Culturing Human Pluripotent and Neural Stem Cells in an Enclosed Cell Culture System for Basic and Preclinical Research

The overall goal of using this equipment is to grow stem cells in defined atmospheric conditions with maximum sterility. What we're going to demonstrate today is the use of an enclosed cell culture facility that allows for exquisite control over cell culture parameters. In our laboratory, we're using this facility for the culture of multipotent stem cells and pluripotent stem cells.

The main advantage of this method is that the cells are exposed to a constant environment no matter where they are as you process them. Whether it be manipulating the cells or in the incubator or on the microscope, their exposure to oxygen CO2 in temperatures constant throughout the cycle. Demonstrating the procedure will be Alexander Stover, a staff scientist in my laboratory.

To begin this procedure, click on a module to adjust the gas concentration. In the new window displaying the current settings, click on the existing oxygen setpoint value below Setpoint and enter the required oxygen concentration for this module. Next, click on the green check mark to confirm the setpoint.

Repeat this state for carbon dioxide and enter a setpoint of 5%carbon dioxide for all modules except for the buffer chambers which are only adjustable for oxygen. Then, monitor the current gas concentrations which are labeled as Process Values to make sure that they reach the new setpoints. Afterward, adjust the gas setpoints of all modules to the appropriate values.

Match the carbon dioxide and oxygen levels of the chambers that will be exposed to one another. For example, adjust the process chamber to 5%oxygen before opening an incubator that grows cells at 5%oxygen. Also adjust the buffer chamber that is used to add items to the process chamber to 5%oxygen.

Subsequently, set the temperature of the process chamber to 37 degrees celsius using the same screen for gas adjustments. After that, set the process chamber floor temperature to 37 degrees celsius. Click on the incubation module banks underneath each incubator and adjust the temperature of the banks to 37 degrees celsius.

In the Laminar Hood, spray all materials being entered into the system with 70%ethanol and allow them to dry. Wipe any flasks or plates of cells gently with a sterile gauze sponge saturated with 70%ethanol. Next, make sure both the outer and inner doors of the buffer chamber are securely closed, then open the outer door and place the items inside before closing it.

Using the software, select the buffer module and click on the Dilution Factor tab. Enter one into the Log Factor box followed by clicking start. Once the graphical interface has stopped flashing Dilution Factor, open the buffer chamber's inner door and transfer the materials to the process chamber.

In order to remove items from the process chamber, first ensure that the buffer chamber has undergone a Dilution Factor. Then, open the inner door, place the items to be removed inside, and close it. After that, open the outer door and remove the items.

Place three petri dishes in the water pan at the base of each incubator. Fill these dishes with sterile water and maintain the water level in these dishes as they critical for maintaining the relative humidity setpoint in the incubator. Within the graphical interface, click on incubator.

Subsequently, click on the existing value for relative humidity under the setpoint and enter 85%Then, click on the green check mark to accept this value. Next, adjust the buffer chambers, the process chamber, and an incubator to the gas setpoints required for the type of cells being introduced. Clean the surface of the process chamber with sterile gauze and a non-flammable disinfectant such as benzalkonium chloride-based product.

Then, clean the gloves and surfaces that are commonly touched such as door handles. Subsequently, clean the surface of the Laminar Flow Hood and buffer chamber with 70%ethanol and allow it to dry. After that, place the flask or plate of cells in the hood and briefly wipe its exterior surface with a sterile gauze sponge saturated with ethanol.

Then, put the flask or plate in the buffer chamber and run a Dilution Factor. Upon completion, immediately transfer the flask or plate to the appropriate incubator. As the incubator is at the back of the process chamber, pull a shelf out into the process chamber for cell placement and avoid opening the incubator doors unnecessarily for extended periods of time.

In this step, prepare the cell culture medium. Allow the medium to equilibrate with the carbon dioxide and oxygen levels present within the system by leaving the media container slightly uncapped for 20 minutes. Afterward, remove the old medium from the wells.

For pluripotent stem cells, remove almost all of the old medium with a small amount of the old medium remaining to prevent desiccation during the feeding process. For neural stem cells, remove half of the old medium. Then, add fresh medium to the cell culture plates and place the plates back into their designated incubator.

Shown here is an image of the iPSCs. Here are the iPSCs live stained with AF594 labeled Trial 160 with a one to 100 dilution in media. All the iPSCs were transduced in the cell production facility in 5%oxygen with Sendai virus.

And here is the phase-contrast image of iPSC-derived neural stem cells which were grown at 5%oxygen in chamber slides. Cells were then fixed in 4%paraformaldehyde and stained with Anti-Human Nestin primary antibody and Alexa Fluor 488 secondary antibody. Once mastered, this set of techniques can be done in two hours if it is performed properly.

While attempting this procedure, it is important to remember to correctly set the gas concentrations before beginning. Following the procedure, other methods like reprogramming and differentiation can be performed in order to answer additional questions such as the affect of hypoxia on pluripotency or multipotency. After its development, this technique paved the way for researchers in the field of stem cell biology to explore clinical cell production for the treatment of pediatric neurodegenerative disorders.