. Mix thoroughly.
Note: If not used immediately, aliquot supplement and store at -20°C. Do not exceed the shelf life of the supplement. After thawing aliquots, use immediately. Do not refreeze.
- Add 25 mL of STEMdiff™ Forebrain Neuron Maturation Supplement and 0.938 mL of 1 M glucose stock solution to 100 mL of BrainPhys™ Neuronal Medium. Mix thoroughly.
Note: If not used immediately, store STEMdiff™ Forebrain Neuron Maturation Medium at 2 - 8°C for up to 4 weeks. Warm medium to 37°C before using and protect from light.
Part II. Coating MEA Plates
- Prepare a suitable volume of 0.1% PEI solution in borate buffer (pH 8.4). Filter sterilize and use immediately.
- E.g. To prepare 10 mL of 0.1% PEI solution, add 200 μL of 5% PEI solution to 9.8 mL of borate buffer (pH 8.4).
- Add 0.1% PEI solution to each well:
- For the whole-well method: Pipette 200 μL of solution into each well to cover the entire growth area.
- For the spot method: Pipette a 5 µL droplet of solution just over the electrode array at the center of each well.
- Incubate the MEA plate for at least 2 hours at room temperature.
- Gently tilt the PEI-coated cultureware onto one side. Aspirate the excess solution, ensuring that the coating is not scratched.
Note: This step is optional for the spot method, in which case it is not strictly necessary to aspirate the 5 µL PEI droplet before Step 5.
- Wash the PEI-coated cultureware 3 times (refer to Table 2 for recommended volumes) by pipetting sterile distilled water toward the edge of each well to avoid scratching the PEI substrate. To remove each wash volume, tilt the plate and aspirate carefully from the bottom edge of the well.
- Replace the plate lid and allow the plate to air dry overnight at room temperature in a biosafety cabinet.
Note: This step may be shortened to ~ 2 hours at room temperature if desired, provided the plate completely dries out during this time.
Table 2. Recommended Volumes for CytoView MEA™ Plates
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