How to Culture Neurons (for beginners)

Do it in your kitchen for under $2000

Introduction

This is a guide to culture neurons. If we can do it in our basement, you can too.

If you want to be able to communicate with the neurons, culture them on an MEA. Here is a good overview of the current state of MEAs.

A neuron culture that was brought up using this procedure

Materials (with links)

• Primary Cortical Rat Neurons

• Pen Strep (100x concentration)

• Neurobasal Plus Medium

• B-27 Supplement (50x concentration)

• GlutaMax-1 Supplement (100x concentration)

• Poly-D-Lysine

• 70% - 100% Isopropyl Alcohol (IPA)

• 1-10 mL pipette

• 100-1000 uL pipette

• 2-20 uL pipette

• Extra pipette tips of each size

• 50mL conical culture tube [autoclavable] x 1

• 15mL conical culture tube [autoclavable] x 1

• Water bath (can maintain 37 degree celsius)

• Cellular culture well trays (~2cm in diameter)

• Circular microscope coverslips (fit inside each well in tray)

• Laminar flow hood

• Multi-Electrode array

• Microscope

• CO2 incubator (find this locally on eBay or FB marketplace)

Soy Broth Reps

To ensure familiarity with the procedures, and to ensure your procedures are sterile, you should run dummy “soy broth” reps before attempting to culture real neurons. In soy broth reps, you follow the procedure, but replace every chemical except pen-strep (you should use real pen-strep) with a soy broth solution. Prepare ~500mL of soy broth solution ahead of time. Make sure to sterilize it through autoclaving or other means.

After you put the plated wells in the incubator, check back in 24 hours. If the solution is cloudy, then the well was compromised and your neurons would have died. Repeat this until you can consistently get clear culture wells before culturing real neurons.

Achieving Sterility

The following is a procedure to create a sterile space in which neurons can be safely cultured without contamination.

1. Empty the inside of the laminar flow hood, and turn on the hood to max airflow. 

2. Wear plastic gloves

3. Use IPA inside of a spray bottle to spray your hands and sleeves with IPA. Also rub the IPA residue on your hands onto the IPA spray bottle you are using to sterilize it.

4. Spray every surface inside of the laminar flow hood with the IPA spray bottle. Make sure every surface is coated with IPA.

5. Place the IPA bottle outside of the laminar flow hood.

The inside of this hood can be thought of as the “sterile domain”. For this reason, everything that goes inside of the hood must be sterilized before entering (including your hands & sleeves!).

Non-Autoclavable Items

1. Spray both of the pipettes with no pipette tip attached (Items 9 & 10) with IPA.

2. The precision pipettes contain a small chamber at their tip which contaminants may be present in. Dip the end of the tipless pipette and suck in 1 mL of IPA and squirt out the IPA multiple times.

3. Spray the outside of the pipettes with IPA one more time, and place them inside of the hood.

4. Spray the water bath (item 13) with IPA. There does not have to be water inside of the water bath at this point in time. Place the water bath inside of the hood and plug it in to a near outlet.

5. Spray the insides and outsides of the cell culture wells with IPA, transfer into the hood. 

6. Spray the insides and the outsides of the MEA with IPA, transfer into the hood.

Autoclavable Items

1. Place 5x 50mL conical culture tubes in the autoclave (item 11) in the autoclave, cap off.

2. Place 3x 15mL conical culture tubes in the autoclave (item 12) in the autoclave, cap off.

3. Place both of the pipette racks (items 9 & 10) in the autoclave, lid off or slightly open.

4. Close the lid of the autoclave and let the materials autoclave at 121 C for >15 minutes.

5. When the autoclaving process is complete, open the autoclave and quickly transfer everything to the laminar flow hood, while IPA-spraying them as they are transferred from the autoclave to the laminar flow hood.

Culturing:

The following steps are derived from the Fisher Scientific guide on how to culture neurons [link].

1. Set the incubator to 37 C, 5% CO2, and >80% humidity. Add a humidity pan.

2. Obtain sterile DI water. Recommended procedure is to autoclave DI water in an autoclavable container.

3. Fill water bath with autoclaved DI water, set to 37 C

While culturing, the following must be obeyed at all times:

1. Any medium containing the neurons must not be vortexed or induced to any sudden motion at any time.

2. Any equipment that is to touch the neurons or the media that they are in must be rinsed with complete neurobasal medium as neurons may stick to these plastic surfaces.

3. All of the following procedures are done inside of the prepared hood.

If the operator has to leave the hood during this procedure at any time, it is okay (unless dealing with time/temperature/pH-sensitive materials like neurons) as long as the hands and sleeves of the operator is sprayed with IPA before re-entering the hood.

Preparing Culture Wells & MEA:

MEAs and culture wells are not always sterile, and should be tested with multiple runs of soy-broth reps. Do not trust your vendor. You may need to develop your own protocols to chemically or thermally sterilize your wells or MEAs.

1. Place cover slips in each of the growth wells if necessary.

2. Fill each of the culture wells and the MEA wells with just enough deionized water to fully cover the bottom surface. Then, add 4.5 microliters of Poly-D-Lysine to each well per square centimeter area. Let the wells sit for at least 1 hour.

3. Put the wells aside in the hood once finished

Preparing Culture Media:

1. Add a ~3-6 mL of complete medium per culture well of Neurobasal Plus Medium into the Complete Neurobasal Plus Medium conical culture tube. You should err on the side of adding too much.

2. Add GlutaMax-I supplement to the “Complete Neurobasal Plus Medium” such that there is 2.5mL of GlutaMax-I per 1L of Neurobasal Plus Medium. The GlutaMax originally has a concentration of 200mM, this will decrease the final concentration to 0.5mM.

3. Add B-27 Plus Supplement to the Complete Neurobasal Medium prepared at a volumetric concentration of 2% (20mL of B-27 Plus Supplement per 1L)

4. Add Pen-Strep to the Complete Neurobasal Medium prepared at a volumetric concentration of 1% (10mL of Pen Strep per 1L of complete medium).

5. Place the “Complete Neurobasal Plus Medium” tube in the water bath to warm it to 37 C.

Plating Neurons:

1. Rinse a 15mL conical culture tube (tube labeled “Complete neurons”) with the Complete Neurobasal Plus Medium pre warmed to 37 degrees celsius. Leave in cell culture hood.

2. If using neurons from liquid nitrogen storage, twist cap slightly to release pressure and then re-tighten the cap. Transfer the vial of neurons to a 37 degrees celsius water bath for thawing as fast as possible. Handle as little as possible. You can use a box of dry ice when transporting the neurons from the freezer to the water bath.

3. Thaw the frozen neuron vial by gently swirling it in the 37 degrees celsius water bath for <2 minutes. The vial should still be cold to the touch. Take from water bath after only “a tiny ice crystal is left”. Transfer to cell culture hood.

4. Disinfect vial with alcohol (described as 70% isopropyl alcohol). Tap the vial gently on the surface of the cell culture hood so that the liquid settles to the bottom of the vial.

5. Rinse a 1-mL pipette tip with the Complete Neurobasal Plus Medium and very gently transfer the neurons from the vial to the prepared 15mL conical culture tube very gently. One drop at a time, per second at fastest. Do not force the pipet into the vial, be very gentle.

6. Rinse the vial which the neurons used to be in with 1mL of the Complete Neurobasal Plus Medium, pre warmed to 37 degrees celsius so you get every bit of the remaining neurons. Again, gently transfer these rinsed neurons and medium into the same conical culture tube mentioned in the last step. One drop at a time. Do not add the total 1mL of medium (used for rinsing) into the conical nozzle at once. Do it slowly, or else the neurons may suffer from osmotic shock.

7. Add 2mL of Complete Neurobasal Plus Medium still at 37 degrees celsius (prepared in steps 1&2) into the same conical culture tube used in step 8. At this point, the conical culture tube should have 4mL worth of fluid in it. Slowly mix the fluid with the 1-mL pipette in step 7 without creating any air bubbles. If you aren’t going to use the pipette in step 7, rinse whatever you are using with the Complete Neurobasal Plus Medium or else the neurons will stick to it.

8. Use volumetric estimates to add 15,000 neurons per cm^2 from the 15mL conical culture tube to each of the MEA/culture wells (1 * 10^6 neurons/4mL -> 60 microliters for 15,000 neurons).

9. In each culture well/MEA well, add 11.5mL of Complete Neurobasal Plus medium per 1mL of “Complete Neuron” fluid added. For example, if a well needs 250,000 neurons and thus 1mL of Complete Neuron fluid is added, there needs to be 11.5 mL of Complete Neurobasal Plus Medium added to make for additional growth medium for the neurons.

10. Transfer the MEAs and the Culture Wells into the incubator, lid on. Make sure the lid is not airtight as it will suffocate the neurons.

11. After 4-24 hours of incubation, renew half the medium in the MEAs and the wells.

To feed the neurons, renew half the medium and replace it with fresh Complete Neurobasal Plus Medium every 3-5 days.