1. Preparation of Media and Reagents
- Thaw the basement membrane matrix coating for culture plates at 4 °C (do not allow the basement membrane matrix to warm up, or it will solidify). Make 1 mL aliquots and store them at -20 °C or -70 °C.
- Reconstitute basic fibroblast growth factor (bFGF) in sterile phosphate-buffered saline (PBS) at 10 μg/mL and make 10 μL aliquots. Store them at 4 °C.
- To a new, unopened, 500 mL bottle of DMEM/F12 with glutamine, add B27 (10 mL), N2 (5 mL), and penicillin-streptomycin (5 mL). Place 50 mL of this neural stem cell (NSC) media in a conical tube and add 10 μL of 10 μg/μL (2 μg/mL final) bFGF. Store the NSC (+) bFGF media at 4 °C.
- To make (-) bFGF media, use the same recipe as described in step 1.3 but do not add bFGF. This media will be used to differentiate NSCs to neurons and to maintain neuronal cultures after differentiation.
2. Lentiviral Constructs
NOTE: Before beginning work with lentiviral constructs, ensure that the lab has been approved to use biosafety level 2 (BSL-2) agents. Furthermore, BSL-2 culture hoods, personal protective equipment (PPE), and disposal methods must be used when working with lentiviral vectors.
- Obtain tau constructs packaged into lentivirus from a preferred source.
3. Culturing Human Neural Stem Cells
NOTE: NSCs are typically seeded at 100,000–150,000 cells/cm2 and most commercially available NSCs are sold as 1 x 106 cells/vial. This protocol has been optimized for 10 cm cell culture dishes (although other sizes of dishes may be used); therefore, if commercially available NSCs are being used, the NSCs may need to be expanded by first being cultured in six-well dishes in order to result in enough cells to seed 10 cm dishes. This protocol can alternatively be adapted for a variety of cell culture dish sizes.
- For the preparation of cell culture plates, remove one aliquot of frozen basement membrane matrix coating for cell culture plates and allow it to thaw at 4 °C (aliquots can be placed at 4 °C overnight the day before the cells are to be seeded). Add 385 μL of basement membrane matrix coating to 5 mL of DMEM/F12 media + penicillin-streptomycin.
NOTE: 5 mL of DMEM/F12 media + penicillin-streptomycin is enough to coat a single 10 cm dish (1 mL of this basement membrane matrix coating solution is sufficient to coat one well of a six-well dish). Alternatively, if the cells are to be fixed and immunostained, or stained for thioflavin, culture cells on glass coverslips in 24-well culture dishes.
- Keep the media and the matrix coating cold before and while adding them to culture dishes. Add the basement membrane matrix coating to culture dishes and place the dishes in an incubator (at 37 °C) for 1 h. For optimal coating, do not incubate the basement membrane matrix for more than or less than 1 h. After 1 h, aspirate the basement membrane matrix coating from the cell cultures dishes. Make sure this coincides with the NSCs being ready to be plated.
NOTE: If cells are not being thawed from frozen stocks, skip step 3.2 and continue with step 3.3.
- If cells are being thawed from frozen NSC stocks, take a vial of frozen cells and warm it in a water bath heated to 37 °C by moving the vial back and forth in the water. Once thawed, spray the vial with 70% ethanol and place it into a cell culture hood. Transfer the cells to ~10 mL of DMEM/F12 + penicillin-streptomycin media and centrifuge the tube at room temperature at 1,000 x g for 5 min. 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.
NOTE: For example, if NSCs are being plated onto a six-well plate—one well on a six-well culture dish has a surface area of 9 cm2—900,000 to 1,350,000 cells are required to seed. So, one vial containing 1,000,000 cells can be resuspended in 2 mL of media and added to a single well of a six-well dish.
- Add enough cells suspended in NSC media to seed the basement membrane matrix-coated culture dishes (100,000 cells/cm2) and change the media every other day (if using a frozen stock, change the media the day after plating and every other day thereafter). Grow the cells until they are 75%–80% confluent.
NOTE: The cells will likely reach 75%–80% confluence shortly after plating, but this may take longer if frozen stocks are used.
- Once the NSCs are 75%–80% confluent, begin neuronal differentiation by removing the NSC (+) bFGF media and replacing it with NSC (-) bFGF media. Culture the cells in this media (replacing the media every other day) for at least 4 weeks.
NOTE: The cells will continue to divide for a few days following the withdrawal of bFGF; therefore, cells may reach 90–100% confluence.
- After culturing for 4 weeks, the cells will have achieved a neuronal fate and will be ready for the treatment of lentivirus.
4. Transduction and Maintenance of Neuronal Cultures
- To transduce neurons with lentivirus, use a titer count of 3.4 x 105 transducible units/cell (a 100% confluent 10 cm dish will contain ~10 million neurons). Dilute the transducible units to the necessary concentration in cell culture media and add them to the cells (use the normal volume of media for the cell culture dish). Two days after adding the lentivirus, wash the cells 1x with fresh (-) bFGF media (without lentivirus) and continue culturing in (-) bFGF media as usual.
- For the post-lentiviral treatment, feed the transduced neurons by changing the cell culture media ([-] bFGF media) every other day. Maintain the cells for ~8 weeks after transduction. Routinely visualize the cells under a light microscope to ensure viability.
NOTE: Sparseness or dendritic breakages are signs that the cells are no longer viable.
