Dorsal Root Ganglia Neurons and Differentiated Adipose-derived Stem Cells: An In Vitro Co-culture Model to Study Peripheral Nerve Regeneration

NOTE: All the experiments involving animals were carried out in accordance with the U.K. Animals (Scientific Procedures) Act, 1986.

1. Experimental Set-up

1. Prior to start tissue and cell harvest, check that all the tools are sterile. Whether necessary, autoclave a pair of sharp surgical scissors, a very fine forceps and a fine standard forceps. Also sterilize each substrate before cell seeding using UV sterilization, ethanol exposure or steam sterilization as appropriate.
2. Preparation of media for stem cell harvest and differentiation
   1. Prepare the stem cell growth medium, containing Minimum Essential Medium (α-MEM) supplemented with 10 % fetal bovine serum (FBS), 200 mM L-glutamine, and 1 % penicillin-streptomycin (PS).
   2. Prepare a 10 mM stock solution of forskolin by dissolving 10 mg of forskolin in 2.436 ml of sterile dimethyl sulfoxyde. Use at final concentration of 14 μM.
   3. Prepare a 35 mg/ml stock solution of retinoic acid by dissolving 50 mg in 1.43 ml of sterile dimethyl sulfoxyde. Use at final concentration of 350 ng/ml.
   4. Prepare platelet-derived growth factor (PDGF) stocks (100 μg/ml) by dissolving 10 μg of lyophilized powder in 100 μl of distilled sterile water. Use at final concentration of 5 ng/ml.
   5. Prepare basic fibroblast growth factor (bFGF) stocks (100 μg/ml) by dissolving 50 μg of lyophilized powder in 500 μl of distilled sterile water. Use at final concentration of 10 ng/ml.
   6. Prepare the stem cell differentiation medium, containing stem cell growth medium supplemented with 14 µM forskolin, 126 ng/ml glial growth factor-2 (GGF-2), 5 ng/mL platelet-derived growth factor (PDGF), and 10 ng/ml basic fibroblast growth factor (bFGF).
3. Preparation of media and stock solutions for neuron harvest and dissociation
   1. Prepare Bottenstein and Sato’s (BS) medium²⁸, by adding 1 % PS and 1 % N2 supplement to Ham’s F12 medium. Calculate the final volume needed as 500 µl per well (if using a 24-well plate).
   2. Prepare collagenase IV stocks in Ham’s F12 medium at the concentration of 1.25 % wt/v. Filter-sterilize the solution and store at -20 °C as 200 µl aliquots.
   3. Prepare bovine pancreatic trypsin stocks in Ham’s F12 medium at the concentration of 2.5 % wt/v, filter-sterilize and store at -20 °C as 200 µl aliquots.
   4. Prepare the nerve growth factor (NGF) stock solution at the concentration of 5 µg/mL in a filter-sterilized 1 mg/ml fatty acid-free bovine serum albumin (BSA) solution in F12 medium and store at -20 C as 200 µl aliquots. Do not filter the NGF after reconstitution.
4. In case no surface modification has been performed, coat coverslips/plates with poly-D-lysine (0.1 mg/ml for 15 min at RT) and/or laminin (2-10 µg/cm² for 2 hr at 37 °C) to support neuron attachment and neurite outgrowth as appropriate.
5. Always warm up media in a water bath at 37 °C before using.

2. Adipose-derived Stem Cell (ASC) Harvest and Differentiation into a SC Phenotype

1. ASC harvest from visceral and inguinal fat of adult male Sprague-Dawley rats
   1. Prior to start, prepare a tube with 10-15 ml of Hanks’ Balanced Saline Solution (HBSS) supplemented with 1% v/v of PS solution and store on ice until fat harvesting.
   2. Terminate the rat by cervical dislocation and decapitation. Shave the rat and make an incision through the abdominal skin, exposing the internal organs and avoiding bleeding. Remove the visceral fat encasing the stomach and intestines (usually characterized by a fatty yellow consistency) and the inguinal fat surrounding the testicles from an adult male Sprague-Dawley rat. Transfer the fat in the tube containing HBSS on ice.
   3. In a biological safety cabinet (class II) finely chop the fat using a pair of scissors and a sterile razor blade until fine consistency is reached and transfer it into a tube containing 15 ml of 0.2 % wt/v collagenase type I solution freshly prepared and filter-sterilized on the day.
   4. Transfer the tube in a water bath at 37 °C and leave the fat tissue to digest in presence of the enzyme for 30 min-1 hr under continuous agitation. Monitor the digestion closely and stop before the tissue is fully dissociated, this will improve cell viability and cell yield. Filter theun-dissociated tissue through a 100 µm cell strainer.
      NOTE: Good tissue digestion will result in homogenous consistency of the fat, visible by eye when gently swirling the tube, acquiring a beige appearance.
   5. Neutralize the enzyme by adding 15 ml of stem cell growth medium containing fetal bovine serum at 37 °C and centrifuge the solution at 160 g for 10 min in order to collect the stromal vascular fraction, including the stem cells, at the bottom of the tube.
   6. At this stage, the pellet can be resuspended in 1 ml of red blood cells lysis buffer to remove blood cell contamination. After resuspension and pipetting for 1 min, add 10 ml of fresh stem cell growth medium and centrifuge at 160 g for 10 min.
   7. Carefully aspirate the supernatant from the tube, taking care of the deposited cell pellet at the bottom. Resuspend the cells in 10 ml of stem cell growth medium, transfer them into a 75 cm² flasks and incubate at 37 °C, 5 % CO₂. Maintain the cells at sub-confluent levels until passage 1-2, changing medium every 3 days.
2. ASC differentiation to a SC phenotype
   1. At passage 1-2, remove the stem cell growth medium from the 75 cm² flask and replace it with 10 ml of fresh medium containing 1 mM β-mercaptoethanol freshly prepared and filter-sterilized on the day. Incubate the cells at 37 °C, 5 % CO₂ for 24 hr. At this stage, it is important that the cells are seeded at low density (30 %) before starting the differentiation.
   2. Wash the cells carefully with HBSS, aspirate and replace it with 10 ml of medium containing 350 ng/ml retinoic acid. Incubate the cells at 37 °C, 5 % CO₂ for 72 hr. Try to minimize exposure of cell medium to light.
   3. After 3 days, wash the cells carefully with HBSS, aspirate and replace it with 10 ml of stem cell differentiation medium (see step 1.2.6). Maintain the cells at sub-confluent levels, changing medium every 3 days.
      NOTE: Following 2 weeks of incubation, ASC are differentiated into SC-like ASC, expressing their characteristic phenotype (as demonstrated by Kingham et al.⁵). They can then be used up until the 10th passage without noticeable changes in behavior²⁹ .

3. Harvest and Dissociation of Dorsal Root Ganglia (DRG) Neurons

1. Terminate the rat by cervical dislocation and decapitation. Shave the rat and lift the skin to expose the spinal column. Using sharp scissors, excide the spinal column taking extra care of the confined organs and blood vessels. Transfer the spinal column in a biological safety cabinet (class II) using a Petri dish and remove any dorsal part.
2. Divide the spinal column in half along the longitudinal axis using sterile and sharp surgical scissors to expose the cord tissue. At this point, it is helpful to cut the spinal column in two smaller segments below the level of the rib cage, to make it easier to handle during the DRG neuron harvest. Using fine forceps, gently remove all the cord tissue, paying attention to not pull and remove the DRG roots. This way the DRG and roots will be exposed within the vertebral canals, still encased in the column. Observe DRG as white filaments coming out directly from the canals.
3. Pull out the entire DRG root (not just the DRG) from the vertebral canals by using very fine forceps, going deep into the vertebral canals and paying attention not to damage the roots of the ganglia. Transfer the DRG into a small petri dish (60 mm²) containing 3-4 ml of Ham’s F12 medium supplemented with 1% PS. If using different animals, use separate dishes.
4. Under a dissecting microscope, clean the DRG of any excess of nerve roots surrounding the ganglia using sterile forceps and a scalpel to reduce glial cell contamination. Transfer the DRG into a small petri dish (35 mm²) with 1.8 ml of fresh F12 medium.
5. Add 200 µl of 1.25 % wt/v collagenase type IV stock solution (final concentration of 0.125%) and incubate the DRG at 37 °C, 5 % CO₂ for 1 hr. Carefully aspirate the medium with a glass pipette, paying attention not to aspirate or damage the DRG. Add fresh F12 medium supplemented with 0.125 % wt/v collagenase type IV enzyme and incubate for 1 hr as previously described.
6. Aspirate the medium and gently wash the DRG with F12 medium. Add then 1.8 ml of F12 medium and 200 µl of trypsin (final concentration of 0.25 % wt/v) and incubate at 37 °C, 5 % CO₂ for 30 min.
7. Remove trypsin and add 1 ml of F12 medium supplemented with 500 µl of FBS to arrest the enzymatic reaction. Aspirate the medium and gently wash the DRG with F12 medium for three times to remove traces of serum.
8. Add 2 ml of fresh F12 medium and carefully transfer the DRG with the medium into a 15 ml tube using a glass pipette. Gently dissociate the DRG neurons by pipetting up and down (about 8-10 times) with the glass pipette (plastic tips can be used as alternative).
9. Allow the pellet to settle at the bottom of the tube and collect the medium in a new tube. Add 2 ml of fresh F12 medium to the tube containing the pellet and repeat the mechanical dissociation with the glass pipette. Repeat this step until the suspension becomes homogeneous (about 3-4 times) and collect all the dissociated DRG in the new tube. This method reduces the stress deriving from the mechanical dissociation and improves the viability of the neurons.
10. Filter the resulting homogenized suspension into a new 15 ml tube using a 100 µm cell strainer to remove un-dissociated neurons and other debris. At this stage, it might be convenient to first filter the cell suspension into a 50 ml tube and then transfer the solution into the smaller tube using a glass pipette. Centrifuge the suspension at 110 g for 5 min.
11. Prepare a 15 % bovine serum albumin (BSA) by adding 500 µl of a 30 % BSA solution to 500 µl of F12 medium. Slowly pipette the solution down the wall of a 15 ml tube to create a gradual protein trail. At this stage, it is helpful to hold the tube at a 45° angle and slowly release the BSA using the numbers on the falcon tube as a reference for the forming “track”.
12. Aspirate the supernatant from step 3.10 leaving 500 µl at the bottom of the tube and resuspend the cell pellet in the same medium. Slowly pipette the suspension along the protein trail previously prepared in step 3.11 (use the numbers on the tube as a reference) and centrifuge at 500 g for 5 min.
13. Aspirate the supernatant and resuspend the pellet in 1 ml of modified BS medium (or a mixed medium for the SC-like ASC/DRG co-culture, as described below in step 4.5.
    NOTE: The volume to re-suspend the DRG neurons can be made up to the actual volume required, depending on the final cell concentration desired and the number of samples to be seeded. One animal will provide enough cells for a 24-well plate experiment.
14. Incubate the seeded samples at 37 °C, 5% CO₂ for 2 hr to allow cell attachment and finally add fresh BS medium supplemented with 50 ng/ml of nerve growth factor (NGF).

4. Direct Co-culture of SC-like ASC and DRG Neurons

1. Maintain SC-like ASC in culture at sub-confluent levels as described in step 2.2.3. Twenty-four hr prior to DRG harvest, aspirate the stem cell differentiation medium and wash the cells with HBSS. Aspirate and incubate in 3 ml of trypsin at 37 °C, 5% CO₂ for 3 min.
2. Check under a light microscope that all the cells have detached from the flask. Gently tap the flask to help detachment. Add 7 ml of medium to stop the trypsin reaction, collect the cell suspension in a 15 ml tube and centrifuge at 110 g for 5 min.
3. Aspirate the supernatant and resuspend the cell pellet in 5 ml of stem cell differentiation medium. Count the cells using a hemocytometer and dilute the cell suspension according to the final concentration required. Ideally, seeding 20,000 SC-like ASC/cm² would guarantee a confluent layer of cells.
4. Seed the SC-like ASC on the substrate and incubate at 37 °C, 5 % CO₂ for 24 hr to allow cell attachment.
5. After 24 hr incubation, aspirate the medium and add the DRG neurons on top of the cells as described in step 3.14 and 3.15. Change the medium to a mixed medium containing 50 % of stem cell differentiation medium and 50 % of BS medium. In addition, reducing the FBS concentration to 1-2.5 % in the final mixed medium helps avoiding the proliferation of contaminating satellite cells derived from the DRG dissociation.
6. Incubate the co-cultured samples at 37 °C, 5 % CO₂ and maintain in culture for the required time for future tests.