Culture of human cell lines by a pathogen-inactivated human platelet lysate

Alternatives to the use of fetal bovine serum (FBS) have been investigated to ensure xeno-free growth condition. In this study we evaluated the efficacy of human platelet lysate (PL) as a substitute of FBS for the in vitro culture of some human cell lines. PL was obtained by pools of pathogen inactivated human donor platelet (PLT) concentrates. Human leukemia cell lines (KG-1, K562, JURKAT, HL-60) and epithelial tumor cell lines (HeLa and MCF-7) were cultured with either FBS or PL. Changes in cell proliferation, viability, morphology, surface markers and cell cycle were evaluated for each cell line. Functional characteristics were analysed by drug sensitivity test and cytotoxicity assay. Our results demonstrated that PL can support growth and expansion of all cell lines, although the cells cultured in presence of PL experienced a less massive proliferation compared to those grown with FBS. We found a comparable percentage of viable specific marker-expressing cells in both conditions, confirming lineage fidelity in all cultures. Functionality assays showed that cells in both FBS- and PL-supported cultures maintained their normal responsiveness to adriamycin and NK cell-mediated lysis. Our findings indicate that PL is a feasible serum substitute for supporting growth and propagation of haematopoietic and epithelial cell lines with many advantages from a perspective of process standardization, ethicality and product safety

IMPACT OF DIFFERENT DOSING STRATEGIES OF NIVOLUMAB IN PATIENTS WITH SOLID TUMORS: ITALIAN SINGLE CENTER ANALYSIS

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Optimization study of FDM 3d printing for the presentation of the architectural models

The prototyping of physical models by 3d printing is today one of the most common methods of presentation or composition of architectural projects, both in the academic and professional fields. In particular, the 3d printing with the FDM method based on PLA is diffused in several laboratories and studies, offering a high-precision physical model of remarkable resistance. Given its increasing use, also a lot of scientific literature has deepened its qualities, practices, and aesthetics, as well as identifying material compositions and procedural expedients increasingly efficient from the ecological point of view, therefore, the study proposes an analysis and the verification of the actual sustainability of the process, focusing phase of estimation, project management, and actual extrusion, in an exclusively ecological perspective, through experimentation and comparison of data. The analysis has led to the specific deepening of the techniques of infill of the printed volume, which affect in particular the consumption of material and its efficiency, therefore identifying optimal solutions and finding critical issues to be taken into account during the life of the prototype

Protective e¤ect of the V1a receptor antagonist SR49059 on brain edema formation following middle cerebral artery occlusion in the rat

Summary There exists no pharmacological treatment for fulminating brain edema. Since evidence indicates that brain aquaporin-4 (AQP4) water channels are modulated by vasopressin V1a receptors, we examined the edema-reducing properties of the selective V1a receptor antagonist, SR49059, following middle cerebral artery occlusion (MCAO). Male Sprague-Dawley rats were randomly assigned to sham procedure, vehicle, or SR49059 infusion at di¤erent dosages (each n ¼ 6, 480 mL/hr, 640 mL/hr, 720 mL/hr) and starting 60 minutes before or after MCAO. After a 2-hour period of ischemia and 2 hours of reperfusion, the animals were sacrificed for assessment of brain water content, sodium, and potassium concentration. Statistics were performed using an ANOVA followed by a Tukey post hoc analysis. SR049059 treatment reduced brain water content in the infarcted area given at 640 mL/hr ( p ¼ 0.036), 720 mL/hr 60 minutes before ( p ¼ 0.002) or 60 minutes after ( p ¼ 0.005) MCAO. The consecutive sodium shift into the brain was prevented ( p ¼ 0.001), while the potassium loss was inhibited only by pre-treatment ( p ¼ 0.003). These findings imply that in ischemia-induced brain edema, the selective V1a receptor-antagonist SR49059 inhibits brain edema and the subsequent sodium shift into brain. This substance o¤ers a new avenue in brain edema treatment and prompts further study into AQP4 modulation

Biochemical analysis of the cerebrospinal fluid: evidence for catastrophic energy failure and oxidative damage preceding brain death in severe head injury: a case report

Objectives: To compare biochemical and clinical parameters in a case of fatal severe traumatic brain injury (TBI) with secondary insult. Design and methods: A TBI patient was catheterized for intracranial pressure (ICP) monitoring and cerebrospinal fluid (CSF) analysis of ascorbate, malondialdehyde, oxypurines, and nucleosides. Results: Oxidative brain damage preceded ATP catabolite increment in the CSF even with ICP below 20 mm Hg. Sustained oxidative stress caused irreversible energy state derangement followed by a refractory ICP rise. Massive oxypurine and nucleoside release was recorded 36 h before brain death. Conclusions: Molecular events, detected by biochemical CSF analysis and preceding modification of clinical parameters in severe TBI with secondary insult, are discussed. (C) 2004 The Canadian Society of Clinical Chemists. All rights reserved

Potency testing of mesenchymal stromal cell growth expanded in human platelet lysate from different human tissues

Mesenchymal stromal cells (MSCs) have been largely investigated, in the past decade, as potential therapeutic strategies for various acute and chronic pathological conditions. MSCs isolated from different sources, such as bone marrow (BM), umbilical cord tissue (UCT) and adipose tissue (AT), share many biological features, although they may show some differences on cumulative yield, proliferative ability and differentiation potential. The standardization of MSCs growth and their functional amplification is a mandatory objective of cell therapies. The aim of this study was to evaluate the cumulative yield and the ex vivo amplification potential of MSCs obtained from various sources and different subjects, using defined culture conditions with a standardized platelet lysate (PL) as growth stimulus

Ion-pairing high-performance liquid chromatographic method for the detection of N-acetylaspartate and N-acetylglutamate in cerebral tissue extracts

An ion-pairing high-performance liquid chromatographic method for the determination of N-acetylaspartate and N-acetylglutamate using a C-18 column and a UV detection at 210 nm wavelength, by means of a diode array detector, is presented. A buffer containing 2.8 mM tetrabutylammonium hydroxide, 25 mM KH2PO4, 1.25% methanol, pH 7.00, is utilized for the isocratic separation of these N-acetylated amino acids, at a how rate of 1 ml/min and a column temperature of 23 degrees C. The suitability of this chromatographic separation (without additional chromatographic steps prior to HPLC assay) to monitor variations both of N-acetylaspartate and of N-acetylglutamate in perchloric acid brain extracts from rats subjected to the impact acceleration model of diffuse brain injury is also reported. According to the data presented, this HPLC method allows the separation of the two N-acetylated amino acids considered from the many possible interfering compounds, commonly present in extracts of cerebral tissue, which have high extinction coefficients at 210 nm wavelength. Values of N-acetylaspartate and N-acetylglutamate determined by this method showed that cerebral trauma negatively affects both compounds, according to the severity of trauma itself. (C) 2000 Academic Press

Single-sample preparation for simultaneous cellular redox and energy state determination

A simple and reliable method for the preparation of biological samples for the evaluation of biochemical parameters representative of the redox and energy states, such as glutathione (GSH), oxidized glutathione (GSSG), oxidized nicotinamide adenine dinucleotide (NAD+), reduced nicotinamide adenine dinucleotide (NADH), oxidized nicotinamide adenine dinucleotide phosphate (NADP+), reduced nicotinamide adenine dinucleotide phosphate (NADPH), coenzyme A (CoASH), oxidized CoASH, ascorbate, malondialdehyde, oxypurines, nucleosides, and energy metabolites, is presented. Fast deproteinization under nonoxidizing conditions is obtained by tissue homogenization in ice-cold, nitrogen-saturated CH3CN + 10 mM KH2PO4 (3:1; v:v), pH 7.40. After sample centrifugation to pellet precipitated proteins, organic solvent removal is performed on clear supernatants by three washings with large volumes of high-performance liquid chromatography (HPLC)-grade chloroform. The remaining aqueous phase, free of solvent and any lipid-soluble substances that may interfere with the further metabolite analysis, is used for the simultaneous ion-pairing HPLC determination of 39 compounds by means of a Kromasil C-18, 250 x 4.6-mm, 5-mum-particle-size column with tetrabutylammonium hydroxide as the pairing reagent. Results obtained by using the present method to prepare different rat tissue extracts demonstrate that it is possible to perform a single tissue preparation only for monitoring, in the same sample, compounds representative of the redox state (through the direct determination of GSH, GSSG, NAW, NADH, NADP(+), NADPH, CoASH, and oxidized CoASH) and of the cell energy state (by the analysis of oxypurines, nucleosides, and energy metabolites). Applicability of this sample processing procedure to quantify variations of the aforementioned compounds under pathological conditions was effected in rats subjected to moderate closed-head trauma. (C) 2003 Elsevier Inc. All rights reserved