Attachment culture of cortical organoids at the microwell air-liquid interface

Summary

The cortical organoid is an efficient model for studying human brain neurodevelopment and neurological disease. However, its three-dimensional structure limits real-time observation of internal physiological changes. Here, we present a protocol for an air-liquid interface attachment culture for cortical organoids. We describe steps for transplanting cortical organoid slices and generating the air-liquid interface. We then detail calcium imaging on organoid external neural networks and immunohistochemical staining on confocal plates.

Graphical abstract

Highlights

• •A method of air-liquid interface attachment culture for cortical organoids
• •A stable fluid environment and effortless optical observation
• •Same-region calcium imaging and immunohistochemical (IHC) cross-analysis

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

The cortical organoid is an efficient model for studying human brain neurodevelopment and neurological disease. However, its three-dimensional structure limits real-time observation of internal physiological changes. Here, we present a protocol for an air-liquid interface attachment culture for cortical organoids. We describe steps for transplanting cortical organoid slices and generating the air-liquid interface. We then detail calcium imaging on organoid external neural networks and immunohistochemical staining on confocal plates.

Before you begin

This protocol uses cortical organoids^1,2,3 as an example. There are no clear restrictions on the type of brain organoids or the culture protocol used, so it can also be applied to brain organoids cultured by other methods (e.g., unguided neural organoids⁴). Determine the sample type, medium type, and whether to slice according to your experimental needs.

Cortical organoid slice generation

Timing: at least 6 weeks

• 1.The generation and culture of cortical organoids in our experiments mainly follow the protocol proposed by X. Qian et al.^1,2 In general, cortical organoids are used after 45 days of culture.
• 2.
  The production of cortical organoid slices mainly follows the protocols of Giandomenico et al.^5,6 and Qian et al.² It can be selected according to the experimental equipment conditions. The final slice thickness can be used within 200–500 μm. See references for the specific protocol and technical details or follow the steps below.

  • a.
    Dissolve 3% (w/v) ultra-low-melting-point agarose in DMEM/F-12 (keep at 45°C to prevent solidification).
  • b.
    Embed 15 cortical organoids in agarose within a disposable base mold (1.5 cm wide, square), then move on ice for 10 min to solidify.
  • c.
    Perform vibrating sectioning by Leica VT 1200S in DMEM/F-12 containing 1 X Penicillin-Streptomycin-Amphotericin B. (Set vibratome as 0.12 mm/s in speed and 1 mm in amplitude.)
• 3.
  Transfer cortical organoid slices in ultra-low attachment six-well plates.

  • a.
    Add the complete medium^1,2 supplementing 1% (w/v) Matrigel to slices and incubate for at least 1 h of static culture.
  • b.
    Then transfer the six-well plate to the orbital shaker (120 rpm) in the incubator, and culture for at least 24 h, but up to 48 h, before the next movement.

Polydimethylsiloxane (PDMS) microwell preparation

Timing: 1 day

• 4.
  Preparation of PDMS raw material.

  • a.
    Use SYLGARD 184 Silicone Elastomer PDMS kit with a 10 to 1 mix ratio, and stir for at least 10 min.
  • b.
    Immediately fill the PDMS (liquid state after mixing) into 50 mL syringes (remove the needles), and expel excess air that has not mixed with the liquid PDMS.
  • c.
    Infuse about 10 mL of liquid PDMS into one 10 cm culture dish, and rotate the culture dish to make the PDMS completely spread on the bottom surface. Check the PDMS is evenly distributed in the culture dish to ensure the consistency of the microwell depth in subsequent experiments.
  • d.
    Place culture dishes containing PDMS horizontally in an oven at 60°C over 8 h.
• 5.
  Microwell generation. (Figure 1).

  • a.
    Transfer the culture dish to a biohazard safety cabinet, use tweezers to peel and remove the cured PDMS from the culture dish, and attach it to a clean steel plate.
  • b.
    Use a 20 mm hole punch to cut out a circular piece of PDMS.
  • c.
    use an 8 mm hole punch to cut a microwell in the middle.
  • d.
    form a ring structure with an outer diameter of 20 mm and an inner diameter of 8 mm (Graphical abstract).
  • e.
    Use tweezers to hold the PDMS ring into a 50 mL centrifuge tube filled with ethanol 75% solution for shaking and washing for at least 5 s.
  • f.
    Then transfer it to a 1000 μL empty pipette tip box. A plurality of PDMS rings are placed at intervals and then autoclaved and dried. Troubleshooting 1.
  • g.
    Use tweezers to attach the PDMS ring to the 12-well confocal plate, and make sure to press out all air bubbles on the attachment surface so that the PDMS is completely adhered to the plate bottom and cannot be horizontally moved.
  • h.
    The inner wall of the PDMS ring and the bottom surface of the confocal plate together form a microwell structure.

CRITICAL: Ensure the tools are clean and dust-free in step 5 to prevent dust from the attachment surface, which can result in incomplete attachment and liquid leakage. Be careful not to reverse the PDMS ring during the operation, and ensure that the PDMS surface attached to the bottom of the confocal plate is the surface generated by the previous culture dish bottom. Troubleshooting 2.

• 6.
  Coating confocal plate microwell.

  • a.
    Add 100 μL of poly-L-ornithine solution to each microwell, ensuring complete coverage of the bottom and the absence of air bubbles.
  • b.
    Add sterilized deionized water to the gap of the confocal plate wells to prevent the microwells from drying out.
  • c.
    Transfer the confocal plates to an incubator and leave for more than 3 h.
  • d.
    Wash the microwells 3 times with sterilized deionized water.
  • e.
    After Aspirating all water, place confocal plates in the biohazard safety cabinet with the lid open until the microwells are dried.
  • f.
    Add 200 μL of neurobasal medium with 1% (w/v) Matrigel dissolved (coating solution) in each microwell, ensuring complete coverage of the bottom and the absence of air bubbles (Graphical abstract).
  • g.
    Transfer the confocal plates to an incubator for more than 1 h.

Figure 1.

Manufacturing process of PDMS ring

(A) Hole punches and tweezer.

(B) Cured PDMS in a 10 cm culture dish.

(C) Use a 20 mm hole punch and an 8 mm hole punch successively to punch holes.

(D) Get PDMS rings.

(E) Use ethanol 75% solution to wash away adhering impurities.

(F) Transfer it to a 1000 μL empty pipette tip box for autoclaving and drying.

(G) PDMS rings in the 12-well confocal plate.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
Rabbit Polyclonal Tubulin β 3 (TUJ1) (1:500)	Abcam	Cat# ab18207, RRID: AB_444319
Chicken Polyclonal Glial Fibrillary Acidic Protein (GFAP) (1:2000)	Abcam	Cat# ab4674, RRID: AB_304558
Goat Polyclonal Synaptophysin (Syn) (1:2000)	RD	Cat# AF5555, RRID: AB_2198864
Rabbit polyclonal postsynaptic density protein 95 (PSD-95) (1:1000)	Thermo Fisher	Cat# 51-6900, RRID: AB_2533914
Chicken polyclonal microtubule-associated protein 2 (MAP2) (1:2000)	Abcam	Cat# ab92434, RRID: AB_2138147
Mouse Monoclonal Vesicular Glutamate Transporter 1 (VGLUT1) (1:500)	Millipore	Cat# MAB5502, RRID: AB_262185
Rabbit monoclonal S100 beta (1:500)	Abcam	Cat# ab52642, RRID: AB_882426
Goat anti-chicken IgY H&L (Alexa Fluor 647)	Abcam	Cat# ab150171, RRID: AB_2921318
Donkey anti-rabbit IgG H&L (Alexa Fluor 488)	Abcam	Cat# ab150073, RRID: AB_2636877
Donkey anti-rabbit IgG H&L (Alexa Fluor 568)	Abcam	Cat# ab175470, RRID: AB_2783823
Donkey anti-goat IgG H&L (Alexa Fluor 488)	Abcam	Cat# ab150129, RRID: AB_2687506
Goat anti-mouse IgG H&L (Alexa Fluor 594)	Abcam	Cat# ab150116, RRID: AB_2650601
Donkey anti-rabbit IgG H&L (Alexa Fluor 647)	Abcam	Cat# ab150075, RRID: AB_2752244
Chemicals, peptides, and recombinant proteins
Corning Matrigel Growth Factor Reduced (GFR) Basement Membrane Matrix, LDEV-free	Corning	Cat# 354230/356230
Polydimethylsiloxane (PDMS)	Dow Corning	Cat# SYLGARD 184 Silicone Elastomer kit
Poly-L-ornithine solution	Sigma	Cat# P4957-50ML
Neurobasal medium	Thermo Fisher	Cat# 21103049
BrainPhys neuronal medium	STEMCELL	Cat# 05790
Fluo-4, AM, cell permeant	Thermo Fisher	Cat# F14201
Triton X-100	Sigma	Cat# X100-100ML
Normal donkey serum	Jackson ImmunoResearch	Cat# 017-000-121
Calcein-AM	Yeasen	Cat# 40747-A
Hoechst 33342	Yeasen	Cat# 40731ES10
DAPI	Sigma	Cat# D9542
Agarose, ultra-low gelling temperature, molecular biology grade	Sigma	Cat# A2576-5G
DMEM/F-12	Gibco	Cat# 11330032
Penicillin/streptomycin/amphotericin B, sterile solution	Leagene	Cat# CA0077
Experimental models: Cell lines
DXR0109B	ATCC	Cat# ACS-1023
H9	Wicell	Cat# Wa09
Software and algorithms
CALIMA	F.D.W. Radstake et al.7	https://aethelraed.nl/calciumimaginganalyser/index.html
Other
Incubator	Esco	Cat# CCL-170T-8
Biological safety cabinet	Esco	Cat# 2010655
Orbital shaker	NEST	Cat# 105008
Vibrating microtomes (vibratomes)	Leica	Leica VT 1200 S
Ultra-low attachment 6-well plate	Corning	Cat# 3471
Sterile microcentrifuge tubes	Axgen	Cat# MCT-150-C-S
12-well confocal plate	Cellvis	Cat# P12-1.5P
10-cm culture dish	Biofil	Cat# TCD010100
Sterile filter pipette tips	Kirgen	Cat# KG1313; KG1212; KG1111
1-Channel mechanical pipette	Eppendorf	Cat# 3120000020, 3120000011, 3120000062 3120000046

Step-by-step method details

Cortical organoid slices transplantation (seed) & air-liquid interface generation

Timing: 3 h

Here we place sliced cortical organoids in microwells to adhere to the bottom surface (e.g., confocal plates) while also being in contact with air, creating a relatively stable fluid environment conducive to neuronal migration and organization.

• 1.
  Cortical organoid slices transplantation (seed).

  • a.
    Transfer cortical organoids from step 3 to a 1.5 mL microfuge tube, aspirate supernatant then replace with complete medium.
  • b.
    Aspirate 180 μL of coating solution from the microtiter wells, use a 200 μL pipette tip with a cut tip (widen the opening over 1500 μm in diameter to keep single cortical organoid undamaged) to aspirate 100 μL of medium containing a single cortical organoid, then pipette into a single microwell.

    Note: Ensure that the bottom of the microwell is kept moist during the transfer process to prevent the coated bottom surface from drying up.

  • c.
    Use a 1000 μL pipette tip nested with a 10 μL pipette tip, then gently push the cortical organoid into the center of the microwell.

    Note: Push with contact on the side of the cortical organoid, do not push perpendicular to the cortical organoid. Do not touch or scratch the coated microwell bottom when moving the cortical organoids.

• 2.
  Air-liquid interface generation.

  • a.
    Use a 100 μL pipette to slowly aspirate medium at the edge of the microwell until the cortical organoids emerge from the liquid surface and are also attached to the bottom surface (Graphical abstract).

    • i.
      While aspirating, constantly adjust the position of the pipette tip at the edge of the microwell, thereby adjusting the position of the cortical organoid in the microwell, keeping it approximately in the center.
    • ii.
      Monitor the amount of liquid in the microwells to prevent drying up and exposure of the coated bottom surface to the air caused by long-time operation or excessive aspiration.
  • b.
    Check that the cortical organoids in all microwells attach to the bottom surface while also in an air-liquid interface environment.
  • c.
    Move slowly to an incubator for static culture. Troubleshooting 3, Troubleshooting 4.

Daily medium change

Timing: 4 h

Since the medium volume in the microwell is only about 50 μL, it is necessary to change medium daily and give sufficient medium for incubation during a period of time. Repeat this step every 24 h (change medium 1 time in 24 h) to maintain the culture of cortical organoids at the air-liquid interface.

• 3.Warm complete medium at 27°C.
• 4.Use a 200 μL pipette to slowly drop 100 μL of the complete medium onto the cortical organoids.

Note: When adding the complete medium, drip droplets one by one; do not create a continuous jet. Avoid blowing off attached cortical organoids.

• 5.Move slowly to an incubator for 2 h of static culture.

Pause point: There is a pause point between step 5 and step 6 for 2 h.

• 6.Repeat step 2 to create the air-liquid interface again. Then move slowly to an incubator for static culture for the remaining 22 h till the next medium change. Troubleshooting 5.

Calcium imaging on cortical organoid external neural networks

Timing: 6 h (excluding imaging time)

• 7.Use the change medium method from step 4 to change 100 μL BrainPhys Neuronal Medium with 5 μM Flou-4. Then move slowly to an incubator for 3 h of static culture.

Pause point: There is a pause point between step 7 and step 8 for 3 h.

• 8.Use a 200 μL pipette to slowly drop 200 μL of the BrainPhys Neuronal Medium onto the cortical organoids.
• 9.Add 500 μL of the BrainPhys Neuronal Medium from the outside of the PDMS ring and use the stable liquid environment in the microwell to protect the cortical organoid from the impact of the liquid flow. Then aspirate the media outside the microwells.
• 10.Repeat step 9 for 3 times. Then move slowly to an incubator for 1 h of static culture.

Pause point: There is a pause point between step 10 and step 11 for 1 h.

• 11.Use a confocal microscope for calcium imaging. If drug experiments are required, use the methods described in steps 9–11 to add and wash drugs.

Immunohistochemical staining on confocal plates

Timing: 12 h (excluding imaging time)

• 12.After step 6 (no calcium imaging) or step 11(calcium imaging), use the change medium method from step 4 to add 1 mL of 4% Paraformaldehyde for 15 min.
• 13.Use the methods described in step 10 to wash samples with 5 mL Phosphate-buffered saline (PBS) 3 times.
• 14.Use tweezers to remove the PDMS ring vertically. Surround the sample with an immunohistochemistry pen, then followed by membrane permeabilization and blocking with 0.1% (w/v) Triton X-100 in normal donkey serum for 1 h at 27°C.
• 15.Samples were incubated with primary and secondary antibodies (see Key resources table) over 8 h at 4°C, respectively.

Note: The calcium ion indicator will flow away under membrane permeabilization, and will not affect the imaging and analysis of immunofluorescence.

• 16.Treatment with DAPI for 30 min, then use FOCM⁸ tissue clearance reagent for 30 min. Finally, the samples were covered with glycerol for storage at −20°C.

Expected outcomes

The air-liquid interface attachment culture method provides excellent conditions for growing cortical organoid external neurons. It also supports and promotes the generation and maturation of the self-organized neural network. On day 3, neurons migrated outward with longer axons and maintained cell activity (Figures 2A and 2B). On day 7, the number and density of external neurons from the cortical organoid increased significantly, accompanied by the early formation of nerve tracts (projections) (Figures 2A and 2C). At the same time, external neurons of cortical organoids were shown to successfully generate diverse cellular interactions and synaptic connections (Figures 2D and 2E). On day 14, external neurons formed a distinct self-organizing structure with robust nerve tracts (projections) and laterally intertwined neuronal connections (Figure 2A).

Figure 2.

Cells inside cortical organoids migrate outward and self-organize based on organoid cell components

(A) The bright-field images of 124-day cortical organoids with attachment culture at the air-liquid interface for 3, 7, and 14 days.

(B) Co-staining of Calcein-AM (live cell) and Hoechst (nuclei) of a 60-day cortical organoid with attachment culture at the air-liquid interface for 3 days.

(C) The calcium indicator Flou-4 images of 130-day cortical organoids with attachment culture at the air-liquid interface for 5 days. Cortical organoid external neural networks generate an organized structure of nerve tracts (projections).

(D) Immunohistochemical staining of 45-day cortical organoids attachment cultured at the air-liquid interface for 7 days reveals diverse cellular interactions. An example shows astrocytes (GFAP) interact with neurons (TUJ-1).

(E) Immunohistochemical staining of 45-day cortical organoids attachment cultured at the air-liquid interface for 7 days reveals the establishment of synaptic structures between neurons. Presynaptic (Syn), postsynaptic (PSD-95), and dendritic (MAP-2). The imaging area from cortical organoid external neural networks. Yellow arrowheads point out representative presynaptic and postsynaptic puncta.

All of the yellow dashed boxes correspond to the magnified areas.

We performed calcium imaging on cortical organoid external neurons and analyzed the data using the software developed by F.D.W. Radstake et al.⁷ The experimental data show the cortical organoid external neuronal network has proper electrophysiological functions, and the activities between neurons have obvious synchronization (Figure 3A). We also give an example of the same-region calcium imaging and immunohistochemical (IHC) cross-analysis, which realizes the subcell-resolution component differentiation on the electrophysiological activity recording area (Figure 3B).

Figure 3.

Same region calcium imaging and immunohistochemical cross-analysis method

(A) Calcium imaging area and analysis process. The exemplified sample is a 190-day cortical organoid with attachment culture at the air-liquid interface for 7 days. Seven regions of interest (ROIs) were selected for an example spike identification process, details in ref. 7. All regions of interest (ROIs) and their spikes event corresponding frame were jointly plotted into a raster plot, red dots represent spike occurrences, showing synchronization in the recorded regions. The total recording time is 6.5 min (6.5 s/frame). Activation rate = all ROIs have spikes / all ROIs. (The yellow dashed line area is only for showing the imaging area, and does not represent the magnifying image of the same sample).

(B) An example of tracking calcium imaging region on immunofluorescence imaging shows subcell-resolution component differentiation on the electrophysiological activity recording area.

Compared to the interior of cortical organoids, external neurons have superior optical imaging conditions (Figures 2B–2D). At the same time, it also partially reproduces the cellular components and interactions inside the cortical organoids (Figures 2D and 2E). Fixed attachment cultures also provide a prerequisite for the tracking of corresponding regions for cross-analysis (Figure 3B). All IHC primary leave-out negative control have shown (Figure 4).

Figure 4.

All IHC primary leave-out negative control in main figures

(A) Primary leave-out negative control of Figure 2D.

(B) Primary leave-out negative control of Figure 2E.

(C) Primary leave-out negative control of Figure 3B.

Despite the after-mentioned limitations, this protocol provides a more stable fluidic environment and fixes the relative position of cortical organoids, compared to traditional air-liquid interface culture. This protocol is well suited for experiments that require frequent medium changes like drug testing, as well as electrophysiological recordings that require component analysis at the subcellular level by IHC validation. If combined with microfluidic-based high-fidelity organoid culture techniques in the future, it is expected to fully overcome its shortcomings. Moreover, the same-region cross-analysis method presented in this protocol has a broad application prospect in neural network analysis, such as synaptic function quantification and excitatory-inhibitory network balance. Thus, we believe that this is a supreme solution under the existing technical means and can be widely used in brain organoid-related research.

Limitations

Currently, this protocol is limited by the low volume of the medium. And daily medium change is required. Although the effect of this protocol on cortical organoid development was not determined, organoid activity was not compromised (Figure 2B). And it is not certain how the external neurons' network properties of these neurons compare to those present in organoids and traditional 2D iPSC-derived neuronal cultures. The specific differences need to be further verified and discussed.

Troubleshooting

Problem 1

The surface of the PDMS and the support surface of the sterilization box stick and damage each other during the sterilization process or it’s unable to dry between surfaces.

Potential solution

• •Change the sterilization box to reduce the contact area between the PDMS and the support surface of the sterilization box.
• •Use tin foil packaging for sterilization (distinguish the upper and lower surfaces of the PDMS ring).

Problem 2

The adhesion on the PDMS surface is poor.

Potential solution

• •Might the upper and lower surfaces are confused, try inverting the PDMS surface. (The upper surface of the PDMS ring is affected by the surface tension during the curing process, and its surface flatness and sealing performance are worse than the bottom surface.)
• •Adjust the mix ratio in steps 4a–4d to 11:1 or even 12:1, and lengthen the curing time in the oven until the PDMS is cured.

Problem 3

Failure to generate an air-liquid interface, media loss, and wells drying out.

Potential solution

• •For a perfect air-liquid interface and correct external neural network generation, the quality of the PDMS microwell preparation (steps 4–6) is very important. Make sure that the bottom of the microwell is well sealed (by adding coating solution) before seeding and check problem 2 solution.

Problem 4

After 24 h of seeding, the cortical organoids are not stably attached to the bottom of the microwells and generate external neural networks (Figure 5).

Figure 5.

Typical examples of neural outward migration at 24 h (1 day) after seeding

Potential solution

• •Check the organoid activity and the above operating procedures. In general, there is no significant improvement with prolonged culture at the air-liquid interface. Because under normal circumstances, there will be sparse outward migration of neurons that can be observed under a microscope 24 h after planting. If this does not occur, the organoids are usually necrotic before seeding, or the coated culture surface is not suitable for organoid attachment. Therefore, there is no need for a longer period of air-liquid interface culture. This phenomenon can be used as a simple condition for judging whether the experiment is successful or not. Typical examples of neural outward migration at 24 h after seeding are shown in Figure 5.

Problem 5

The bottom of the wells was found to dry out and the neurons died during daily fluid changes.

Potential solution

• •Pay attention to maintaining the humidity of the culture environment, add sterilized water into the gap of the multi-well plate, and check the remaining water volume in the water basin in the incubator.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Shaohua Ma (ma.shaohua@sz.tsinghua.edu.cn).

Materials availability

This study did not generate any unique reagents.

Data and code availability

This study did not generate/analyze datasets/code.