Our protocol combined several techniques that have proven effective for long-term organoid maintenance. This is useful for studying aging in age-related diseases where clinical manifestations are outside the developmental period. The main advantage of this technique is that it does not require any specialized equipment or reagents.
It's heavily based on materials commonly found in the lab. Aging and neurodegenerative diseases have become a particular point of interest as our current modeling methods mostly focus on established disease phenotypes rather than the early developmental phases. Embryonic stem cells and iPSCs are very delicate cells.
First-time users should be gentle and have patience. Since these are long-term cultures, one should be patient from the beginning To begin, spray 70%ethanol on a suture strand and prepare PLGA microfilaments by fraying the suture strand with the blunt end of the scalpel. Place the fiber under a microscope.
And using a ruler, begin cutting the fiber into fragments of 500 micrometer to one millimeter long strands. Cut about 25 millimeters of the fiber in total and add the filaments in a 15-milliliter tube containing one milliliter of Antibiotic-Antimycotic solution. Next in the hood, dilute the fiber solution with 10 milliliters of DMEM/F-12 and vortex to mix well.
Then, add 20 microliters of the fiber solution to three wells of a 96-well plate. Using a bright-field microscope, count and average the fibers per well. Dilute or concentrate the wells with the fiber solution to average 5 to 10 microfilaments per well.
Once the previously cultured induced pluripotent stem cells have reached 70 to 80%confluency, check the cells under a microscope at 10 or 20x magnification. Ensure the colonies display less than 10%area of spontaneous differentiation. Aspirate the medium with a pipette and wash the cells once with DPBS.
Then, add 500 microliters of cell detachment solution or 0.5 millimolar EDTA to dissociate the colonies, and incubate at 37 degrees Celsius for three to five minutes. Next, add one milliliter of fresh E8 media to each well and pipette gently to detach all cells. Transfer the entire cell suspension into a 15-milliliter centrifuge tube, and add another one milliliter of fresh E8 media.
Then, spin down the cells at 290 g for three minutes at room temperature. After discarding the supernatant, resuspend the cell pellet in one milliliter of the E6 media supplemented with 50 micromolar ROCK inhibitor and count the cells using a hemocytometer. Prepare a cell suspension of the desired seating density in E6 media supplemented with the ROCK inhibitor.
Then, add 150 microliters of the cell suspension into each well of a 96-well ultra-low attachment plate. Next, centrifuge the plate at 290 g for three minutes at room temperature to force aggregate the cells. Then, incubate the plate at 37 degrees Celsius in a humidified atmosphere with 5%carbon dioxide for embryoid body formation.
After 24 hours of incubation, transfer the plate to the hood and carefully aspirate 120 microliters of the media using a pipette, taking care not to aspirate the embryoid body by lowering the pipette tip too far into the well. Next, add 150 microliters of fresh E6 media supplemented with two micromolar XAV939, and the SMAD inhibitors to each well. Transfer the plate back to the incubator and replace the medium daily with freshly prepared E6 supplemented with the inhibitors.
At day seven of incubation, check whether all the embryoid bodies have reached a diameter of 550 to 600 micrometers and display a smooth, clear edge. At this stage, they are ready to be embedded in an extracellular matrix. To embed the organoids, prepare dimpled embedding sheets by placing a four-inch long thermoplastic ceiling film sheet on an empty P200 box.
Then, use a 15-milliliter conical tube or a 500-microliter micro-centrifuge tube and gently press down the film sheet into the holes to make 12 or the desired number of dimples. Next, spray 70%ethanol on the film sheet and let it dry inside the hood with the UV light on for at least 30 minutes. Meanwhile, thaw a sufficient quantity of basement membrane matrix on ice.
Then, using a wide bore P200 tip, transfer one embryoid body from the 96-well plate to each dimple. Remove as much media as possible with a normal pipette tip but do not let the embryoid body dry. Next, with a regular pre-chilled P200 tip, add approximately 30 microliters of undiluted membrane matrix to each organoid, ensuring that the embryo body is as close as possible to the center of the droplet.
Once all embryoid bodies are embedded in the matrix, place the film sheet in a sterile Petri dish. Then, incubate the Petri dish at 37 degrees Celsius for 10 minutes in a humidified atmosphere with 5%carbon dioxide to allow the membrane matrix to solidify. For organoid differentiation, add five milliliters of the prepared differentiation media with B-27 without vitamin A to one well of an ultra-low attachment 6-well plate.
Prewarm the plate to 37 degrees Celsius in an incubator. Once the membrane matrix has solidified, transfer the embedded embryoid bodies to the 6-well plate by pushing out the dimples from the back of the film sheet. If necessary, use one milliliter of media from the well and pip it onto the sheet to detach the droplets from the film sheet.
The embedded embryoid bodies in the membrane matrix differentiated towards cerebral organoids displaying distinct budding formations by day in vitro 10. At day in vitro 30, organoids are further matured using either dimple or sandwich embedding. Long-term culture shows cerebral organoids grown to significant sizes.
At day in vitro seven, embryoid bodies display SOX2 positive neural rosettes, scattered immature neurons, and neural progenitor cells. Whereas by day in vitro 120, they exhibit mature neuronal markers such as MAP2 and NeuN. These cytoskeletal markers can be used in conjunction with other postmitotic markers such as doublecortin and synapsin to probe for synaptic plasticity and other age-related declines, as well as additional brain tissue like astrocytes and glia.
Ensuring that the edges of the tissue remain intact is critical. To minimize harmful shear force, piping should be done slowly, for example, while changing media and organoid embedding. The slowed organoid growth allows us to study the progress of disease in early cellular manifestations.
Organoid creation paves the way of early detection in treatment for many diseases.
