Generating an Ultra-Low-Density Neuronal Culture Using a High-Density Neuronal Feeder Layer

1. 24-well Plates Preparation for High-Density Neuron Cultures

NOTE: Perform the following steps (2-3) the day before the planned harvest of embryos.

1. Bring two 24-well plates into the cell culture hood and open the individual packaging.
2. Connect an 18 G syringe needle to a 1 ml plastic single-use syringe.
3. Hold the syringe and aim the needle tip at ~1 mm left to the center of the bottom of the well. Apply moderate force (~250 g) to etch the bottom of the well (~1 mm long). A groove will be formed, and the plastic materials displaced will form elevated support above the flat bottom well surface. Etch another ~1 mm-long parallel groove at ~1 mm to the right of the center of the bottom in a similar way.
4. Repeat this step for all the wells of the two 24-well plates. The two 24-well plates used for high-density culture are now ready for coating with poly-D-lysine (see step 2.8 below).

2. Coverslips Preparation for Low-Density Neuron Cultures

1. Use 12-mm diameter #1 round glass.
2. Place 50 coverslips inside a 100 mm diameter sterile plastic petri dish and submerge coverslips in 20 ml 70% ethanol solution. Place this petri dish on a rotating platform with moderate (70 rpm) speed rotation for an hour. Remove 70% ethanol and then replace with a fresh batch of 70% ethanol. Rotate and wash for another hour.
3. Discard 70% ethanol cleaning solution. Add sterile water (filtered through 0.2 µm filter unit). Rinse the coverslips rigorously by rotating the petri dish by hand. Rinse three times, each time replacing with fresh sterile water.
4. Place the petri dish with coverslips inside a sterile cell culture hood with laminar flow. Aspirate the rinsing water through the vacuum line and add 10 ml filtered sterile water to immerse the coverslips. The coverslips should be sterile, and the surface should be clean enough for poly-D-lysine coating.
5. Open two 24-well plates from their individual packing and place them in the hood.
6. Turn on the vacuum line in the hood. Connect a 200 µl pipette tip to the vacuum line and use a scissor to cut the tip to create a larger opening.
7. Use a fine-tip #5 tweezers to pick up coverslips from the petri dish, one at a time, and use the vacuum line to completely dry the coverslip. Then place one coverslip into each of the wells of the 24-well plate. The 50 cleaned coverslips should be enough to fill each of the wells of the two 24-well plates.
8. Dilute the poly-D-lysine stock solution (1 mg/ml) into a working solution (0.1 mg/ml) with a borate buffer (pH 8.5). For the four 24-well plates (including two high density plates with etched bottom), prepare 30 ml of total working solution.
9. Add 300 µl 0.1 mg/ml poly-D-lysine work solution to each well with glass coverslips. Make sure the coverslips are completely immersed in solution. If not, use the #5 tweezer tip to press down coverslips against the bottom of the well, and use the tip to guide poly-D-lysine solution to cover all the surface of the coverslips. Visually inspect all the coverslips to make sure no air was trapped between the plate and coverslips.
10. Add 300 µl 0.1 mg/ml poly-D-lysine work solution to each well of the high-density culture plate with etched bottom. Rotate by hand to spread the solution to cover the entire bottom of the well.
11. Return all the four plates with poly-D-lysine to the culture incubator, and incubate O/N.

3. Washing and Pre-conditioning Plates for Culture

NOTE: The following steps (3-5) are carried out on the day of tissue harvest.

1. After incubation/coating of coverslips and 24-well plates O/N, bring all four plates (two with donor coverslips, two acceptor high density plates with etched plastic bottom) into the culture hood. Use the vacuum line to remove the poly-D-lysine solution.
2. Immediately following the removal of the poly-D-lysine solution, add ~1 ml sterile water to each well using a plastic transfer pipette. After all the wells are filled with water, let stand for 10 min. Remove water by vacuum, then add 300 µl Wash medium in each well to cover the bottom of the well (with or without glass coverslips). Do not let the poly-D-lysine coating dry out.
3. Return all four plates to the cell incubator. The plates are ready to use once the dispersed hippocampal neurons are prepared.

4. Plating of Neurons and Long-Term Co-culture

1. Bring the two 24-well plates with etched bottoms for high density neurons into the cell culture hood. Remove the 300 µl precondition medium by vacuum. Plate 0.6 ml high density cell suspensions at 250,000 neurons/ml.
2. Bring the other two 24-well plates with coverslips into the culture hood and remove the 300 µl precondition medium by vacuum. Plate 0.6 ml low density cell suspensions at 10,000 neurons/ml on the coverslips inside the two 24-well plates.
3. Return all four 24-well plates to the incubator. Let sit for 2 hr.
4. Flip the low-density coverslips with adhering neurons to the high-density culture. Make sure the neurons are facing each other (i.e., not separated by the glass coverslip). Then return the two plates with co-culture to the incubator.

5. Co-culture Sustaining

1. Feed co-cultures by adding 300 µl fresh Feed medium (see Materials) at day in vitro (DIV) 5. There is no need to remove 50% of the original complete culture medium, as reported by many other studies. The feeding medium also contains 15 µM cytosine arabinoside (Ara-C, to yield a final concentration of 5 µM) to minimize the chance of glia contamination and proliferation.
2. Following this initial feeding, add 300 µl fresh Feed medium without Ara-C to the well every week. Should the culture be kept for more than one month, replace half of the medium with fresh Feed medium every week beyond one month time point (with the first medium half-replacement at DIV33). Morphologically, both high density and low-density neurons look healthy up to three months (the longest time observed by the experimenters).