Selective loss of microvesicles is a major issue of the differential centrifugation isolation protocols

Microvesicles (MVs) are large extracellular vesicles differing in size, cargo and composition that share a common mechanism of release from the cells through the direct outward budding of the plasma membrane. They are involved in a variety of physiological and pathological conditions and represent promising biomarkers for diseases. MV heterogeneity together with the lack of specific markers had strongly hampered the development of effective methods for MV isolation and differential centrifugation remains the most used method to purify MVs. In this study, we analysed the capacity of the differential centrifugation method to isolate MVs from cell-conditioned medium using flow cytometry and TEM/AFM microscopy. We found that the loss of MVs (general population and/or specific subpopulations) represents a major and underestimate drawback of the differential centrifugation protocol. We demonstrate that the choice of the appropriate rotor type (fixed-angle vs swinging-bucket) and the implementation of an additional washing procedure to the first low-speed centrifugation step of the protocol allow to overcome this problem increasing the total amount of isolated vesicles and avoiding the selective loss of MV subpopulations. These parameters/procedures should be routinely employed into optimized differential centrifugation protocols to ensure isolation of the high-quantity/quality MVs for the downstream analysis/applications.

Autologous platelet-rich plasma action on skin autografts in horses

ABSTRACT: This study evaluated the action of autologous platelet-rich plasma (PRP) on cutaneous wounds, containing skin autografts, in the gluteal region of horses. Seven healthy horses were used. Two 6 x 6cm cutaneous wounds were produced on each side of the gluteal region. Eight days after wound induction, grafts were performed with skin fragments harvested from the neck, as well as the application of PRP, prepared by double-centrifugation protocol. Wounds with autografts on the left side received PRP (group T), and those with autografts on the right side did not receive treatment (group C). Macroscopic and microscopic evaluations were performed, considering the integration of autografts and retraction of wound edges, as well as neovascularization, inflammatory infiltrate, young fibroblasts, collagenization, reepithelization and autografts integration. There was no difference between the groups (P > 0.05) in relation to most macroscopic and microscopic variables. However, neovascularization was significantly greater (p = 0.0191) in group T, on the 14th day after grafting. It is concluded that PRP favors the process of skin repair with autografts in horses, since it increases the neovascularization in the initial phase of wound healing. Furthermore, the PRP seems to positively influence the integration of the skin autografts and the retraction of the wound edges.

THE G-FORCE CONUNDRUM IN PRF GENERATION- MANAGEMENT OF A PROBLEM HIDDEN IN PLAIN SIGHT

ABSTRACTAimA force of 400g at 2700 RPM results in an optimum leucocyte and platelet-rich fibrin (L-PRF). Most of centrifuges with varying characteristics generate a g-force in excess of 700g at 2700 RPM. In this context, the study explores the effect of the original centrifugation protocol and a modified protocol tailor-made to lower the RPM to generate a g-force of ~400g on platelet concentration, clot size and growth factors release in L-PRF prepared in two different commercially available centrifuges.Materials and Methods25 subjects each were assigned to the following groups; R1 and R2 where L-PRF was obtained from two laboratory swing-out centrifuges (Remi 8C® & Remi C854®, Mumbai, India) respectively. PRF was obtained from each subject within a group using two protocols; Original (O) protocol: conforming to the original centrifugation cycle (2700 RPM for 12 min) and Modified (M) protocol. Clot size, growth factor estimation and platelet counts were measured at 20, 40 and 60 mins from all the L-PRF clots.ResultsAt the third time period (40–60min), there were no significant differences in clot sizes with the original protocol (p=0.09), but a highly significant difference was noticed with the modified protocol in both the centrifuges (p=0.001). Our results showed an increased concentration of VEGF and EGF with modified protocol than with original protocol with both the centrifuges (p=0.001). By the end of second and third time periods, more platelet concentration was observed with modified protocol than with the original protocol in both the centrifuges (p=0.001)ConclusionThis study infers that the centrifuge type and RCF can affect the quality and quantity of cells and growth factors and an optimum relationship between g-force and RPM should be maintained in order to obtain L-PRF with adequate cell viability and optimum growth factor release.

Effect of Myo-inositol and N-acetyl-L-cysteine on processed human spermatozoa for use in modern methods of fertility treatment

Background and aims: Today, the prevalence of structural and functional disorders of sperm and subsequently the possibility of occurrence of autism, spontaneous abortion, and infertility has increased dramatically. This study aimed to investigate the potential effects of myoinositol and N-acetyl-L-cysteine (NAC) on the improvement of processed spermatozoa indices. Methods: In this study, 63 patients with male factor infertility were studied in Hazrat Zahra Infertility Center of Shahrekord. To do liquefaction, the semen sample was incubated at 37°C for 20 minutes. The Density Gradient Centrifugation protocol was used to process sperm. The processed sperm samples were incubated at 37°C for 20 minutes in four groups of control, myo-inositol (2 mg/mL), NAC antioxidant (10 Mm/L) and myo-inositol+NAC. All biological parameters of sperm were evaluated using Makler slide, SQA-V sperm analyzer gold version 2.48, TUNEL assay, and Toluidine blue staining. Results: In the present study, 63 men aged 22-58 years with infertility complaints were studied. The NAC group and the myo-inositol group exhibited significantly higher motility in comparison with the control group (P<0.001). The NAC group also had a significantly higher number of TUNEL-negative cells and a lower number of TUNEL-positive cells than the control group (P<0.01), but in other variables, the differences were not statistically significant. The morphology, S0, S1, S2 and staining types in the intervention groups did not show any significant difference compared to those in the control group (P > 0.05). Conclusion: NAC and myo-inositol were able to improve certain indices such as sperm motility and integrity; therefore, the quality of sperm could be improved by these drugs to treat infertility.

Does Double Centrifugation Lead to Premature Platelet Aggregation and Decreased TGF-β1 Concentrations in Equine Platelet-Rich Plasma?

Blood-derived autologous products are frequently used in both human and equine medicine to treat musculoskeletal disorders. These products, especially the platelet-rich plasma (PRP), may contain high concentrations of growth factors (GFs), and thus improve healing in several tissues. Nevertheless, the procedures for preparation of PRP are currently non-standardized. Several protocols, which are based on distinct centrifugation patterns (rotation speed and time), result in PRPs with different characteristics, concerning platelet and GFs concentrations, as well as platelet activation. The aim of the present study was to compare two different protocols for PRP preparation: protocol (A) that is based on a single-centrifugation step; protocol (B), which included two sequential centrifugation steps (double-centrifugation). The results here reported show that the double-centrifugation protocol resulted in higher platelet concentration, while leukocytes were not concentrated by this procedure. Although platelet activation and aggregation were increased in this protocol in comparison to the single-centrifugation one, the TGF-β1 concentration was also higher. Pearson’s correlation coefficients gave a significant, positive correlation between the platelet counts and TGF-β1 concentration. In conclusion, although the double-centrifugation protocol caused premature platelet aggregation, it seems to be an effective method for preparation of PRP with high platelet and TGF-β1 concentrations.

Production and evaluation of leukocyte- and thrombocyte-rich fibrin membranes in birds

The aim of this study was the preparation and histological evaluation of Leukocyte- and Thrombocyte-Rich Fibrin (L-TRF) membranes obtained from the blood of four bird species. Forty adult healthy birds were divided into four groups of equal size: G1 – macaws, G2 – domestic chickens, G3 – parrots, G4 – toco toucans. A total of 0.5 mL of blood was collected from each bird, put into a glass tube without anticoagulant and centrifuged at 3000 rpm for 10 min. L-TRF membranes produced after compression of the clot were processed for histological analysis. The ratio of thrombocytes/area was not significantly different among Groups G2, G3 and G4, but a significant difference was found between Groups G1 and G2 with the highest thrombocyte concentration/area in G1. The groups did not differ statistically in the number of leukocytes/area. The fibrin-to-cells ratio did not vary statistically among Groups G1, G2 and G3, but this ratio was significantly higher in Group G4 than in the other groups. The thrombocyte-to-leukocyte ratio was the highest in Group G1, but it did not differ among Groups G2, G3 and G4. In conclusion, the centrifugation protocol allowed the production of L-TRF membranes in the four bird species studied. Histologically, cell ratios were analogous in domestic chickens and parrots, and macaws had the highest ratio of thrombocytes.

EVALUATION OF CENTRIFUGING REGIMES FOR THE PURPOSE OF OPTIMIZING THE PLATELET RICH PLASMA HARVESTING PROTOCOL

Aim: Based on the classical principles, to determine the optimal conditions for centrifugation, PRP harvesing (platelet-rich plasma). To conduct a quantitative assessment of the substrate obtained under different conditions of centrifugation. Materials and methods. Based on the basic principles of obtaining platelet-rich plasma (PRP) by centrifuging in containers with an anticoagulant followed by phase separation to obtain the final substrate, the efficiency of the technique under the conditions of single and double centrifugation as well as under different conditions of acceleration and centrifugation was evaluated. Blood for follow-up was collected from 20 healthy volunteers (11 men, 9 women) average 25.3±4.1 in syringes of LuerLock design with ACD-A anticoagulant solution, and centrifuged. Centrifugation was carried out under controlled conditions using a centrifuge with rotating bowls of the rotor. Centrifugation was performed at an acceleration of 100-400g in time intervals up to 20 minutes. Activation of the substrate was performed with calcium chloride solution. Quantitative evaluation of platelets of whole blood and the final substrate of PRP was carried out with a semi-automatic analyzer. Results. The obtained results demonstrate the maximum level of harvesting efficiency when performing double centrifugation in the 150g×15 min+250g×10 min mode. Subject to this centrifugation protocol, it is possible to obtain a substrate that complies with the standardized requirements for PRP. The maximum level of an increase in the number of platelets in the substrate in comparison with whole blood is determined at the level of ×4.36 with concentration (volume reduction) x5 in comparison with the volume of whole blood. Conclusions. This study demonstrated the advantage of double centrifuging modes over single modes. According to the results of the study, it was possible to determine the conditions for an optimal double-centrifugation mode (acceleration and duration), which allows us to achieve the most efficient concentration of the substrate.