This study investigates the impact of long-term organ graft storage on its metabolic profile, which is crucial for assessing organ function and potential rejection. The methodology involves the use of solid-phase microextraction (SPME) to isolate metabolites from the organ graft.
Long-term storage of an organ graft affects its metabolic profile, which is an indicator of organ function and early-stage organ rejection.
To isolate these metabolites, begin by taking a thin solid-phase microextraction or SPME probe coated with a biocompatible sorbent. Immerse the probe in a hydroalcoholic cleaning solution to hydrate the coated sorbent and loosen up any bound impurities.
Agitate the probe to remove the impurities. Next, place the probe in an activation solution and agitate again. Chemicals in the activation solution modify the probe surface, enhancing its adsorption potential. Rinse the probe with water and sterilize it to remove microbial contaminants.
Now, insert the sterile and activated probe into the organ graft such that the tissue matrix covers the entire sorbent surface. The sorbent material attracts free polar and non-polar metabolites from the graft, enabling adsorption onto its surface. Retract the probe and rinse it with water to remove any residual tissue.
Then, place the probe into a desorption solution, which interacts with the adsorbed metabolites. Agitate the probe to separate and solubilize the captured metabolites into the desorption solution. Finally, remove the probe and process the resultant metabolite mixture for subsequent analysis.
Start by preparing a preconditioning mixture composed of 1 to 1 methanol and water. Pipette 1 milliliter of the solution into each 2-milliliter glass vial and place one probe in each vial. Agitate the vials on a vortex agitator at 1,200 rpm for 1 hour. Then, rinse the probes with LC-MS grade water and sterilize them according to the standard surgical sterilization protocol or in sterile processing department.
When ready to extract the sample, open the sterile packaging and insert two probes directly into the kidney cortex for 10 minutes per time point, making sure that the entire length of the coating is covered by the tissue matrix. Make sure to keep track of the time of sampling for each probe. Retract the probe by pulling it out from the tissue and immediately rinse the coating with LC-MS grade water to remove any remaining blood, making sure to rinse away from the surgical site.
To transport the probes, place them in separate vials and close them. Then, place the vials in a box filled with dry ice or liquid nitrogen. Store the samples at negative 80 degrees Celsius or immediately proceed with desorption.
Prepare a desorption solution composed of acetonitrile and water for metabolomic analysis and another composed of isopropanol and methanol for lipidomic analysis. Add 100 microliters of the solution to inserts in the 2-milliliter labeled vials and place one probe in each vial. Agitate the vials at 1,200 rpm for 2 hours. Then, remove the probes from the vials and proceed with LC-MS analysis.
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