The overall goal of this study is to show how to implement automated procedures for culturing and differentiating human IPS cells into neuronal lineages, as well as their automated imaging. Standardized automated procedures allow low experimental variation while ensuring high phenotypic reproducibility. In addition, the system can be adapted for the development of new protocols.
Demonstrating the procedures will be Joachim Tager and Elisangela Bressan, post-docs from our lab. In the graphic user interface of the automated system, click resource instrument process view and select the resource instrument process. Click run instrument process and run the run HEPA hood and reloading processes.
Select the resources to be loaded, open the door and load the cell culture plates and disposable tips in the appropriate positions as indicated by the popup images. Then decontaminate the door with 70%ethanol before closing and run the decontamination process. The system will be sterilized by UV radiation for 30 minutes.
To load the plates with culture medium or dissociation reagent, execute the run HEPA hood step and open the door of the liquid handling station. After decontamination with 70%ethanol, place the reservoirs on the deck to the assigned position and de-lid them. Close the door of the liquid handling station, then run the InitHepaHood resource instrument process to power down the HEPA hood.
To execute an automated culture method, in the calendar view of the graphic user interface, click add process step and select the cell line to be used in the experiment. Use the wizard to select the project and mark the batch to be used. Click the right facing arrowhead and navigate to the next page of the wizard to select the process step to be executed.
In the last page of the wizard, schedule the experiment and set the appropriate parameters details variables necessary for running the method. Then click OK.After loading a 50 milliliter tube with the human IPS cell suspension, load coated culture plates for receiving the cells on the shelf and execute the seeding of plates from tubes method. To count the cells in the Brightfield imaging cytometer, automatically prepare a 384-well counting plate with cell suspension on the deck and use the robotic arm to transfer the 384-well counting plate from the deck to the Brightfield imaging cytometer.
After counting, the system will return the plate to its original position and transfer a coated culture or assay plate from the shelf to the pipetting deck. The system will then seed the cells in the user-defined number and volumes suitable for the plate and mix by pipetting. After seeding, the plate will be moved to the on-deck shaker for 10 seconds at 500 revolutions per minute for cell distribution before being transferred to the carbon dioxide incubator.
To assess the automated confluence, execute the check confluency method and select a batch that contains at least one culture plate and no assay plates. In the parameters detail section, enter iPSCf_2020 for image acquisition in the imaging analysis settings. The system will then transfer the first plate from the incubator to the Brightfield imaging cytometer and image the cell confluence.
To change cell culture or assay plate media, execute the media change of culture plates method and select a batch containing only culture plates. The system will transfer the plates to the deck and tilt the plates to allow aspiration of the supernatant. The supernatant will be discarded to the waste collection module and discard the tips and 12 milliliters of fresh medium will be added to each plate.
The system will then re-lid the plates and return the plates to the cell culture incubator. Alternatively, to change medium of assay plates, execute media change of assay plates method. To subcultivate the cells, execute the subcultivation of adherent cells method and select the batch containing the culture plates that need subcultivation.
After discarding the medium, the system will wash the cells one time with eight milliliters of PBS per plate before adding eight milliliters of 0.5 millimolar EDTA to the cells. After eight minutes on the deck with tilting option, the EDTA will be replaced with 12 milliliters of fresh medium per plate. The system will shake the plates at 2, 000 revolutions per minute for one minute to dislodge the colonies before triturating the cells with five cycles of pipetting to break up the colonies into 50 to 80 micron clumps.
The cells will then be transferred to a 50 milliliter tube on the deck before seeding them in a one-to-seven split ratio. For automated high content, high throughput cell imaging, execute the imaging method and select a batch that contains at least one assay plate and no culture plates. The system will then transfer an assay plate to the automated confocal microscope for imaging.
Be sure to select the correct batch and plate IDs while performing the required process steps. The hiPSC cultures should be monitored daily for growth and analyzed for their percentage of confluency in the Brightfield imaging cytometer. After passaging and manual or automated system culture, the cells exhibit a typical stem cell and pluripotency marker expression.
Neurons differentiated in the automated culture system demonstrate a similar morphology and neuronal network organization to neurons cultivated manually. After six days of differentiation, automatically-differentiated cortical neurons are positive for neuron-specific class III beta-tubulin and upper cortical layer marker expression. After eight days, the cells also expressed microtubule-associated protein 2, the neural cell adhesion molecule, and synapsin I, as well as cortical neuron markers.
Very low or no expression of these markers is observed in hiPSC. This system can also be used to establish a live cell automated neurite outgrowth assay that allows neurite lengths to be measured over 11 days of differentiation without manual intervention. Using the automated culture system to perform medium changes over a 65-day culture period facilitates hiPSC differentiation into midbrain dopaminergic neurons with the expected cellular organization and morphology.
In addition to the neurite outgrowth, other automated phenotypic assays relevant to study neurodegeneration, for example, TDP-43 translocation RNA foci and alpha-synuclein fibril uptake, can be explored using this high throughput, high content format.
