The overall goal of this procedure is to set up adipose-derived stem cell and dorsal root ganglion or DRG neuron co cultures for studying in vitro nerve regeneration. This is accomplished by first obtaining undifferentiated adipose-derived stem cells from rat fat tissue. In the second step, the cells are differentiated into schwan like cells using a cocktail of growth factors.
Next, the DRG neurons are harvested from a rat spinal cord and associated in the final step, the neurons are seated onto the differentiated stem cells to generate the in vitro co-culture system. Ultimately, the cells are stained for neuronal and glial markers to facilitate neurite growth, quantification and imaged by scanning electron microscopy for morphological assessment. This method can help to answer different question in the regenerative medicine field, so such as how adipose the right stem cell interact with do ug organal neurons on different biomaterials?
The reason demonstration of this method is critical because this involves steps that are difficult to learn due to the low accessibility and the small size of the tissues In a biological cabinet. Begin by using a pair of scissors and a sterile razor blade to finely chop the visceral and inguinal fat harvested from adult male spro dolly rats into a fine consistency. Next, transfer the resulting fat slurry into a tube containing 15 milliliters of freshly prepared filter, sterilized collagenase type one solution and placed a tube into a 37 degree Celsius water bath under continuous agitation for 30 to 60 minutes.
Monitor the digestion closely stopping before the tissue is fully dissociated to improve the cell viability and yield. When the tissue is ready, filter it through a 100 micron cell strainer. A good digestion will result in a homogenous fat consistency that appears beige with gentle swirling.
Neutralize the digestive enzymes with 15 milliliters of 37 degrees Celsius stem cell growth medium supplemented with FBS. Then spin down the cells to collect the stromal vascular fraction. Aspirate the supinate starting from the top layer of fat.
Resuspend the pellet in one milliliter of red blood cell ISIS buffer with pipetting. After one minute, stop the lysis by adding 10 milliliters of fresh, medium and centrifuge to cells again. Now carefully aspirate the S supernatant.
Resuspend the cells in 10 milliliters of medium and seed the cells in 75 square centimeter. Flasks at 37 degrees Celsius and 5%carbon dioxide. Maintain the cells at sub confluent levels until the first or second passage at which the cells are ready for differentiation into schwan like cells by treatment with beta more cap ethanol and retinoic acid to obtain the DRG neurons after harvesting the spinal cord.
Begin by removing any dorsal parts and then use sterile and sharp surgical scissors to divide the spinal column in half along the longitudinal axis, exposing the cord tissue. Cut the spinal column into two smaller segments below the level of the rib cage, and then use fine forceps to gently remove all the cord tissue. Taking care not to pull and remove the DRG roots, which appear as white filaments coming directly out of the canals.
Once all of the core tissue has been removed, use very fine forceps to pull the entire DRG root, reaching deep into the vertible canals and taking care not to damage the roots of the ganglia. Place the DRG into a 60 square millimeter pet tree dish containing three to four milliliters of hams F 12 medium supplemented with antibiotics. Then under a dissecting microscope, use sterile forceps and a scalpel to clear away any excess nerve roots surrounding the ganglia to reduce satellite cells contamination.
Next, transfer the DRG into a 35 square millimeter Petri dish containing 1.8 milliliter of fresh F 12 medium and add 200 microliters of collagenase. Type four stock solution. Incubate the DRG for one hour and then carefully aspirate the medium with a glass pipette.
Taking care not to aspirate or damage the DRG. Repeat the collagenase step and wash the DRG with F 12 medium. After the second wash, add 1.8 milliliters of F 12 medium and 200 microliters of trypsin to the cells.
Removing the trypsin and adding one milliliter of medium supplemented with 500 microliters of FBS to arrest the enzymatic reaction. Then aspirate the medium and gently wash the DRG with F 12 medium three times to remove all traces of the serum. Now feed the cells with two milliliters of fresh F 12 medium and use a glass pipette to carefully transfer the cell suspension into a 15 milliliter tube.
Pipette up and down eight to 10 times to gently dissociate the neurons and then allow the palate to accumulate at the bottom of the tube when the palate has settled. Transfer the supinate into a new tube and add two milliliters of fresh F 12 medium to the palate. Repeating the mechanical dissociation and medium transfer into the suspension becomes homogenous.
Then rerate the cell suspension three to four times followed with filtration of the resulting homogenized suspension through a 100 micron cell strainer into a new 50 milliliter tube. Centrifuge the cells in a 15 milliliter tube while they are spinning slowly pipette freshly prepared. 15%bovine serum albumin down the inside of a 15 milliliter tube, held at a 45 degree angle to create a gradual protein trail.
Using the numbers on the tube as a reference for the forming track, then aspirate the snat from the cells, saving the last 500 microliters for resus, suspending the pellet and slowly dispense the cells along the protein trail into the new tube. After spinning down the cells, again, resuspend the pellet in one milliliter of modified BS medium and seed the cells in a small volume of medium. In a 24 well plate for two hours when the cells have attached add fresh BS medium supplemented with 15 nanograms per milliliter of nerve growth factor following dissociation.
The DRG cells can also be used for co-culture with the swan cell-like adipose stem cells by seeding them on top of the pre-seed cells. These images demonstrate the importance of schwan cell-like adipose stem cells for DRG Neurite sprouting in a co-culture model using polycaprolactone films as substrates. No neurites were observed on untreated surfaces in the absence of swan cell-like adipose stem cells, whereas formation of the neurites were clearly improved in the co-culture system.
On average, the number of neurites per cell body increases significantly in the presence of stem cells. Indeed, as these images illustrate, the ability of DRG neurons to sprout neurons occurs preferentially in conjunction with differentiated stem cells as indicated by the yellow arrows rose. So after watching this video, should now be able to obtain lib derive stem cells and to dissociate DGen neurons.
But also you should be able to set up an model to study in the peripheral average degeneration.
