This article details a method for injecting fluorescently labeled induced pluripotent stem cell-derived neural progenitor cells (NPCs) into human cerebral organoids. The technique minimizes tissue disruption and allows for effective cell transplantation, which is crucial for neural cell therapy.
Begin with fluorescently labeled induced pluripotent stem cell-derived neural progenitor cells or NPCs.
Resuspend the cells in an extracellular matrix hydrogel to create a uniform cell-matrix mixture.
Incubate the mixture on ice.
Next, transfer a human cerebral organoid into a dish.
Remove excess media and place the dish under a microscope for the injection procedure.
Dispense the cell-matrix mixture onto a pre-chilled, sterile glass slide, and aspirate it using a pre-chilled syringe.
Slowly inject the mixture onto the organoid surface to minimize tissue disruption.
Let the organoid rest briefly, then add media, transfer the organoid to a multi-well plate, and incubate.
The hydrogel solidifies, anchoring the NPCs at the injection site. Track the NPCs using fluorescence microscopy.
Over time, the NPCs proliferate and migrate to distant regions of the organoid, where they differentiate into immature and mature neurons.
This enables reliable cell transplantation with minimal organoid disruption, suitable for neural cell therapy.
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 percent carbon dioxide with a complete medium change every second day.
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