A proprietary GMP human platelet lysate for the expansion of dermal fibroblasts for clinical applications.

Recent years have witnessed the introduction of ex vivo expanded dermal fibroblasts for several cell therapy and tissue-engineering applications, including the treatment of facial scars and burns, representing a promising cell type for regenerative medicine. We tested different in-house produced human platelet lysate (HPL) solutions against fetal bovine serum as supplements for in vitro fibroblast expansion by comparing cell yield, molecular marker expression, extracellular matrix (ECM) generation, genomic stability and global gene expression. Our in-house produced HPL supported fibroblast growth at levels similar to those for FBS and commercial HPL products and was superior to AB human serum. Cells grown in HPL maintained a fibroblast phenotype (VIM+, CD44+, CD13+, CD90+), ECM generation capacity (FN+, COL1+) and a normal karyotype, although gene expression profiling revealed changes related to cell metabolism, adhesion and cellular senescence. The HPL manufacturing process was validated within a GMP compliant system and the solution was stable at -80ºC and -20ºC for 2 years. Dermal fibroblasts expanded in vitro with HPL maintain a normal karyotype and expression of fibroblast markers, with only minor changes in their global gene expression profile. Our in-house produced GMP-HPL is an efficient, safe and economical cell culture supplement that can help increase the healthcare activity of blood transfusion centers through the re-use of transfusional plasma and platelets approaching their expiration date. Currently, our HPL solution is approved by the Spanish Agency of Medicines and Medical Devices and is being used in the manufacture of cell therapy products.Abbreviations: AB plasma: plasma group AB; ABHS: AB Human Serum; ABHS+GF: AB Human Serum supplemented with growth factors; ANOVA: Analysis of variance; ATMPs: Advanced Therapies for Medicinal Products; CPE: cytopathic effect; DEGs: Differentially expressed genes; DMEM: Dulbecco's modified Eagle's Medium; ECM: Extracellular matrix; ELISA: enzyme-linked immunosorbent assay; FBS: Fetal bovine serum; FDR: False discovery rate; FGF: Fibroblast growth factor; GMP: Good manufacturing practice; HPL: Human platelet lysate; HPL-CM: commercial human platelet lysate; MSCs: mesenchymal stem cells; NEAA: non-essential amino acids; P/S: penicillin/streptomycin; PBS: phosphate buffered saline; PC: leukodepleted platelet concentrate; PCR: polymerase chain reaction; PDGF: Platelet-derived growth factor; PDGFRA: Platelet-derived growth factor receptor alpha; qPCR: quantitative polymerase chain reaction; RNA: Ribonucleic acid; RT: Room temperature; TAC: Transcriptome analysis console; TGF-ß: Transforming growth factor beta.

Inactivation of human plasma alters the structure and biomechanical properties of engineered tissues.

Fibrin is widely used for tissue engineering applications. The use of blood derivatives, however, carries a high risk of transmission of infectious agents, necessitating the application of pathogen reduction technology (PRT). The impact of this process on the structural and biomechanical properties of the final products is unknown. We used normal plasma (PLc) and plasma inactivated by riboflavin and ultraviolet light exposure (PLi) to manufacture nanostructured cellularized fibrin-agarose hydrogels (NFAHs), and then compared their structural and biomechanical properties. We also measured functional protein C, prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT) and coagulation factors [fibrinogen, Factor (F) V, FVIII, FX, FXI, FXIII] in plasma samples before and after inactivation. The use of PLi to manufacture cellularized NFAHs increased the interfibrillar spacing and modified their biomechanical properties as compared with cellularized NFAH manufactured with PLc. PLi was also associated with a significant reduction in functional protein C, FV, FX, and FXI, and an increase in the international normalized ratio (derived from the PT), APTT, and TT. Our findings demonstrate that the use of PRT for fibrin-agarose bioartificial tissue manufacturing does not adequately preserve the structural and biomechanical properties of the product. Further investigations into PRT-induced changes are warranted to determine the applications of NFAH manufactured with inactivated plasma as a medicinal product.

Brain Organoids to Evaluate Cellular Therapies.

Animal models currently used to test the efficacy and safety of cell therapies, mainly murine models, have limitations as molecular, cellular, and physiological mechanisms are often inherently different between species, especially in the brain. Therefore, for clinical translation of cell-based medicinal products, the development of alternative models based on human neural cells may be crucial. We have developed an in vitro model of transplantation into human brain organoids to study the potential of neural stem cells as cell therapeutics and compared these data with standard xenograft studies in the brain of immunodeficient NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice. Neural stem cells showed similar differentiation and proliferation potentials in both human brain organoids and mouse brains. Our results suggest that brain organoids can be informative in the evaluation of cell therapies, helping to reduce the number of animals used for regulatory studies.

Retrieval of germinal zone neural stem cells from the cerebrospinal fluid of premature infants with intraventricular hemorrhage.

Intraventricular hemorrhage is a common cause of morbidity and mortality in premature infants. The rupture of the germinal zone into the ventricles entails loss of neural stem cells and disturbs the normal cytoarchitecture of the region, compromising late neurogliogenesis. Here we demonstrate that neural stem cells can be easily and robustly isolated from the hemorrhagic cerebrospinal fluid obtained during therapeutic neuroendoscopic lavage in preterm infants with severe intraventricular hemorrhage. Our analyses demonstrate that these neural stem cells, although similar to human fetal cell lines, display distinctive hallmarks related to their regional and developmental origin in the germinal zone of the ventral forebrain, the ganglionic eminences that give rise to interneurons and oligodendrocytes. These cells can be expanded, cryopreserved, and differentiated in vitro and in vivo in the brain of nude mice and show no sign of tumoral transformation 6 months after transplantation. This novel class of neural stem cells poses no ethical concerns, as the fluid is usually discarded, and could be useful for the development of an autologous therapy for preterm infants, aiming to restore late neurogliogenesis and attenuate neurocognitive deficits. Furthermore, these cells represent a valuable tool for the study of the final stages of human brain development and germinal zone biology.

Nanostructured fibrin agarose hydrogel as a novel haemostatic agent.

Blood loss remains a major concern during surgery and can increase the morbidity of the intervention. The use of topical haemostatic agents to overcome this issue therefore becomes necessary. Fibrin sealants are promising haemostatic agents due to their capacity to promote coagulation, but their effectiveness and applicability need to be improved. We have compared the haemostatic efficacy of a novel nanostructured fibrin-agarose hydrogel patch, with (c-NFAH) or without cells (a-NFAH), against two commercially available haemostatic agents in a rat model of hepatic resection. Hepatic resections were performed by making short or long incisions (mild or severe model, respectively), and haemostatic agents were applied to evaluate time to haemostasis, presence of haematoma, post-operative adhesions to adjacent tissues, and inflammation factors. We found a significantly higher haemostatic success rate (time to haemostasis) with a-NFAH than with other commercial haemostatic agents. Furthermore, other relevant outcomes investigated were also improved in the a-NFAH group, including no presence of haematoma, lower adhesions, and lower grades of haemorrhage, inflammation, and necrosis in histological analysis. Overall, these findings identify a-NFAH as a promising haemostatic agent in liver resection and likely in a range of surgical procedures.