To date, the cerebral organoids differentiated from pluripotent stem cells is an vitro 3D model closest to the native human brain tissue. Additionally, cerebral organoids are equally suitable for modeling various central nervous system pathologies, testing pharmacologically-active substances, and also for use in regenerative medicine. Meanwhile, this technology is still at the initial stage of development.
The protocol can be divided into two groups depending on the method of obtaining aggregates and the further cultivation with differentiation. The first includes protocol and schemes of differentiation, different in the cultivation time and differentiation inducers with subsequent maturation of organoid in the shaker in special low-adhesion devices like market tubes or plates. Another group includes using a special bioreactors.
Analysis of various protocols has shown that organoids should be cultivated under conditions with circulating nutrient medium. However, the absence of simple and inexpensive systems for obtaining organoid with a standard size and morphology require the development of such devices for scaling the process. In this work, we describe our method for obtaining and testing simple and inexpensive mini bioreactors that make it possible to obtain large quantities of high-quality organoids.
Cut the sterile 15 milliliters centrifuge tubes into the rings, 7 or 8 millimeters in height. Autoclave the rings. Break low-aggregations on treated or microbiological Petri dishes into crumbs.
Dissolve about 1 grams of plastic crumbs in 10 milliliters of chloroform overnight to prepare a liquid plastic. Make a plastic knob in the center of a sterile ultra low-adhesion 6 centimeters Petri dish. There are two equally suitable ways.
The first, put the autoclave in a plastic ring on a center and apply half milliliters of liquid plastic to the inside of the ring. The second, without any plastic rings drop half milliliters of liquid plastic on the center of the Petri dish. Leave the dishes open for 2 or 3 hours in a laminar flow hood until complete dryness.
Cultivate induced pluripotent stem cells in the medium for pluripotent stem cells, up to 75 or 90%confluence in the 35 millimeters Petri dishes, pre-coated with a matrix dissolved in cold Dulbecco Modified Eagle Medium, and Fisher-12 medium. Prepare medium A plus serum replacement. Cultivate induced pluripotent stem cells for 1 day in the medium with serum replacement.
Change medium for pluripotent stem cells to a serum replacement medium at the day zero of differentiation. Prepare medium A.Change the medium to medium A at day 2 of differentiation. Cultivate cells in medium A for 2 weeks, refreshing medium in Petri dishes every 2 day.
At day 14, start the formation of spheroid using a special 24-well culture plate which contains approximately 1, 200 microwells in each well. Prepare a 24-well culture plate with microwells. Add to each well 1 milliliter of medium A, centrifuge briefly at 1, 300g for 5 minutes in a swinging-bucket rotor fitted with plate holder.
Control under the microscope that there are no bubbles in microwells. Prepare the medium B.Remove the medium from the Petri dish. For the cell detachment, treat the cells with 1.5 milliliters EDTA solution prepared on PBS.
Control the cell detachment under the microscope. Harvest the cell into 15 milliliter tube. Add 5 milliliter mixed Dulbecco and Fisher medium in the tube towards the cells.
Centrifuge at 200g for 5 minutes. Remove the supernatant and re-suspend cells in 2 milliliter of medium B.Transfer the cells suspension containing 1 billion cells into each well of a 24-well plate with microwells. Gently pipette cells up to and down several times and centrifuge briefly 100g for 1 minute to capture cell in microwells.
Control under the microscope that the cell are evenly distributed in microwells. Incubate the plate overnight for cell aggregation in spheroids. The next morning, day 15, check under the microscopes the quality of spheroids, which are transparent and smooth if healthy.
Carefully collect the spheroids from each well into a 15 milliliter tube, leave the spheroids to precipitate by gravity for 2 and 3 minutes, and then remove the supernatant. Add to the spheroids 2 milliliters of the matrix, thawed and ex tempore on ice. Mix gently by pipetting and incubate at room temperature for 30 minutes.
To wash excess of the matrix, add to the tube 8 milliliters of medium B.Pipette gently, then centrifuge the tube for 1 minutes at 100g. Remove the supernatant. Add to the tube 20 milliliters of medium B, pipette gently, then split the spheroid suspension between mini bioreactors.
Then place the mini bioreactors into a 15 centimeter Petri dish, which prevents the evaporation of water and contamination. Put the Petri dish with mini bioreactors on a orbital shaker. Cultivate the organoids at rotation rate 70, 75rpm.
On day 16, prepare medium C.Transfer organoid into 50 milliliter tube. For 5 minutes, let them fall to the bottom. Aspirate the supernatant and add 5 milliliters of the medium C.Rejoin the organoids in the mini bioreactors.
Cultivate spheroids in medium C for 2 weeks, refreshing the medium every 2 days. During these 2 weeks, select about 100 spheroids per mini bioreactors for the following cultivation. On day 30, prepare medium D.Change cultivation medium to medium D, which is a maturation medium.
Refresh cultivation medium every 2 or 3 days for 3 weeks. Then use the medium D dissolved in F and GDNF. After 1 month of consecutive cultivation in medium A and B, organoids standardized in size and morphology are obtained.
As it grows, they need to change the medium more often, up to 4 times a week also increases. After 2 months of cultivation, they reach 4 and 5 millimeter in diameter. And after another month, 5 and 6 millimeter and the growth stops.
The figure shows the appearance of organoid enzyme, clear sections painted with hematoxylin and eosin. Immunohistochemical analysis for 1 month shows the presence of SOX2 positive clusters of progenitor cells, including inside central part. In the peripheral of the organoids, glial fibrillary acidic protein, microtubule-associated protein 2, and thiazine hydrolase positive.
Cells are concentrated, which corresponds to mature form of neurons. In some cases, clusters of whites on necrotic area are formed in the central part. This indicates the limitation of this protocol for obtaining larger organoids.
Most likely, the limiting factor of growth is the rate of diffusion of nutrient and oxygen. The various media and cell type of spheroids and testing our system for some kinds of organoids. Lots of brain and retina, combined organoids from liver carcinoma cells and mesenchymal stem cells, combined organoids from capital blast mesenchymal stem calls and the hematopoietic stem cells differentiation from induced pluripotent stem cells and intestine organoids.
Was it variable in the initial composition of cells? Differentiation factors, culture, and medium, extracellular matrix, and possibly also the gas mixture composition, the rate of the platform rotation, and other parameters. It will be possible to obtain organoid with the same shape, size, or morphology model in various organs and tissues.
The use of mini bioreactors proposed into this work.
