We present for the first time, a new approach for reproducible and scalable generation, of IPS cells derived at Cerebellar Organoids, and a chemical defined conditions, using single use bioreactors. To obtain cells for seeding the bio-reactor, add one milliliter of cell detachment medium, to each well of a six well plate of human iPSC's. Incubate the plate at 37 degrees Celsius for seven minutes, until gentle shaking easily detaches the cells, from the well.
Using a P 1000 micropipette, pipet the cell detachment medium, up and down, until the cells dissociate into single cells. Next add two milliliters, of complete cell culture medium to each well, this will inactivate enzymatic digestion. Then use the pipette, to gently transfer the cells to a sterile conical tube.
Centrifuge the tube at 210 times gravity for three minutes. Remove the supernatant, and re-suspend the cell pellet in culture medium. Count the cells using a hemocytometer, and trypan blue dye.
See the bioreactor vessel with the iPSC's, at a density of 250, 000 cells per milliliter. Insert the bioreactor vessel, into the Universal Base Unit, in an incubator at 37 degrees Celsius, 95%humidity and 5%carbon dioxide. To promote iPSC aggregation, set the agitation rate of the Base Control Unit to 27 RPM.
On day one, with day zero being the day of seeding the bioreactor, place the bioreactor, and the Base Unit in a sterile flow hood, then use a serological pipette, to collect a one milliliter sample, of the cell suspension. Plate the cell suspension in an ultra low attachment, 24 well plate. Use a microscope, to confirm that iPSC derived aggregates have formed.
If so, capture images of the aggregates at 40 X or 100 X.Continue culturing the iPSC's and the bioreactor, until the average diameter of the aggregates, is 100 micrometers, then replace 80%of the medium, with fresh MT01 without rock inhibitor. When the aggregates, reach 200 to 250 micro meters in diameter, let all the organoids settle, at the bottom of the bioreactor, then replace all the spent medium, with gfCDM differentiation medium. Place the base unit and the bioreactor in the incubator and decrease agitation to 25 RPM.
After removing the supernatant from the stored organoids, add one milliliter of 15%sucrose to the pellet, and mix well by swirling gently. After incubating the organoids overnight, at four degrees Celsius, remove the sucrose solution, then add one milliliter of 15%sucrose, 7.5%gelatin, and quickly mix by gently swirling. While the organoids are incubating at 37 degrees Celsius fill a plastic container halfway with the 15%sucrose 7.5%gelatin solution, and allow it to solidify.
After the organoids have been incubating for one hour, use a Pasteur pipette, to place a drop of the organoid sucrose gelatin mixture, on top of the solidified sucrose and gelatin. After waiting 15 minutes for the drop to solidify, fill the remainder of the plastic container, with more 15%sucrose, 7.5%gelatin, allow it to solidify completely at room temperature and then incubate it for 20 minutes at four degrees Celsius. Cut the gelatin into a cube, with the organoids in the center, fix the cube to a piece of cardboard, with a drop of OCT compound, then placed 250 milliliters of Isopentane, in a 500 milliliter cup, fill an appropriate container, with liquid nitrogen.
Using forceps and thick gloves, carefully place the cup of Isopentane, on the surface of the liquid nitrogen. Isopentane and liquid nitrogens, are hazardous reagents. The use of these two reagents, require personal protective equipment, including thick gloves and an adequate ventilation.
When the Isopentane, has cooled to negative 80 degrees Celsius, place the gelatin cube in the Isopentane until it freezes. A well frozen cube, produces a metallic sound when lightly tapped with forceps, indicating that it is ready to be stored. Wash the microscope slides containing the organoid sections, with 50 milliliters 1X PBS for five minutes, then transfer the slides to a Coplin jar, containing fresh 1X PBS.
Transfer the slides to a Coplin jar, containing 50 milliliters of freshly prepared glycine and incubate for 10 minutes at room temperature, then transfer the slides to a Coplin jar, containing 50 milliliters of 0.1%Triton, and permeabilized for 10 minutes at room temperature. Wash the slides twice with 1X PBS, for five minutes each time. Prepare the immunostaining dish, with three millimeter paper, soaked in 1X PBS.
Dry the slides with a tissue all around the slices and place them on the three millimeter paper. With a Pasteur pipette, cover each slide with 0.5 milliliters of blocking solution. After incubating for 30 minutes at room temperature, remove excess blocking solution and drive the slides with a tissue, all around the slices.
Place 50 microliters of diluted primary antibody, on each slide and cover them with cover slips. Lace the slides, in the previously prepared immunostaining dish, and incubate them at four degrees Celsius. After overnight incubation, transfer the slides to a Coplin jar, with 50 milliliters of TBST, letting the cover slips fall off, then wash the slides three times with TBST, for five minutes each time.
Place 50 microliters, of diluted secondary antibody on each slide, and cover with the cover slips. Place the slides, in the previously prepared immunostaining dish, incubate for 30 minutes at room temperature, protected from light. Transfer the slides to a Coplin jar again, and wash them three times with 50 milliliters of TBST, for five minutes each time.
Using a Pasteur Pipette, at 0.5 milliliters of dappy solution, over the whole surface of each slide and incubate for five minutes at room temperature. After 24 hours in the bioreactor, iPSC's efficiently formed spheroid shaped aggregates. The morphology was well-maintained until day five, with a gradual increase in size.
Demonstrating a high degree of homogeneity. A quantitative analysis by microscopy, also revealed normal distribution, of aggregate sizes by day one, and both cell lines attained the optimal aggregate size by day two. After the desired aggregate diameter was achieved, neural commitment was induced, and then generation, of different cerebellar progenitors was promoted.
During differentiation organoids, showed a more pronounced epithelialization similar to neural tube-like structures with luminal space. Additionally, the organoid diameter distribution, was homogeneous during the initial cerebellar commitment until day 14. Immunofluorescence analysis supports, that an efficient neural commitment, of the iPSC derived organoids, is already achieved, by day seven of differentiation.
Immunostaining also demonstrated, efficient cerebellar commitment and efficient maturation of cerebellar organoids. Furthermore, after 80 days in the bioreactor, live dead staining of organoids showed high cell viability, and no evidence of necrotic areas. This technique represents an important tool, for studying pathological pathways, involved in the degeneration of the cerebellar observed in of and for evaluating the therapeutic effect of new drugs.
