Dec 19, 2019
CULTURING i3NEURONS (Basic Protocol 6)
- Michael S. Fernandopulle1,
- Ryan Prestil1,
- Christopher Grunseich1,
- Chao Wang2,
- Li Gan2,
- Michael E. Ward1
- 1National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland;
- 2Gladstone Institute of Neurological Disease, Gladstone Institutes, San Francisco, California
- Neurodegeneration Method Development CommunityTech. support email: ndcn-help@chanzuckerberg.com
External link: https://doi.org/10.1002/cpcb.51
Protocol Citation: Michael S. Fernandopulle, Ryan Prestil, Christopher Grunseich, Chao Wang, Li Gan, Michael E. Ward 2019. CULTURING i3NEURONS (Basic Protocol 6). protocols.io https://dx.doi.org/10.17504/protocols.io.5jpg4mn
Manuscript citation:
Fernandopulle, M. S., Prestil, R., Grunseich, C., Wang, C., Gan, L., & Ward, M. E. (2018). Transcription-factor mediated differentiation of human iPSCs into neurons. Current Protocols in Cell Biology, e51. doi:https://doi.org/10.1002/cpcb.51
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: July 17, 2019
Last Modified: December 19, 2019
Protocol Integer ID: 25935
Keywords: i3LMN, i3Neurons, iPSC, iPSC-derived neurons, transcription factor-mediated differentiation
Abstract
Following Day 3 replating, i3Neurons 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.
Cortical Neuron Culture Medium (CM) is sufficient to promote the maturation and longterm maintenance of i3Neurons in culture. i3Neurons 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 Basic Protocol 5) 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 i3Neuron attachment, viability, and successful outgrowth.
Attachments
Materials
- Freshly-prepared 1× poly-L-ornithine (PLO) or polyethyleneimine (PEI) solution (see Table 3)
Poly-L-ornithine solutionMerck MilliporeSigma (Sigma-Aldrich)Catalog #P4957
Poly-D-lysine hydrobromideMerck MilliporeSigma (Sigma-Aldrich)Catalog #P7405
Poly(ethyleneimine) solutionMerck MilliporeSigma (Sigma-Aldrich)Catalog #03880
Boric acidMerck MilliporeSigma (Sigma-Aldrich)Catalog #B6768
Sodium tetraborateMerck MilliporeSigma (Sigma-Aldrich)Catalog #221732
Sodium chloride Merck MilliporeSigma (Sigma-Aldrich)Catalog #S7653
Sodium hydroxide solutionMerck MilliporeSigma (Sigma-Aldrich)Catalog #S2770
Note
Polymers should be resuspended to recommended concentration, filter sterilized, and diluted to 1× for use.
Borate buffer may be prepared as described here and filter sterilized, or may be purchased commercially.
| Component | Amount per 50 ml | |
| Poly-L-ornithine (PLO), 10× stock | ||
| PLO | 50 mg (per 50 ml) | |
| Borate buffer (see below) | to 50 ml | |
| Poly-D-lysine (PDL), 1× | ||
| PDL | 50 mg (per 50 ml) | |
| Borate buffer (see below) | to 50 ml | |
| Polyethyleneimine (PEI), 10× stock | ||
| PEI | 1 ml | |
| Borate buffer (see below) | 49 ml (per 50 ml) | |
| Borate buffer | ||
| Boric acid (100 mM) | 3.09 g (per 500 ml) | |
| Sodium tetraborate (25 mM) | 4.77 g (per 500 ml) | |
| Sodium chloride (75 mM) | 2.19 g (per 500 ml) | |
| Milli-Q water | to 500 ml | |
| Sodium hydroxide (1 M) | As needed to adjust pH to 8.4 |
Table 3: Poly-L-Ornithine (PLO), Poly-D-Lysine (PDL), and Polyethyleneimine (PEI) Solutions
- Laminin
Laminin Mouse Protein, NaturalThermo Fisher ScientificCatalog #23017015
- Cortical Neuron Culture Medium (CM; see Table 4)
BrainPhys™ Neuronal Medium 500 mL
STEMCELL Technologies Inc.Catalog #5790
B-27 SupplementGibco - Thermo Fisher ScientificCatalog #17504044
Recombinant Human/Murine/Rat BDNFpeprotechCatalog #450-02
Recombinant Human NT-3peprotechCatalog #450-03
Laminin Mouse Protein, NaturalThermo Fisher ScientificCatalog #23017015
Note
Prepare medium in sterile biosafety cabinet; medium should then be aliquotted to add additional supplements fresh; warm to 37°C before use.
| Component | Amount per 50 ml | Final concentration | |
| BrainPhys neuronal medium | 49 ml | ||
| B27 supplement, 50× | 1 ml | 1× | |
| BDNF (10 μg/ml) in PBS containing 0.1% IgG and protease-free BSA (store at −80°C) | 50 μl | 10 ng/ml | |
| NT-3 (10 μg/ml) in PBS containing 0.1% IgG and protease-free BSA (store at −80°C) | 50 μl | 10 ng/ml | |
| Laminin (store at −80°C; stock concentration 1 mg/ml; thaw on ice and dispense with chilled pipets) | 50 μl | 1μg/ml |
Table 4: Cortical Neuron Culture Medium (CM)
- Additional reagents and equipment for general iPSC culture (Basic Protocol 1) and counting cells (Phelan & May, 2015)
Safety warnings
Please see SDS (Safety Data Sheet) for hazards and safety warnings.
Coating dishes
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.
Incubate dishes for at least 01:00:00 at Room temperature .
Note
For best results, incubate dishes overnight in a 37 °C incubator.
Aspirate coating solution.
Wash dishes with sterile water.
Repeat twice for three total washes.
Note
Four or more total washes are recommended for PEI-coated plates.
Aspirate water.
Let dishes dry completely in a biosafety cabinet (typically requires 00:30:00 to 01:00:00 ).
Note
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.
Coated and dried dishes should be used immediately or wrapped in aluminum foil and stored at 4 °C for up to 1 week.
Note
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 1 Mass Percent ) slowly on ice and use chilled pipets to dispense in order to reduce polymerization. Dilute laminin stock to 10 Mass Percent in cold PBS, and add one half culture volume of this solution to the polymer-coated, washed, and dried dishes. Incubate for at least 02:00:00 at 37 °C before aspirating and plating neurons directly. This is optional for PLO and highly recommended for PEI.
Plating cells
Plating cells
Prepare CM in a sterile biosafety cabinet.
Note
Doxycycline (final concentration of 2 Mass Percent ) 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).
Place CM in 37 °C water/bead bath for approximately 00:20:00 or until warm to the touch.
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).
Note
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
Culture maintenance
Check cells daily under a phase-contrast microscope, paying particular attention to cell debris and morphological changes.
Note
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.
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.
Note
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.
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 for
biochemistry, 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 (< p3 post-harvest). Astrocytes can also be frozen in aliquots after expansion and thawed immediately prior to co-culture with partially differentiated neurons at d3. If directly co-culturing astrocytes, we recommend a ratio of 2 neurons:1 astrocyte. See Support Protocol 7 for detailed
information.