Comparative Evaluation of the Efficacy of Human Umbilical Cord Mesenchymal Stem Cell-Derived Exosomes and Autologous Serum Treatments in Corneal Epithelial Wound Healing

Project no. 30827 · Status: Kapanmış (Yayın bekleniyor) · 2024

Summary

In ophthalmology, the need for treatments that facilitate re-epithelialization is still important. For this purpose, advanced treatment applications such as autologous serum and umbilical cord serum containing growth factors and cytokines that play a key role in epithelial regeneration processes are used to accelerate healing. In addition to the use of exosomes as vesicles carrying therapeutic molecules to selectively target disease-related cells and mechanisms, it has been shown that mesenchymal stem cell-derived exosomes contain various cytokines and growth factors (e.g. IL-6, IL-10, HGF, TGFβ1) that are known to contribute to immunoregulation. In ocular diseases, although it is thought that MSC-derived exosomes would be as effective as transplanted MSCs in preventing inflammation and damage, there are not many studies in the literature to elucidate the therapeutic capabilities and in vivo mechanisms of action of MSC-derived exosomes on the corneal epithelium. The limited number of existing studies have used a mouse model, and exosome therapy used in these models has been shown to reduce corneal scarring, inflammation and fibrosis, while supporting corneal epithelial regeneration. On the other hand, rabbits are more advantageous to use than mice and rats, especially for ocular surface studies. The reason for this is that the globe is larger, ocular surface tests can be applied more safely, and the ocular surface biochemical properties they have are more suitable for drug studies. Our aim with this project is to demonstrate the effectiveness of mesenchymal stem cell-derived exosome therapy, which has started to take its place in clinical practice in other fields of medicine, in an experimental corneal wound model and to complete its preclinical research. In this way, the infrastructure for clinical research on the way to productization will be completed. In this study, first Wharton Jelly-derived MSC-derived exosomes will be prepared in accordance with the legal activity permit. Within the scope of the study, cords belonging to 4 different pregnant women who gave birth at term by elective cesarean section at the Ege University Department of Obstetrics and Gynecology will be collected. Then, the cord samples will be washed with some cold phosphate buffer saline (PBS) in a sterile container to completely clean the clot residues and blood on them. The entire cord will first be cut lengthwise and two arteries and one vein in the cord will be dissected to reveal the Wharton jelly region. Then, MSC isolation will be performed with the explant culture method detailed below. After the dissection process, the tissue pieces will be placed in 300 cm2 flasks with the Wharton Jelly side facing the ground. Approximately 2 cm areas will be left between the tissue pieces. After this process, the flasks will be incubated in incubators at 37°C and the tissue pieces will be allowed to stick to the flask surface. After the flasks reach 80-90% confluency, the exosome-rich media (Condition media) in the flask will be collected for exosome isolation. The cells that synthesize the relevant exosomes will also be removed by trypsinization and turned into samples for characterization analyses. Flow cytometric commercial antibodies will be used for the characteristic analysis of the isolated MHCs. According to the kit protocol, the cells will be labeled with four different antigen antibodies (CD44, CD73, CD90 and CD105) according to the kit procedures. As the negative antibody group, CD34, CD45, CD11b, CD19, CD14, CD79a and HLA-DR, which are antigens that should not be synthesized in mesenchymal stem cells, will be used. After the Condition Medium (CM) collected for exosome isolation in MHCs is centrifuged at 300 g, +4°C for 10 minutes, the pellet will be removed and centrifuged again at 2,000 g, +4°C for 20 minutes. After this process, ultracentrifugation processes will be carried out. Condition media will be transferred to ultracentrifuge tubes and centrifuged at 10,000 g at +4°C for 30 minutes. After centrifugation, the supernatant will be collected and ultracentrifuged at 100,000 g at +4°C for 70 minutes. After ultracentrifugation, the isolated exosomes will be suspended with isotonic sodium chloride. The suspended exosomes will be passed through 0.22 µm pore diameter filters against any large particles. Protein concentration will be determined using Pierce BCA Protein Assay Kit according to the manufacturer's instructions. First, to create a standard graph, serum albumin proteins will be prepared at different concentrations (0 µg/ml, 125 µg/ml, 250 µg/ml, 500 µg/ml, 750 µg/ml, 1000 µg/ml, 1500 µg/ml, 2000 µg/ml) and pipetted into 96-well microplates in 25 µl volumes. Then, 25 µl volumes of exosome samples will be pipetted into the other wells of the 96-well microplates. In accordance with the kit manufacturer's instructions, 200 µl of the prepared BCA working reagent will be pipetted into the wells containing the standards and samples. The prepared microplate will be incubated at 37°C for 30 minutes to ensure that the reaction takes place. Purple - dark purple color changes will occur in the wells containing the protein samples. Spectrophotometric measurement will be made to obtain this change as numerical data. In the animal experiment model, there will be 3 experimental groups including the control group in the grouping of rabbits. A total of 16 New Zealand rabbits will be included in the study, 4 in the control group and 6 in the other groups. All rabbits will be given 35-50 mg/kg ketamine and 5-10 mg/kg xylazine intramuscularly (IM) or subcutaneously (SC) for sedation. After topical anesthesia (proparacaine hydrochloride), under sterile conditions, 5% povidone iodine will be applied to the right eyes of animals in all groups, and then the corneal epithelium in the central 6.5 mm circular area will be mechanically debrided completely to create an experimental corneal wound model. Exosomes at a concentration of 1 × 106/μl will be applied to the right eyes of rabbits in Group 1, autologous serum in Group 2, and artificial tears in Group 3. All drops will be applied in 4 doses per day (1 drop at 6-hour intervals). Preservative-free antibiotic drops will be applied to the debrided eyes of all groups 4 times per day to prevent secondary infections. The clinical appearance of the corneas of all groups will be photographed every day for 6 days, and the duration of corneal re-epithelialization, corneal re-epithelialization area, and amount of corneal opacity will be evaluated. Chemosis, conjunctival hyperemia, and eyelid edema findings will be evaluated and scored between 0 and 3 (0=no findings, 1=mild findings, 2=moderate findings, 3=severe findings). At the end of a total of 7 days of follow-up, rabbits will be sacrificed with intravenous high-dose anesthetic administration. After the eyes are enucleated while preserving the globe integrity, they will be placed in 10% formaldehyde and transferred for examination under light microscopy. After fixation, the samples embedded in paraffin and sectioned will be stained with hematoxylin eosin dye. The corneal surface will be examined semi-quantitatively under light microscopy in terms of wound healing. Edema, mononuclear inflammatory cell infiltration, fibroplasia, neovascularization, and reactive regenerative changes will be evaluated and scored. Digital photographs of the sections will be taken and recorded. The normality and homogeneity analyses of the obtained data will be performed, the appropriate statistical method will be determined and analyzed with the SPSS program (SPSS Inc., Chicago, IL, USA). When p<0.05, the changes will be considered statistically significant.

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