In this protocol we detail an in vitro model of human tauopathy. This method fills a materials need which may be useful for drug screening and disease mechanism studies. The main advantage of this technique is that the cell culture paradigm allows for a more rapid assessment of tau toxicity and potential tau therapeutics compared to animal studies To begin this procedure thaw the basement membrane matrix coating for culture plates at four degrees Celsius.
Make one-milliliter aliquots and store them at minus 20 or minus 70 degrees Celsius. Next, reconstitute the basic fibroblast growth factor in sterile PBS at a concentration of 10 micrograms per milliliter. Make 10-microliter aliquots and store them at four degrees Celsius.
Then set out a new unopened 500-milliliter bottle of DMEM-F12 with glutamine. Add 10 milliliters of B27, five milliliters of N2, and five milliliters of penicillin-streptomycin to prepare neural stem cell media. Place 50 milliliters of the NSC media into a conical tube, and add 10 microliters of the prepared bFGF.
Store this NSC plus bFGF media at four degrees Celsius. First, remove an aliquot of frozen basement membrane matrix coating for cell culture plates from the freezer and it allow to thaw at four degrees Celsius. Add 385 microliters of the thawed basement membrane matrix coating to five milliliters of DMEM-F12 media containing penicillin-streptomycin.
If cells are being thawed from frozen NSC stocks, take a vial of frozen cells from the freezer and warm it in a water bath heated to 37 degrees Celsius by moving the vial back and forth in the water. After this, spray the vial with 70%ethanol. Then under a cell culture hood, transfer the cells to approximately 10 milliliters of DMEM-F12 media containing penicillin-streptomycin.
Centrifuge at room temperature at 1, 000 times g for five minutes. Then aspirate the media and resuspend the cells in NSC media. Dilute the cells in NSC media to obtain the appropriate seeding density for the cell culture vessel that is being used.
Add enough cells suspended in NSC media to seed the basement membrane matrix coated culture dishes. Grow the cells at 37 degrees Celsius with 5%carbon dioxide until they are 75 to 80%confluent, making sure to change the media every other day. To transduce neurons with lentivirus, use a titer count of 340, 000 transducible units per cell.
Dilute the transducible units in cell culture media to the necessary concentration, and add them to the cells. Two days after adding the lentivirus, wash the cells once with fresh media that does not contain bFGF. Continue culturing the cells at 37 degrees Celsius with 5%carbon dioxide in media without bFGF.
Maintain the cells for about eight weeks after transduction, making sure to change the cell culture media every other day. Use a light microscope to routinely visualize the cells and ensure viability. After transduction, remove the culture dishes from the incubator making sure to keep the lid on them to keep the culture well sterile.
Use a fluorescent microscope capable of live cell imaging to visualize the cells under a 10X objective with an excitation of 514 nanometers and an emission filter of 527 nanometers. In this study neurons transduced with tau-RDLM-YFP lentivirus are fluorescently tagged with YFP. RDLM-transduced cultures are seen to display aggregates after transduction.
These inclusions stain positive for thioflavin. Neuronal differentiation is confirmed by immunolabeling of the neuron-specific marker beta-tubulin III in cultures. It is important to remember that fluorescently tagged secondary antibodies should have an excitation emission spectrum that does not overlap with that of YFP.
Although yellow fluorescence tau aggregates will be visible in the absence of staining, thioflavin should also be used for imaging in order to confirm that the fluorescent signal is aggregated tau and not cellular debris. Following generation of tau overexpressing cells, a number of assessments of neuronal health and tau aggregation can be performed. For example, we have found that these cells display axonal degeneration.
In addition to cell culture studies, we have used this method to examine the pathogenicity exosomal-derived tau.
