Organoid Seeding Protocol on Chips for MEA recordings

Organoid Seeding Protocol on Chips

MEA Chip Preparation

Please refer to the supplier’s protocol for chip surface preparation.
For MaxOne single-well chips (Maxwell Biosystems), the preparation includes:

• Cleaning with 1% Terg-a-zyme solution for a minimum of 2 hours at room temperature.
• Washing thoroughly with distilled water.
• Sterilizing with 70% ethanol.
• Drying under laminar airflow in a sterile cell culture hood.

Poly-L-Ornithine Hydrobromide Coating

Dissolve Poly-L-Ornithine hydrobromide (Sigma P3655) in distilled water at 20 μg/mL and incubate for at least 5 hours at 37 °C (up to overnight).
Prepare sufficient solution to cover the entire chip surface (typically 1 mL per Maxwell MEA chip).
Incubate at 37 °C, then remove excess solution before proceeding.

Laminin Coating

Aspirate the Poly-L-Ornithine solution and add Laminin (25 μg/mL in 1× PBS; Santa Cruz, sc-29012).
A volume of 50 µL per chip is sufficient to cover the active electrode area.
Incubate for at least 5 hours at 37 °C, or overnight if preferred.

Pre-conditioning (Pre-incubation)

Without necessarily aspirating the Laminin solution (as the volume is small), proceed to pre-conditioning:
Supplement 1 mL of the culture medium normally used to maintain organoids  with Matrigel (Corning 354234) at a ratio of 5 µL Matrigel per 1 mL of medium, and add it to te chip. Use cold medium directly from the refrigerator to prevent Matrigel precipitation. This step adds a third coating layer, as Matrigel deposits on the chip surface.

To minimize medium use, an alternative is to apply 50 µL of medium containing 1 µL Matrigel only to the chip center, where the organoids will attach.
Incubate for a minimum of 30 minutes (preferably up to 2 hours) at 37 °C before placing organoids.

Organoid Placement

To transfer organoids:

• Sterilize scissors with ethanol under the hood.
• Cut the end of a pipette tip to widen it and prevent damage during transfer.
• Gently pipette the organoid onto the center of the chip, over the active electrode area.

If the organoid is not centered, carefully reposition it with an uncut pipette tip, avoiding direct contact with the chip surface to prevent damage.

Organoid Attachment

Organoid attachment is highly sensitive to mechanical disturbance. Handle chips carefully to minimize vibration.
After seeding, gently transfer chips to the incubator and verify that the organoids remain centered.
If repositioning is required, open the chip lid inside the incubator and adjust with a pipette tip.
Close the incubator door carefully and slowly, to prevent vibrations that can disrupt adhesion.
Depending on conditions, organoids typically adhere within a few hours to 2–3 days.
Avoid vibrations during this period and until firm attachment is confirmed.

Medium Change

Organoids must always remain covered with culture medium; do not remove all the medium during medium change. For example, remove 500 µL and replace with an equal or slightly larger volume of fresh medium. We have consistently observed partial evaporation from MEA chips; therefore, it is recommended to add slightly more medium than is removed to compensate (e.g., remove 500 µL and add 600 µL). Always monitor and adjust for evaporation visually.

For spontaneous activity recordings, it is recommended to use BrainPhys medium (Stem Cell Technologies #05790); use it as the base medium to prepare the medium in which normally organoids are maintained. Approximately one week after seeding, if the organoids are attached, remove half the volume (500 µL) and add 500 µL of LTSM prepared with BrainPhys as the base.
Repeat this medium exchange once or twice per week.

 

Activity

In our hands, cortical organoids typically exhibited detectable spontaneous activity 2–3 weeks after plating, corresponding to 1–2 weeks after gradual transition to BrainPhys-based medium.

Tips

• Organoid Size:
  In our experience, larger organoids adhered poorly and were more sensitive to vibrations. Fragmenting them into smaller pieces using sterile tweezers before transfer improved attachment.
• Alternative Methods:
  Alternative attachment strategies may be explored depending on experimental needs. Some recording protocols use a tissue holder to secure organoids during short-term (“acute”) recordings, after which they are returned to their culture plates. Another approach involves placing the organoid on the chip, aspirating most of the medium so it settles and attaches in the center, and then gently re-adding medium after ≤5 minutes to prevent drying.
• Handling Chips:
  To reduce vibration when movin them in and out of the incubator, place chips within larger culture plates (2–3 chips per plate). Keeping them enclosed also provides better protection against contamination.

For further details and ordering codes, please refer to the original article: FGF8-mediated gene regulation affects regional identity in human cerebral organoids (https://doi.org/10.7554/eLife.98096)