With the growing interest in cell therapies, new methods to evaluate the functionality of these cells are needed. This protocol allows in vitro long-term assessment of these cell engraftment into a neural context. The main advantages of this technique is that all the components come from a human origin.
Also, the engraftment can be easily tracked, and the environment from the organoid can be easily changed, which allows testing different drugs or kind of treatments. Demonstrating this technique will be Iliana Ibanez, a talented PhD student in my lab. Begin by removing the single cell suspension of induced pluripotent stem cells or iPSC-derived neural progenitor cells, or NPCs, from the ice and centrifuging them at 300g for five minutes at 4 degrees Celsius.
Remove the supernatant and resuspend the cell pellet in 3 milligrams per milliliter of solubilized basement membrane matrix to obtain a final volume of two microliters per injection. Immediately place the suspension back on the ice until use. Transfer the pre-chilled syringe from the 20 degrees Celsius freezer into an ice bucket for further use.
Next, remove the brain organoid plate from the incubator and transfer the organoid into a 35 millimeter dish using a wide bore tip. To stabilize the organoids and facilitate the injection, remove the medium as much as possible without damaging the organoid. Place the 35 millimeter dish containing the organoid under a dissecting microscope to facilitate the injection.
Once the organoids are ready to inject, gently resuspend the EGFP labeled NPC cells using a chilled P20 pipette tip, and move two microliters of these cells onto a pre-chilled sterile glass slide. Take a pre-filled insulin syringe from the ice and slowly draw up the 2 microliters of cell suspension with the bevel of the needle facing down. Open the lid of the dish containing the organoid before focusing the microscope on it.
Hold the dish with one hand, place the bevel of the needle up. And with the other hand, inject the cell slowly into the organoid surface. After injecting the cells, place the lid back into the dish and incubate the injected organoid for one to two minutes.
Then, gently add 500 microliters of the organoid medium to the plate before transferring the organoid into a 24-well plate with a wide bore tip. Incubate the organoid at 37 degrees Celsius and 5%carbon dioxide with a complete medium change every second day. Load a non-injected or negative control organoid in a fluorescence microscope.
And set the illumination intensity between 1 and 2 and exposure time between 80 and 100 milliseconds for minimal autofluorescence. Next, load the positive control organoid and raise the exposure time to ensure that labeled cells are visible. Reposition the organoid with a wide bore pipette tip to find the enhanced green fluorescent protein or EGFP plus region for injection.
Remove the medium completely from the organoid before loading the organoid. And image it with the selected settings. Once the imaging is complete, immediately add a fresh medium to prevent the organoid from drying out.
After repeating the medium removal and imaging for all the organoids, return the organoids to normal incubation and medium changes. Repeat the imaging at desired intervals. Place the slide in a Coplin slide staining jar filled with toluene for two minutes, and repeat this step.
Then, transfer the glass slide to a glass Coplin slide staining jar filled with 100%ethyl alcohol for two minutes. And repeat this step before transferring the slide to a Coplin jar filled with water for two minutes. Repeat the wash with water.
Then, place a plastic container in the water bath, ensuring the plastic does not touch the bottom of the bath. Pour the citrate buffer inside the plastic container and let it reach 95 to 100 degrees Celsius. Once the buffer reaches the desired temperature, place the slides inside the buffer, and loosely cover the container with a lid, leaving the slides inside the water bath for 30 to 40 minutes.
After incubation, remove the plastic container from the water bath and let it cool at room temperature for 20 minutes. Wash the slides three times for two minutes each with PBS. Remove the PBS with a paper tissue without touching the sample.
Prepare the permeabilization buffer and fill a glass slide staining jar with it. Immerse the slides in the permeabilization buffer and incubate for 10 minutes. Repeat three washes of PBS for two minutes each.
Then, prepare staining mix to cover each sample and add 25 microliters to the organoid slide before incubating the slide at room temperature for one hour. After incubation, repeat three washes of PBS for two minutes each, and remove the salts by rinsing the slide with distilled water inside a glass slide staining jar. Next, add 10 microliters of liquid mounts and DAPI to each sample before imaging the slide using the fluorescence microscope.
To the glass Coplin staining jar filled halfway with 2X quenching buffer, add 45 milliliters of PBS, and place the slides inside this jar. Incubate the jar overnight at 4 degrees Celsius. Use fluorescence microscopy to check if the fluorophores were effectively quenched before repeating the staining and imaging.
A clear dose dependence of GFP fluorescence was present on input cell number, with consistent EGFP plus cell patch detection at 10, 000 cells and above. The injected organoids and controls imaged for EGFP positivity showed the persistence of the injected site throughout the four month tracking period. Additional EGFP positive cell patches appeared nine days post transplantation and persisted until the study endpoint, indicating the migration of the cells and integration at their new sites.
At a higher magnification, clear neural morphology was observable with long projections into the organoid, confirming the integration of the injected cells. The initial single round staining confirmed the presence of EGFP plus cells at the injection site, including a mixture of cells retaining NPC status, and those that had differentiated towards a neural fate. For the control and injected organoids, very few Nestin plus NPCs were observed, with a majority of TUJ1 plus immature mature neurons.
The two round staining gave more detail, revealing mature neurons around most of the outer region of the organoid, with areas of immature neurons toward the middle. Astrocytes were present in the injected and control organoids, and were interspersed around the outer edges. The slice for which the two round staining was performed in the injected organoid showed a small satellite colony of EGFP plus cells far from the injection site that had adopted the phenotype of mature neurons.
Some of these colonies appear to be near astrocytes. However, no EGFP plus cells with complete overlap to the GFP staining suggested that they were adjacent rather than generating the astrocytes themselves. When you are injecting the organoid is that you have to avoid applying too much force or doing it too fast, as you can destroy the organoid completely.
So, after life tracking, the organoids can be associated and used for flow cytometry analysis, or single cell sequencing approaches. This will allow us to know the molecular state of the cells. This technique could be extremely useful for advancing the cell therapy for neurodegenerative diseases, also for modeling brain tumors or metastasis.
