TITLE: CULTURING i3NEURONS (Basic Protocol 6)
AUTHORS: Michael S. Fernandopulle, Ryan Prestil, Christopher Grunseich, Chao Wang, Li Gan, Michael E. Ward

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Following Day 3 replating, i</span><span style = "vertical-align:super;">3</span><span>Neurons should be cultured for at least 1 week before lysis, fixation, or other experimental endpoint, and they remain viable for at least 1 month with proper maintenance. This protocol covers medium conditions, coating of tissue culture dishes with synthetic polymers, recommended plating densities, and maintenance procedures for long-term culture and for specific experimental applications.</span></div><div class = "text-block"><span>Cortical Neuron Culture Medium (CM) is sufficient to promote the maturation and longterm maintenance of i</span><span style = "vertical-align:super;">3</span><span>Neurons in culture. i</span><span style = "vertical-align:super;">3</span><span>Neurons express general markers of cortical neurons, as well as specific pre- and postsynaptic markers of glutamatergic excitatory cortical neurons. Since these cells are post-mitotic after 3 days of differentiation (see </span><a href="https://www.protocols.io/view/basic-protocol-5-differentiation-of-i3neurons-5h6g39e" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Basic Protocol 5</span></a><span>) and prefer neuron-conditioned medium to fresh medium, maintenance conditions for these cultures are minimal. Generally, half-medium changes every 7 days with fresh, pre-warmed CM are sufficient for culturing beyond d10 (7 days after replating). Neuron attachment and growth also require a strongly adhesive substrate. Coating plates with synthetic polymers such as poly-L-ornithine (PLO) is necessary for i</span><span style = "vertical-align:super;">3</span><span>Neuron attachment, viability, and successful outgrowth.</span></div></div>

[STEPS]
?. [Coating dishes]
Prepare stock solutions of PLO or PEI (see Table 3).
?. [Coating dishes]
Add one half culture volume of the coating solution prepared in step 1 to the tissue culture dishes to be used for plating the Day 3 partially differentiated i3Neurons. Gently tilt plate to ensure full coverage.
?. [Coating dishes]
Incubate dishes for at least  at .
0 Room temperature
For best results, incubate dishes overnight in a   incubator.
?. [Coating dishes]
Aspirate coating solution.
?. [Coating dishes]
Aspirate water.
?. [Coating dishes]
Coated and dried dishes should be used immediately or wrapped in aluminum foil and stored at  for up to 1 week.
4 °C
Faster neurite outgrowth, increased neuronal survival, and reduced clumping for longterm culture may be achieved by a secondary coating with laminin prior to plating.Thaw concentrated laminin stock solution (approximately  ) slowly on ice and use chilled pipets to dispense in order to reduce polymerization. Dilute laminin stock to   in cold PBS, and add one half culture volume of this solution to the polymer-coated, washed, and dried dishes. Incubate for at least   at   before aspirating and plating neurons directly. This is optional for PLO and highly recommended for PEI.
?. [Plating cells]
Prepare CM in a sterile biosafety cabinet.
Doxycycline (final concentration of  ) can be added to this medium at the discretion of the experimenter. Neurogenin-2 requires only 24 hr of expression to induce irreversible differentiation to neurons, so the 3-day differentiation period in IM medium should be sufficient; however, prolonged induction of NGN2 has not been shown to have significant effects on neuronal health or maturation. If other doxycycline-inducible transgenes are present in the cell line, it may be desirable to restrict expression of these cassettes to prevent toxicity and/or accommodate future induction time points.We have found that several basal media (e.g., DMEM/F12, Neurobasal A, and Brainphys), when properly supplemented (+B27/BDNF/NT3/laminin), work well for i3Neuron culture. We favor BrainPhys medium, since it facilitates synapse formation and spontaneous electrical activity, but alternative basal medium/supplement combinations may be superior for specific applications or individual iPSC lines, and other neuronal medium formulations are also worth considering (e.g., B27 Plus, Invitrogen).
?. [Plating cells]
From a thawed cryovial (see Basic Protocol 1) or freshly dissociated 3-day differentiated cells, resuspend in the appropriate amount of medium. Typical cell counts and medium volumes are as follows:a. 96-well plate (imaging): 1–5 × 104 cells in 100 μl medium/well.b. 8-well chamber slide (imaging): 0.3–1.5 × 105 cells in 250 μl medium/well.c. 6-well plate (biochemistry): 1.5–2×106 cells in 1.5 ml medium/well (supplement to 2 to 3 ml one day after plating).d. 10-cm dish (biochemistry): 1–1.2 × 107 cells in 8 ml medium (supplement to 10 to 12 ml one day after plating).e. 15-cm dish (biochemistry): 3–3.5 × 107 cells in 18 ml medium (supplement to 20 to 22 ml one day after plating).
Biochemistry applications typically require a high concentration of cells for a given surface area. Thus, these experiments require a greater volume of medium than would be required for an imaging experiment. However, after splitting, cells typically adhere better to a new plate with a lesser volume of medium compared to a greater volume. Thus, neurons plated on 6-well, 10-cm, or 15-cm dishes for biochemistry should be plated with 1.5 ml, 8 ml, and 18 ml of medium, respectively. These volumes should then be supplemented to the final volumes above on the day after plating.
?. [Culture maintenance]
Check cells daily under a phase-contrast microscope, paying particular attention to cell debris and morphological changes.
High levels of cell debris and/or cell clumping often indicate a problem with either the dish coating or culture medium. If this is seen, remove half volume of culture medium and replace with full volume of medium (50 % additional medium). If additional fresh medium does not result in less debris the next day, there has likely been insufficient coating or the coating medium was expired.
?. [Culture maintenance]
Biweekly half-medium changes (i.e., every 3 to 4 days) are effective for sustaining dense cultures (e.g., biochemistry applications). Weekly half-medium changes are sufficient to sustain long-term cultures at moderate densities (e.g., microscopy applications). Use the appropriate serological pipet or micropipet to slowly aspirate a measured volume from each well, and very gently replace with fresh medium.
Neurons tend to easily dissociate from the dish, so any aspiration or dispensing of medium directly onto cells is not recommended. Take care to aspirate and dissociate by tilting the dish so that medium accumulates on one side. Then, aspirate/dispense with the pipet directed toward the wall of the dish (i.e., away from the cells at the bottom). Should a full medium aspiration be necessary (i.e., for a PBS wash), initially add the medium dropwise in the middle of the well with the plate tilted until a small pool forms in the corner, at which point add the rest of the medium dropwise down the well wall onto this pool. This reduces shear forces on the edges which can cause whole wells to detach in a sheet.
?. [Culture maintenance]
Optional: Supplementation with astrocytes or astrocyte-conditioned medium have been shown to improve the overall health and electrophysiological activity of i3Neurons in long-term cultures. To maintain culture purity forbiochemistry, astrocytes can be supplemented using various commercially available Transwell dish inserts. Alternatively, half-medium changes as described above can be replaced with half-medium changes of astrocyte-conditioned medium (i.e., medium extracted from independent astrocyte cultures and sterile filtered to ensure no cell or debris carryover). Primary astrocytes expanded in DMEM + 10 % fetal bovine serum (FBS) from post-natal (P0-3) mice or rats are sufficient for these purposes, provided that they are low-passage (Support Protocol 7 for detailedinformation.
?. [Coating dishes]
Wash dishes with sterile water.
?. [Coating dishes]
Repeat twice for three total washes.
Four or more total washes are recommended for PEI-coated plates.
?. [Coating dishes]
Let dishes dry completely in a biosafety cabinet (typically requires  to ).
To accelerate the drying process, stand the dishes on their sides and lean them against the back of the BSC. Lids may also be left askew to allow better airflow. In particular, PEI requires complete drying to prevent toxicity.
?. [Plating cells]
Place CM in  water/bead bath for approximately  or until warm to the touch.
37 °C