Production of Human Neurogenin 2-Inducible Neurons in a Three-Dimensional Suspension Bioreactor

Using bioreactors to translate induced pluripotent stem cells or IPSC culture protocols from 2D to 3D enables the generation of cells in high numbers for large-scale applications such as high-throughput screenings. This fast neuronal differentiation protocol in a physiological 3D environment improves starter cell interactions and gives high cell yield over a shorter duration with a low batch-to=batch variation thereby reducing cost and time. The European Bank of Induced Pluripotent Stem Cells is applying similar approaches for the rapid and cost-efficient generation of differentiated cells across multiple lineages, including other brain cells and cardiomyocytes.

Begin pre-cultivation when the human-induced pluripotent stem cell, or human iPSC culture, is 60 to 80%confluent. Aspirate the medium completely from the human iPSCs and gently rinse the cells with 1X DPBS twice. Add two milliliters of prewarmed trypsin EDTA solution to the six-centimeter Petri dish and incubate the cells for three minutes at 37 degrees Celsius in an incubator.

Then, gently tap the dishes to facilitate cell detachment or incubate for one to two minutes longer if cells are not getting detached. Then, resuspend the cells in each dish by adding five milliliters of feeder-free IPSC maintenance medium with ROCK inhibitor. Transfer the cell suspension to a 15-milliliter or 50-milliliter tube and mix gently by pipetting to ensure cell singularization.

Determine the cell numbers in 100 microliters of cell suspension using an automated cell counter and transfer the desired volume of cell suspension into a 50-milliliter tube. Centrifuge the cells at 300 G for three minutes. Next, aspirate the supernatant and resuspend the cells in two milliliters of feeder-free IPSC maintenance medium with ROCK inhibitor.

Fill each 50-milliliter tube with 18 milliliters of the medium. Then, dispense 20 milliliters of cell suspension into each bioreactor tube. Place the tubes into the bioreactor system and set the cultivation parameters for an unlimited duration.

Start the pre-cultivation program via the bioreactor display. To change the media the next day, let the aggregate settle in the bioreactor tubes for about five minutes before carefully aspirating the supernatant. Add 15 milliliters of fresh feeder-free IPSC maintenance medium without ROCK inhibitor per tube and continue the cultivation in the bench-top bioreactor for 24 hours.

To start neuronal differentiation, let the aggregate settle in the bioreactor tubes and carefully aspirate the supernatant from the cells, leaving about five milliliters of supernatant in the tube. Then, add 35 milliliters of neural induction medium consisting of neurobasal medium and two micrograms per milliliter of doxycycline. Place the tubes back into the bench-top bioreactor and continue cultivation.

Perform media changes every day for two days, as demonstrated previously. After aspirating the supernatant as previously shown, transfer the aggregates to a sterile 15-milliliter or 50-milliliter tube and gently rinse the aggregates two times with DPBS. Carefully aspirate the supernatant as much as possible without disturbing the aggregates.

Then add two to five milliliters of prewarmed cell dissociation enzyme to the pellet, depending on the pellet size, and incubate the cells for approximately 10 minutes at 37 degrees Celsius in a water bath. Gently resuspend the sedimented aggregates every two minutes until the aggregates dissociate. Add prewarmed neurobasal medium, three times the volume of the previously added cell dissociation enzyme, and resuspend the cells carefully to ensure cell singularization.

Determine the cell numbers and transfer the corresponding volume of cell suspension for cryopreservation into a 15-milliliter or 50-milliliter tube. Centrifuge to the cells at 300 G for three minutes. Aspirate the supernatant and gently resuspend the cell pellet in the corresponding volume of the freezing medium containing 10%dimethyl sulfoxide.

Aliquot the cell suspension in suitable vials for cryopreservation. Transfer the vials immediately to a pre-chilled, slow-rate freezing container filled with 2-propanol and place the container at minus 80 degrees Celsius overnight. Place the vials at minus 150 degrees Celsius the next day for long-term storage.

After the adherent cultures of human iPSCs were detached, singularized, and transferred into suspension, aggregates formed within 24 hours and continued to grow. After two days of transgene induction, early neurons could be cryopreserved for subsequent maturation. Persistent proliferation of the human iPSCs during the initial days in suspension was observed, and the cell culture number peaked after two days of induction.

However, prolonged cultivation for more than four days in suspension did not improve cell yield, as the aggregates became increasingly resistant to enzymatic singularization. Further, compared to day zero, the aggregate diameter increased by 50%on day two and almost doubled on day five. Although increasing diameter limits nutrient supply to the aggregates, the viability of cells was not affected on day two or five of differentiation.

The temporal gene expression profile as well as immunochemical staining for the neuronal markers beta three tubulin and microtubule-associated protein, or MAP2, confirmed the neuronal cell identity of the day-two cryopreserved cells. Additionally, neuronal cultures were enriched in microtubule-associated protein tau transcripts, or MAPT, and showed a concomitant decline in the expression of the pluripotency regulating transcription factor POU5F1 A dense neuritic network was formed within the first week after thawing, which also suggested an increasing maturation of the neuronal cultures. This protocol lays the foundation for translation into large-scale and fully automated bioreactors, which show a further significant increase in cell output capacity for future large-scale applications.

Once proven with neurogenin 2, the use of linear determining transcription factors to accelerate differentiation has been applied to many different cell types and diseases to facilitate iPSC modeling.