Exosomes might react to vesicles secreted by a cell between 14 and 200 nanometer in size, and split from in the intracellular cargo and communication, which comes to the smallest subgroup of extracellular vesicles. Exosomes which are formed as a result of the differentiation of the endosome, originate from the cell membrane. The stem cells can be isolated from different sources such as bone marrow or adipose tissues.
but adult tissue dried stem cells have higher immunogenic potential Cons springing the size of exosomes, it's told that it's made an important role in central nervous system disease because it can pass through to the blood brain barrier. To begin, wash the seeded Wharton's jelly mesenchymal stem cells with five milliliters of PBS. Then add five milliliters of trypsin EDTA solution before placing the flask in an incubator.
After incubation, collect the cells and centrifuge at 1, 500 G for five minutes. Then remove the supernatant and add one milliliter of DMEM-F12 containing 10%FBS to the tube. Wash cultured cells with PBS.
After washing, add serum free DMEM-F12 medium to the cells and place it in the incubator. To isolate the exosomes, collect the serum free media and centrifuge at 300 G at four degrees Celsius for 10 minutes. Transfer the supernatant to another tube and process it by serial centrifugation at higher speeds.
After the final centrifugation, slowly remove the supernatant and dissolve the pellet in one milliliter PBS. Mix by vortexing and proceed with ultracentrifugation at 110, 000 G for 70 minutes at four degrees Celsius. Add three milliliters of PBS to the exosome suspension collected earlier by ultracentrifugation and sterilize the diluted suspension by filtration using a 0.22 micron filter.
Then transfer 500 microliters of the sterilized exosome suspension to another tube. Next sequentially, add 500 microliters of 0.5 milligrams per milliliter dopamine solution and saponin into the sterilized suspension. After incubation ultracentrifuge the suspension.
The sample can either be used for NTA and DLS analysis to characterize dopamine loaded exosomes or stored at minus 20 degrees Celsius until further use. In the NTA analysis, the average size of Wharton jelly derived exosomes was determined to be 98 nanometers and dopamine loaded exosomes to be 110 nanometers. The numbers of nanoparticles revealed by NTA and DLS analyses were consistent with each other.
The zeta potential of Wharton jelly derived exosomes was measured as minus 15.7 and dopamine loaded exosomes as minus 17.6 millivolts. The HPLC analysis of the dopamine loaded exosomes detected the peak for dopamine at 6.45 minutes. The cumulative drug release profile revealed that 74.8%of encapsulated dopamine was released from the exosomes within the first eight hours.
The cytotoxicity of the dopamine loaded and free exosomes on fibroblasts was investigated. Although the dopamine loaded exosomes did not demonstrate any cytotoxic effects, saponin decreased the fibroblast viability. The MTT assay showed increased cell viability when the fibroblasts were treated with the dopamine loaded exosomes.
The statistical significance of the results was evaluated by performing one-way anova. Additionally, the cytotoxic effects of different concentrations of free exosomes were investigated on the fibroblasts. The fibroblast viability was higher with free exosomes at the concentration of 25 microliters per milliliter.
It's really important for the central nervous system is the patient sizes, the play role in the processes such as plasticity, neuro response, side to side communication, and neurogenesis in the brain. In addition, thanks to surface of exosomes, it says, has been observed that they can in interact with with target cell drug and activity delivered during the drug Is a completion. For the first time in the study, we developed a new drug formulation by encapsulating dopamine into stem cell derived exosomes.
He proposed that this formulation will be so promising for treatment of Parkinson's disease.
