Search

The extracellular matrix (ECM) obtained by decellularization provides scaffolds with the natural complex architecture and biochemical composition of the target organ. Whole kidney decellularization by perfusion uses the vasculature to remove cells leaving a scaffold that can be recellularized with patient-specific cells. However, decellularization and recellularization are highly complex processes that require intensive optimization of various parameters. In pursuit of this, a huge number of animals must be sacrificed. Therefore, we used precision-cut kidney slices (PCKS) as a source of natural scaffolds, which were decellularized by immersion in chemical reagents allowing the examination of more parameters with less animals. However, chemical reagents have a damaging effect on the structure and components of the ECM. Therefore, this study aimed at investigating the effects of physical treatment methods on the effectiveness of PCKS decellularization by immersion in chemical reagents (CHEM). PCKS were treated physically before or during immersion in chemicals (CHEM) with high hydrostatic pressure (HHP), freezing–thawing cycles (FTC) or in an ultrasonic bath system (UBS). Biochemical and DNA quantification as well as structural evaluation with conventional histology and scanning electron microscopy (SEM) were performed. Compared to decellularization by CHEM alone, FTC treatment prior to CHEM was the most effective in reducing DNA while also preserving glycosaminoglycan (GAG) content. Moreover, while UBS resulted in a comparable reduction of DNA, it was the least effective in retaining GAGs. In contrast, despite the pretreatment with HHP with pressures up to 200 MPa, it was the least effective in DNA removal. Histological scoring showed that HHP scaffolds received the best score followed by UBS, FTC and CHEM scaffolds. However further analysis with SEM demonstrated a higher deterioration of the ultrastructure in UBS scaffolds. Altogether, pretreatment with FTC prior to CHEM resulted in a better balance between DNA removal and structural preservation.

Background: Immune cell dysfunction is a central part of immune paralysis in sepsis. Granulocyte concentrate (GC) transfusions can induce tissue damage via local effects of neutrophils. The hypothesis of an extracorporeal plasma treatment with granulocytes is to show beneficial effects with fewer side effects. Clinical trials with standard GC have supported this approach. This ex vivo study investigated the functional properties of purified granulocyte preparations during the extracorporeal plasma treatment. Methods: Purified GC were stored for up to 3 days and compared with standard GC in an immune cell perfusion therapy model. The therapy consists of a plasma separation device and an extracorporeal circuit. Plasma is perfused through the tubing system with donor immune cells of the GC, and only the treated plasma is filtered for re-transfusion. The donor immune cells are retained in the extracorporeal system and discarded after treatment. Efficacy of granulocytes regarding phagocytosis, oxidative burst as well as cell viability and metabolic parameters were assessed. Results: In pGC, the metabolic surrogate parameters of cell functionality showed comparable courses even after a storage period of 72 h. In particular, glucose and oxygen consumption were lower after extended storage. The course of lactate dehydrogenase concentration yields no indication of cell impairment in the extracorporeal circulation. The cells were viable throughout the entire study period and exhibited preserved phagocytosis and oxidative burst functionality. Conclusion: The granulocytes demonstrated full functionality in the 6 h extracorporeal circuits after 3 days storage and in septic shock plasma. This is demonstrating the functionality of the system and encourages further clinical studies.

Background: Use of donor granulocyte concentrate (GC) has been limited due to its short storage time of 6-24 h, which is partially due to residual red blood cells (RBCs) and platelets and the resulting lactate production leading to an acidotic milieu. To increase this storage time, we developed a closed system procedure compatible with standard blood bank technologies to remove RBC and platelets and to enrich the GC. Methods: Standard GCs (sGCs) were sedimented, washed twice with 0.9% sodium chloride (NaCl), and resuspended in blood group-identical fresh frozen plasma. The resulting purified GCs (pGCs) were then stored in platelet bags at a cell concentration of about 5 × 107 ± 1.8 × 107 leukocytes/ml without agitation at room temperature for up to 72 h. Cell count and viability, pH, blood gases, phagocytosis, and oxidative burst were monitored daily. Results: A significant reduction in RBC (98%) through sedimentation, and platelets (96%) by washing, purified the white blood cell (WBC) population and enriched the granulocytes to 96% of the WBC in the pGC. After 72 h of storage, over 90% of the initial WBC count of pGC remained, was viable (>97%), and the granulocytes exhibited a high phagocytosis and oxidative burst functionality, comparable to sGC after 24 h. Conclusion: Purification extends the maximum storage period of GC from 24 to 72 h and may therefore improve the availability of GC and its clinical use.

Additional file 1. Information collected form each study, additional information used. Figure S1. Geometry of the network and included studies for the analysis of 1-month overall survival. Figure S2. League table of 1-month overall survival. Figure S3. Cumulative ranking curves and SUCRA values for 1-month overall survival. Figure S4. Rankograms for 1-month overall survival. Figure S5. Geometry of the network and included studies for the analysis of 3-month transplant-free survival. Figure S6. League table of 3-month transplant-free survival. Figure S7. Ranking of treatments for 3-month transplant-free survival. Figure S8. Rankograms for 3-month transplant-free survival. Figure S9. Geometry of the network and included studies for the analysis of 1-month transplant-free survival. Figure S10. League table of 1-month transplant-free survival. Figure S11. Ranking of treatments for 1-month transplant-free survival. Figure S12. Rankograms for 1-month transplant-free survival. Figure S13. Risk of bias assessment for overall survival. Figure S14. Risk of bias assessment for transplant-free survival. Table S1. Quality of evidence. Table S2. Assessment of hepatic encephalopathy in the included studies. Table S3. Assessment of bilirubin reduction in the included studies. Table S4. Assessment of ammonia reduction in the included studies. Table S5. Assessment of creatinine reduction in the included studies. Figure S15. Forrest plots for 3-month overall survival. Figure S16. Forrest plots for 1-month overall survival. Figure S17. Forrest plots for 3-month transplant-free survival. Figure S18. Forrest plots for 1-month transplant-free survival. Figure S19. Funnel plot and Egger’s test for 3-month overall survival. Figure S20. Funnel plot and Egger’s test for 1-month overall survival. Figure S21. Cummulative ranking curves and SUCRA for methodology-based evaluation. Figure S22. Methodology-based evaluation league tables.