The overall goal of this protocol is to provide neurobiologists and neurovirologists with an easy, reproducible method to generate differentiated human neurons for the use in subsequent in vitro assays. This method can help to answer key questions in infectious disease research, such as determining the neuronal responses to infection or drug treatment. It can also be used to assess proteomic or genomic changes associated with specific experimental conditions.

The main advantage of this technique is that it provides an easy to follow protocol that reproducibly yields homogenous cultures of fully differentiated human neurons. Generally, individuals new to this method will struggle, because of the sensitivity of these cells to distruptions, such as tripsinization, triteration, or exposure to suboptimal environmental conditions. To begin this procedure, allow the media to warm and equilibrate in the incubator, in order to establish a proper pH balance before use.

Then, split the cultures of the maintenance phase when the cells reach 70 to 80%confluency. To passage the cells from a T-75 flask, aspirate off the media. Then, rinse the flask with 10 milliliters of 1X PBS.

Next, aspirate the PBS, and add 2.5 milliliters of 0.05%or 1X tripsin EDTA. After that, incubate the cells for two to three minutes. Then, tip the flask gently to release the cells from the surface.

Subsequently, add 10 milliliters of basic growth media and triterate the cells one to two times. Next, spin bound the cells for two minutes at 1000 times G.Afterward, aspirate the media. Resuspend the palate in five milliliters of basic growth media.

For normal plating in the T-75 flask, dilute the cells to a total volume of 20 milliliters, or count and plate the cells for differentiation. Here is the schedule for differentiation. On day zero, count the cells using a hemocytometer.

Then, dilute the cell suspension to 50, 000 cells per milliliter, using basic growth media. Plate two milliliters of cells per 35 millimeter dish, for a total of 100, 000 cells per dish. Subsequently, place the cells in the incubator at 37 degrees Celsius.

On day one, add RA to the warmed and equilibrated media. Gently aspirate the old media and discard. Next, add two milliliters of differentiation media number one, with RA, to each 35 millimeter dish, and return to the incubator.

On day seven, gently aspirate the old media and discard. Add 200 microliters of warmed tripsin EDTA to each 35 millimeter dish. Then, place the cells in the incubator for two to three minutes, or until they are visibly lifted from the plate, as observed under a microscope.

Quench the tripsin by adding two milliliters of differentiation media number one, with RA to each 35 millimeter dish, and use the media to rinse the remaining neuronal cells off the plate. Then, transfer the full volume to a 50 millileter conical tube. Gently triterate up and down slowly with a 10 milliliter plastic pipette no more than five times.

After that, aliquot two milliliters of the cell suspension into the fresh 35 millimeter dishes, before returning them to the incubator. On day 10, gently aspirate off the media and discard. Add 200 microliters of warmed tripsin to each 25 millimeter dish, and incubate at room temperature for approximately one to two minutes, or until the neurons are visibly lifted from the dish as observed under a microscope.

Day 10 of the protocol is particularly difficult, because of the sensitivity of the cells at this stage of differentiation. It is imperative to minimize the time the cells are in tripsin to approximately one minute at room temperature, and then put the cells immediately back into the incubator. Then, quench the tripsin by adding two milliliters of differentiation media number two to each 35 millimeter dish, and use the media to rinse the remaining neuronal cells off the plate.

Afterward, transfer the contents to a 50 milliliter conical tube. Gently triterate up and down slowly with a 10 milliliter plastic pipette, no more than five times. Dispense two milliliters of cell suspension into ECM-coated 35 millimeter dishes before returning them to the incubator at 37 degrees Celsius.

On day 11, gently aspirate off the old media and discard. Slowly add two milliliters of differentiation media number three with RA to each 35 millimeter dish, before returning them to the incubator. On day 18, change the media to fresh differentiation media number three with RA once every three days to maintain neuron health.

Do not allow neurons to be exposed to air for an extended period of time. After seven days of differentiation, there is an increase in the number of processes on the neuronal-like cells, and there are fewer epithelial-like cells present. Following the first passage, the neuronal cell bodies clump together, and their processes appear shorter, highlighting the stress that the cells encounter during tripsinization and replating.

Prior to plating onto the ECM-coated plates on day 10, longer processes are evident on the differentiating neurons. On day 11, following the final passage, the neurons are visibly stressed. This can result in an apparent decrease in the total number of cells, and clumping of the remaining cell bodies.

However, the cells will recover over the next seven days. On day 18, the cells are terminally differentiated into neurons that have healthy, elongated projections. While attempting this procedure, it's important to remember to limit the amount of time the cells are in tripsin or exposed to air.

It is also important to ensure that all reagants are always prepared fresh. Following this procedure, other methods, such as infection with a pathogenic agent, can be performed to assess subsequent changes in morphology, transcript levels, or protein expression. Thanks for watching, and good luck.