The success in the clinical uses of PRPs, such as PRGF, could be due to their proliferative effects on cells like Mller glia

The success in the clinical uses of PRPs, such as PRGF, could be due to their proliferative effects on cells like Mller glia. It has also been suggested that PRGF induces neuroprotection by activating the anti-apoptotic PI3K/Akt signaling pathway, and/or through SNT-207707 reducing caspase-3, promoting proliferation and survival in main neuronal cultures. main cell cultures of porcine RGCs and Mller cells, as well as on co-cultures of these two cell types. Moreover, the inflammatory component of PRGF was analyzed and the cytokines in the different PRGFs were quantified. In addition, we set out to determine if blocking the inflammatory components of PRGF alters its effect on the cells in culture. The presence of PRGF compromises RGC survival in real cultures and in co-culture with Mller cells, but this effect was reversed by heat-inactivation of the PRGF. The detrimental effect of PRGF on RGCs could be in part due to the presence of cytokines and specifically, to the presence of pro-inflammatory cytokines that compromise their survival. However, other factors are likely to be present in the PRGF that have a deleterious effect on the RGCs since the exposure to antibodies against these cytokines were insufficient to protect RGCs. Moreover, PRGF promotes Mller cell survival. In conclusion, PRGF hinders the survival of RGCs in the presence or absence of Mller cells, yet it promotes Mller cell survival that could be the reason of retina healing observed in the treatments, with some cytokines possibly implicated. Although PRGF could stimulate tissue regeneration, further studies should be performed to evaluate the effect of PRGF on neurons and the implication of its potential inflammatory role in such processes. 20) and blood (5) were obtained at a local slaughterhouse and the eyes were transported to the laboratory on ice in CO2-impartial medium (Life Technologies, Carlsbad, CA, United States) with 0.1% gentamicin. The retinas were obtained from the eyes 1C2?h after enucleation. All animal experimentation adhered to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Human and Pig PRGF This study was carried out by qualified staff in strict accordance with the tenets of the Helsinki Declaration on Biomedical Research Involving Human Subjects. Before blood collection, signed informed consent was obtained from all subjects once the SNT-207707 nature of the study and the possible consequences of the study had been explained to them. Human blood samples were obtained through antecubital vein puncture and PRGF was obtained as explained previously (Anitua et al., 2015a), with some minor modifications. Briefly, human (3) DUSP8 and porcine (5) blood was collected in 5?ml tubes containing 3.8% (wt/vol) sodium citrate. Samples were centrifuged at 460?g for 8?min at room temperature and the plasma portion containing platelets was separated, avoiding the buffy coat and erythrocytes. The plasma portion (1?ml) was reconstituted for 1?h at 34C with 50?l calcium chloride (Braun Medical, Melsungen, Germany) in glass tubes, and the supernatant released was collected after centrifugation at 460?g for 15?min. Finally, part of the total volume of the PRGF obtained was heat-inactivated at 56C for 60?min, following a previously published protocol (Anitua et al., 2014a), and both the samples (PRGF and inactive PRGF) were filtered through a filter with a 0.2?m pore size of (Fisher Scientific, Madrid, Spain), aliquoted and stored at ?80C. Cell Culture Retinal cell cultures were prepared according to the method reported previously (Garcia et al., 2002; Ruzafa et al., 2018), with the following minor modifications. Three types of cultures were used: 1) RGCs cultured in B27-supplemented Neurobasal-A medium (Life Technologies, Carlsbad, CA, United States); 2) a co-culture of RGCs and Mller cells in B27-supplemented Neurobasal-A medium with 10% fetal bovine serum (FBS: Life Technologies, Carlsbad, CA, United States); and 3) Mller SNT-207707 cell cultures in DMEM (Life Technologies, Carlsbad, CA, United States) supplemented with 10% FBS. 1% L-glutamine (2?mM) and 0.1% gentamycin (50?mg/ml) were added to all media. The retinas were dissected out and 8?mm diameter pieces were obtained with a dissecting trephine.