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.This work was supported by preclinical research funds from the Regional Government of Andalusia through the Andalusian Initiative for Advanced Therapie

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

Acknowledgments The authors are grateful to Echevarne Laboratories for the coagulation experiments and to Gloria Carmona and Rosario Sánchez Pernaute as well as all members of the Unidad de Producción y Reprogramación Celular (UPRC) for technical help and support. All figures were created through BioRender.com accessed on 1 September 2021.Funding This research was funded by the Spanish Plan Nacional de Investigación Científica, Desarrollo e Innovación Tecnológica (I+D+i) from Ministerio de Ciencia, Innovación y Universidades (Instituto de Salud Carlos III), grants FIS PI17/0391, RTC-2017- 6658-1, PI20/0317 and ICI19/00024 (BIOCLEFT), co-financed by Fondo Europeo de Desarrollo Regional ERDF-FEDER, European Union and PE-0395-2019 from Consejería de Salud y Familias, Junta de Andalucía, Spain.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.Spanish Plan Nacional de Investigación Científica, Desarrollo e Innovación Tecnológica (I+D+i) from Ministerio de Ciencia, Innovación y Universidades (Instituto de Salud Carlos III), grants FIS PI17/0391, RTC-2017- 6658-1, PI20/0317 and ICI19/00024 (BIOCLEFT)Fondo Europeo de Desarrollo Regional ERDF-FEDEREuropean UnionPE-0395-2019 from Consejería de Salud y Familias, Junta de Andalucía, Spai

Preclinical evaluation of the safety and potency of neural stem cells from the germinal zone (Gz-NSC) for the treatment of intraventricular hemorrhage (IVH) consequences.

Motivation: Intraventricular hemorrhage (IVH) is a common cause of morbidity and mortality in premature infants with no available treatment. After IVH, there is a rupture of the germinal zone into the ventricles that entail the loss of neural stem cells (Gz-NSC). These Gz-NSC can be retrieved from the cerebrospinal fluid (CSF) of IVH patients, obtained after the therapeutic neuroendoscopic lavage performed in these patients to decrease intracranial pressure and that is usually discarded. We have found that Gz-NSC have the potential to differentiate into neuroblasts, oligodendrocyte precursors and few astrocytes when grafted into human brain organoids from iPSCs and mouse brains (1,2). We are evaluating the safety and efficacy profile of CSF-derived Gz-NSC in order to develop a cell therapy for IVH patients.Methods: To examine the differentiation potency of Gz-NSC, we used immunofluorescence assays, fluorescence microspy techniques and computer analysis (ImageJ) to expand previous data, increase sample size, and quantify cell differentiation of grafted Gz-NSC cells in mouse brains and human brain organoids derived from iPSCs.To study the safety profile, flow cytometry assays were carried on to analyze Gz-NSC cell proliferation (Ki67) and immunogenicity (CD80,CD86,CD40, major histocompatibility complex class II (MHC-II)).Results: Based on the immunofluorescence assays, we have found less cells expressing doublecortin, an immature neural protein, and more cells expressing parvalbumin, an interneuron marker, in human brain organoids compared to animal models, suggesting that host can influence cell fate.On the other hand, in order to study the immunogenicity of the Gz-NSC (safety profile), we have analyzed the expression of MHC-II and co-stimulatory molecules (CD80, CD86, CD40) in Gz-NSC before and after in vitro differentiation. Flow cytometry assays revealed Gz-NSC do not express co-stimulatory molecules and express different levels of MHC-II that are reduced when differentiated in vitro, which decreases the probability of an immune response in a future Gz-NSC based cell therapyConclusions: Taking into account that Gz-NSC have the potency to differentiate to a wide range of cerebral cell linages in both, human organoids and animal models, and are weakly immunogenic, an autologous Gz-NSC cell therapy could be a promising opportunity for IVH patients to overcome some of the neurocognitive problems associated to their condition

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

Eficacia y seguridad del uso de un hidrogel de fibrina y agarosa nanoestructurado como parche hemostático en cirugía hepática

INTRODUCCIÓN: La pérdida de sangre en pacientes sometidos a resección o traumatismo hepático es una de las principales causas de morbilidad y mortalidad perioperatoria. En este contexto, los agentes hemostáticos tópicos resultan una herramienta esencial, aunque cualquier mejora de éstos en términos de reducción del tiempo de hemostasia y/o resangrado, facilidad de uso y aplicabilidad, y mejora de respuesta inflamatoria y tisular, podría representar un gran impacto no sólo en los costes hospitalarios sino también en la calidad de vida de los pacientes. Durante los últimos años, los hidrogeles de fibrina y agarosa nanoestructurados (NFAH) se han utilizado como sustitutos artificiales de tejidos. Los resultados preclínicos obtenidos en animales despiertan especial interés en biomedicina al demostrar cualidades de biocompatilibilidad y biodegradabilidad. Estos NFAH fabricados con plasma humano, agarosa tipo VII, ácido tranexámico y cloruro cálcico están compuesto principalmente de fibrina, un polímero cuya función principal es bloquear la pérdida masiva de sangre y participar en la reparación de tejidos dañados tras la ruptura vascular. Por tanto, surge la hipótesis que el NFAH tenga efecto hemostático, no sólo por un potencial efecto bioactivo debido a su composición sino también por un efecto biomecánico protector de la herida. OBJETIVO: El objetivo general de esta Tesis Doctoral es estudiar la eficacia y la seguridad de este NFAH como parche hemostático en cirugía hepática. METODOLOGÍA: Para ello se fabricaron hidrogeles de fibrina y agarosa nanoestructurados con célula (c-NFAH) y sin células (a-NAH) que se aplicaron a dos modelos experimentales de resección hepática en rata: un modelo de resección moderado (con una incisión de 0,5 cm de longitud) y uno severo (con una incisión de 1,5 cm de longitud). Dos agentes hemostáticos comerciales ampliamente utilizados en cirugía hepática, Hemopatch y Tachosil, fueron incluidos en los estudios. Las variables analizadas fueron tiempo en alcanzar la hemostasia (cese de sangrado) tras la aplicación del hemostático, así como la presencia de hematomas, adherencias postoperatorias, factores de inflamación en sangre (CRP, IL-1β Y TNF-α) y parámetros histológicos al momento del sacrificio (hemorragia, inflamación y necrosis). La temperatura corporal e incremento de peso fueron también analizados durante el periodo de estabulación de los animales. Adicionalmente, se realizaron estudios de preservación de los NFAH que permitieran mantener sus propiedades hemostáticas a largo plazo. RESULTADOS/CONCLUSIONES: Los tiempos de hemostasia presentados por los NFAH fueron significativamente menores a los obtenidos por los agentes hemostáticos comerciales Hemopatch y Tachosil en ambos modelos de resección hepática, demostrando una eficacia hemostática superior. Los animales tratados con apósitos NFAH presentaron una respuesta tisular e inflamatoria favorable (hematomas, adherencias, inflamación e histología), en ocasiones mejor a la presentada por animales tratados con los hemostáticos comerciales. De forma global, el apósito a-NFAH resultó ser el mejor agente hemostático del estudio y la congelación resultó ser un método adecuado de preservación que mantuvo la estructura tridimensional del apósito a- NFAH. Los resultados obtenidos en esta Tesis Doctoral demuestran que NFAH posee excelentes propiedades hemostáticas, con ventajas como su biocompatibilidad, propiedades biomecánicas y bajo coste, y creemos que este nuevo producto será parte del arsenal de agentes hemostáticos usados en múltiples procedimientos quirúrgicos en el futuro

Co-adaptation of seed dormancy and flowering time in the arable weed Capsella bursa-pastoris (shepherd's purse)

Rémy Bernard, Bost Jean-Pierre, Tranoy Laurence. Chronique gallo-romaine. In: Revue des Études Anciennes. Tome 105, 2003, n°2. pp. 589-649

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.This work was supported by preclinical research funds from the Regional Government of Andalusia through the Andalusian Initiative for Advanced Therapies.Peer reviewe

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

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-β: Transforming growth factor beta