Development of Combinatorial Therapeutics for Spinal Cord Injury using Stem Cell Delivery

In this study, a combinatorial cell delivery platform is developed for spinal cord injury using decellularized tissue-derived hydrogels. The efficacy of decellularization and cell viability in the composite hydrogels is analyzed to determine their potential as a 3D culture platform. The results will allow further investigation of therapeutic potentials for spinal cord injury.

Recently, in the field of spinal cord injury repair, decellularization has been extensively utilized in the context of hydrogels. This process removes all the cellular and nuclear debris from tissues or organs to prevent a negative immune response, while preserving the extracellular matrix components. Now that the composite nerve hydrogels are optimized, characterization of the platform will be continued to maximize the neuro-regenerative behavior of ASCs for spinal cord repair.

This work will contribute to combinatorial therapeutic development for spinal cord injury repair, especially enhancing stem cell therapy for potential clinical translation. To begin, thaw the frozen spinal cord at four degrees Celsius in the refrigerator for 18 to 24 hours before decellularization. Using sterile scissors, carefully remove the dura mater.

Cut the spinal cord into small pieces, about one centimeter in length. Place three pieces into a 50-milliliter tube. Rinse the spinal cord with deionized water at four degrees Celsius for 18 to 24 hours at 60 RPM.

Next, rinse the spinal cord with 0.025%trypsin EDTA, followed by PBS for 15 minutes twice. Then, rinse the spinal cord with the following solutions for decellularization. Finally, rinse the spinal cord with deionized water for one hour twice, followed by PBS for one hour.

Then, lyophilize the spinal cord at 0.01 millibar and 56 degrees Celsius for three days before storing it until use. Thaw the previously frozen porcine sciatic nerve at four degrees Celsius in the refrigerator for 18 to 24 hours before decellularization. Now, cut the sciatic nerve into small pieces, approximately one centimeter in length.

Place three pieces into a 50-milliliter tube. Rinse the sciatic nerve with deionized water for seven hours. Then, rinse the sciatic nerve with the following solutions for decellularization.

Then, lyophilize it at 0.01 millibar and 56 degrees Celsius for three days before storing it until use. Begin by using autoclave scissors to chop the previously decellularized porcine spinal cord and sciatic nerve into powder. Digest the tissues separately in 0.01 normal hydrochloric acid solution containing one milligram per milliliter of pepsin at a concentration of 15 milligrams per milliliter.

Place a magnetic bar in the solution and stir at 500 RPM for at least four days at four degrees Celsius to generate pregel solutions. Mix sciatic nerve and spinal cord pregel. Then, dilute the hydrogel to the desired concentration using M199 media and PBS.

Use one normal sodium hydroxide and hydrochloric acid to adjust the pH to 7.4. Once the pH is adjusted, resuspend human adipose-derived stem cells in the pregel at a density of 1 million cells per milliliter. Dilute the pregel to 12 milligrams per milliliter using PBS.

Place it into the wells, and then place the PDMS lid onto the pregel. Incubate the plate for 30 minutes at 37 degrees Celsius. Then, add PBS to the wells.

Remove the PDMS lid and the solution. Add ASC growth media to ASC-laden hydrogels and incubate at 37 degrees Celsius for culturing.