Toxicological evaluation of S. involucrata culture: Acute, 90-day subchronic and genotoxicity studies.

Saussurea involucrata is an endangered plant that is used in traditional Chinese medicine. Through the use of plant cell culture techniques, preparations of Saussurea involucrata (S. involucrata) cell cultures have been developed and used to generate medicinal preparations. There have been few evidence-based analyses of the toxicological effects of S. involucrata culture conducted to date. Here, we conducted the experiments designed to assess the acute, subchronic, and genotoxic toxicological effects of S. involucrata culture. The genotoxic study was assessed through Ames, marrow micronucleus, and sperm malformation assays. The acute toxicity was assessed by orally administering in rats and mice at dose of 7500 mg/kg. Subchronic toxicity studies were then conducted by administering rats at doses of 500, 1000, or 1500 mg/kg for 90 days. No genotoxicity was observed at any tested dose levels, nor was any evidence of acute toxicity detected in treated mice or rats. Similarly, subchronic study of S. involucrata culture administration was not associated with any changes in rat food intake, weight, hematological parameters, organ weight, or organ histology. Then, we determined that the no observed adverse effect level of S. involucrata culture was greater than 1500 mg/kg in our 90-day toxicity study.

A possible injectable tissue engineered nucleus pulposus constructed with platelet-rich plasma and ADSCs in vitro.

BACKGROUND: Injectable tissue engineered nucleus pulposus is a new idea for minimally invasive repair of degenerative intervertebral disc. The platelet-rich plasma (PRP) and adipose-derived stromal cells (ADSCs) could be harvested from autologous tissue easily. PRP contains numerous autologous growth factors and has reticulate fibrous structure which may have the potential to make ADSCs differentiate into nucleus pulposus-like cells. The goal of this study was to explore the feasibility of constructing a possible injectable tissue engineered nucleus pulposus with PRP gel scaffold and ADSCs. METHODS: After identification with flow cytometry, the rabbit ADSCs were seeded into PRP gel and cultured in vitro. At the 2nd, 4th, and 8th week, the PRP gel/ADSCs complex was observed by macroscopy, histological staining, BrdU immunofluorescence, and scanning electron microscopy. The glycosaminoglycans (GAG) in the PRP gel/ADSCs complex were measured by safranin O staining with spectrophotometry. In PRP gel/ADSCs complex, gene expression of HIF-1α, aggrecan, type II collagen were tested by RT-PCR. The injectability of this complex was evaluated. RESULTS: Macroscopically, the complex was solidified into gel with smooth surface and good elasticity. The safranin O dye was almost no positive staining at 2nd week; however, the positive staining of extracellular matrix was enhanced obviously at 4th and 8th week. The HE staining and SEM demonstrated that the cells were well-distributed in the reticulate scaffold. BrdU immunofluorescence showed that ADSCs can survive and proliferate in PRP gel at each time points. The level of GAG at 4th week was higher than those at 2nd week (P < 0.05), and significant difference was also noted between 4th and 8th week (P < 0.05). HIF-1α, aggrecan, type II collagen gene expression at 4th week were much more than those at 2nd week (P < 0.05), and significant differences were also noted between 4th and 8th week (P < 0.05). The flow rate of complex was 0.287 mL/min when passed through the 19-gauge needle with the 100 mmHg injection pressure. CONCLUSIONS: Our preliminary findings suggest that the PRP gel make it possible for rabbit ADSCs differentiated into nucleus pulposus-like cells after coculture in vitro. According to the results, it is a better feasible method for construction of autologous injectable tissue engineered nucleus pulposus.

Exon Skipping in Directly Reprogrammed Myotubes Obtained from Human Urine-Derived Cells.

Duchenne muscular dystrophy (DMD), a progressive and fatal muscle disease, is caused by mutations in the DMD gene that result in the absence of dystrophin protein. To date, we have completed an investigator-initiated first-in-human study at the National Center of Neurology and Psychiatry based on the systemic injection of the morpholino oligonucleotides which are prone to exon-53 skipping. For the effective treatment of DMD, in vitro testing with myoblasts derived from DMD patients to screen drugs and assess patient eligibility before undertaking clinical trials is thought to be essential. Very recently, we reported a new MYOD1-converted urine-derived cell (UDC) treated with the histone methyltransferase inhibitor (3-deazaneplanocin A hydrochloride), as a cellular model of DMD. The new autologous UDC might show phenocopy of the disease-specific phenotypes of DMD, leading to the application of precision medicine in a variety of muscle-related diseases. In this article, we describe a detailed protocol for efficient modelling of DMD muscle cells using MYOD1-converted UDCs along with reverse transcriptase polymerase chain reaction (RT-PCR), Western blotting, and immunocytochemistry to evaluate the restoration of dystrophin mRNA and protein levels after exon skipping.

Quercetin-Induced miR-369-3p Suppresses Chronic Inflammatory Response Targeting C/EBP-ß.

SCOPE: Dendritic cells (DCs) are the most potent antigen-presenting cells that play an important role in the crosstalk between the innate and the adaptive immune response. Quercetin exposure is identified as an effective strategy to suppress the inflammatory response induced by LPS. METHODS AND RESULTS: In this study, using a next-generation sequencing analysis, the effect of quercetin on microRNAs (miRNAs) expression in DCs is examined. A signature of 113 miRNAs that are differentially regulated in LPS-stimulated DCs after quercetin exposure is defined. It is demonstrated that the loss of function of miR-369-3p in LPS-stimulated DCs during quercetin exposure led to an increase of CCAAT/enhancer binding protein ß (C/EBP-ß) mRNA and protein and its downstream targets tumor necrosis factor-α (TNF-α) and interleukin 6 (IL6). Conversely, it is shown that the ectopic induction of miR-369-3p without quercetin suppresses the inflammatory response of LPS reducing C/EBP-ß, TNF-α, and IL6 production. In vivo, oral administration of quercetin in dextran-sulfate-sodium-induced colitis induces miR-369-3p expression. CONCLUSIONS: These findings indicate that quercetin-induced miR-369-3p regulates the inflammatory cascade in chronic inflammatory response and present promising therapeutic implications.

Buoyancy-Driven Gradients for Biomaterial Fabrication and Tissue Engineering.

The controlled fabrication of gradient materials is becoming increasingly important as the next generation of tissue engineering seeks to produce inhomogeneous constructs with physiological complexity. Current strategies for fabricating gradient materials can require highly specialized materials or equipment and cannot be generally applied to the wide range of systems used for tissue engineering. Here, the fundamental physical principle of buoyancy is exploited as a generalized approach for generating materials bearing well-defined compositional, mechanical, or biochemical gradients. Gradient formation is demonstrated across a range of different materials (e.g., polymers and hydrogels) and cargos (e.g., liposomes, nanoparticles, extracellular vesicles, macromolecules, and small molecules). As well as providing versatility, this buoyancy-driven gradient approach also offers speed (<1 min) and simplicity (a single injection) using standard laboratory apparatus. Moreover, this technique is readily applied to a major target in complex tissue engineering: the osteochondral interface. A bone morphogenetic protein 2 gradient, presented across a gelatin methacryloyl hydrogel laden with human mesenchymal stem cells, is used to locally stimulate osteogenesis and mineralization in order to produce integrated osteochondral tissue constructs. The versatility and accessibility of this fabrication platform should ensure widespread applicability and provide opportunities to generate other gradient materials or interfacial tissues.

Comparison of two substrate materials used as negative control in endothelialization studies: Glass versus polymeric tissue culture plate.

The endothelialization of synthetic surfaces applied as cardiovascular implant materials is an important issue to ensure the anti-thrombotic quality of a biomaterial. However, the rapid and constant development of a functionally-confluent endothelial cell monolayer is challenging. In order to investigate the compatibility of potential implant materials with endothelial cells several in vitro studies are performed. Here, glass and tissue culture plates (TCP) are often used as reference materials for in vitro pre-testing. However, a direct comparison of both substrates is lacking.Therefore, a comparison of study results is difficult, since results are often related to various reference materials. In this study, the endothelialization of glass and TCP was investigated in terms of adherence, morphology, integrity, viability and function using human umbilical vein endothelial cells (HUVEC).On both substrates an almost functionally confluent HUVEC monolayer was developed after nine days of cell seeding with clearly visible cell rims, decreased stress fiber formation and a pronounced marginal filament band. The viability of HUVEC was comparable for both substrates nine days after cell seeding with only a few dead cells. According to that, the cell membrane integrity as well as the metabolic activity showed no differences between TCP and glass. However, a significant difference was observed for the secretion of IL-6 and IL-8. The concentration of both cytokines, which are associated with migratory activity, was increased in the supernatant of HUVEC seeded on TCP. This result matches well with the slightly increased number of adherent HUVEC on TCP.In conclusion, these findings indicate that both reference materials are almost comparable and can be used equivalently as control materials in in vitro endothelialization studies.

Liquid chromatography/mass spectrometry based detection and semi-quantitative analysis of INSL5 in human and murine tissues.

RATIONALE: Insulin-like peptide 5 (INSL5) is a hormone produced by enteroendocrine L-cells in the colon that has recently been implicated in the control of metabolic homeostasis. However, research into its physiology has been hindered by the reported unreliability of commercially available immunoassays and additional detection assays would benefit this emerging field. METHODS: Peptides from purified murine L-cells and homogenates from both human and mouse colonic tissues were extracted by precipitating larger proteins with acetonitrile. Untargeted liquid chromatography/tandem mass spectrometry (LC/MS/MS) analyses, followed by database searching, were used to detect and identify various INSL5 gene derived peptides and characterise their precise sequence. A similar approach was developed to quantify INSL5 levels in primary intestinal culture supernatants after purification and concentration by solid-phase extraction. RESULTS: Mass spectral analysis of purified enteroendocrine cells and tissue homogenates identified the exact sequence of A and B chains of INSL5 endogenously expressed in L-cells. Differences in the endogenously processed peptide and the Swissprot database entry were observed for murine INSL5, whereas the human sequence matched previous predictions from heterologous expression experiments. INSL5 was detected in the supernatant of human and mouse primary colonic cultures and concentrations increased after treatment with a known L-cell stimulus. CONCLUSIONS: The first LC/MS/MS-based method capable of the detection and semi-quantitative analysis of endogenous INSL5 using MS-based techniques has been demonstrated. The methodology will enable the identification of stimulants for INSL5 secretion from murine and human primary colonic epithelial cultures.

Fundamentals of diffusion MRI physics.

Diffusion MRI is commonly considered the "engine" for probing the cellular structure of living biological tissues. The difficulty of this task is threefold. First, in structurally heterogeneous media, diffusion is related to structure in quite a complicated way. The challenge of finding diffusion metrics for a given structure is equivalent to other problems in physics that have been known for over a century. Second, in most cases the MRI signal is related to diffusion in an indirect way dependent on the measurement technique used. Third, finding the cellular structure given the MRI signal is an ill-posed inverse problem. This paper reviews well-established knowledge that forms the basis for responding to the first two challenges. The inverse problem is briefly discussed and the reader is warned about a number of pitfalls on the way.

Quantitative profiling of 4'-geranyloxyferulic acid and its conjugate with l-nitroarginine methyl ester in mononuclear cells by high-performance liquid chromatography with fluorescence detection.

Oxyprenylated natural products were shown to exert in vitro and in vivo remarkable anti-cancer and anti-inflammatory effects. This paper describes a rapid, selective, and sensitive HPLC method with fluorescence detection for determination of 4'-geranyloxyferulic acid (GOFA) and its conjugate with l-nitroarginine methyl ester (GOFA-L-NAME) in mononuclear cells. Analytes were extracted from cells using methanol and eluted on a GraceSmart RP18 analytical column (250×4.6mm i.d., 5µm particle size) kept at 25°C. A mixture of formic acid 1% in water (A) and methanol (B) were used as mobile phase, at a flow-rate of 1.2mL/min in gradient elution. A fluorescence detector (excitation/emission wavelength of 319/398nm for GOFA and GOFA-L-NAME), was used for the two analytes. Calibration curves of GOFA and GOFA-L-NAME were linear over the concentration range of 1.0-50µg/mL, with correlation coefficients (r2)≥0.9995. Intra- and inter-assay precision do not exceed 6.8%. The accuracy was from 94% to 105% for quality control samples (2.0, 25.0 and 40µg/mL). The mean (RSD%) extraction recoveries (n=5) for GOFA and GOFA-L-NAME from spiked cells at 2.0, 25.0 and 40.0µg/mL were 92.4±1.5%, 94.7±0.9% and 93.8±1.1%, for GOFA and 95.3±1.2%, 94.8±1.0% and 93.9±1.3%, for GOFA-L-NAME. The limits of detection and quantification were 0.3µg/mL and 1.0µg/mL for GOFA and GOFA-L-NAME. This method was successfully applied to measure GOFA and GOFA-L-NAME concentrations in a mononuclear cells.

Spatial Chemical Stimulation Control in Microenvironment by Microfluidic Probe Integrated Device for Cell-Based Assay.

Cell-cell interactions play an important role in the development and function of multicellular organisms. To investigate these interactions in detail, it is necessary to evaluate the behavior of a cell population when the minimum number of cells in the population is stimulated by some chemical factors. We propose a microfluidic device integrated with microfluidic probe (MFP) functionality; this device is capable of imparting a chemical stimulus to cells within a microenvironment, for cell-based assays. The device contains MFP channels at the walls of the cell culture microchannels, and it can control a localized chemical stimulation area at the scale of a single cell to a few cells using MFP fluid control in a microspace. The results of a finite element method-based simulation indicated that it is possible to control the chemical stimulation area at the scale of a single cell to a few cells by optimizing the MFP channel apex width and the flow ratio. In addition, localized cell staining was demonstrated successfully using a spatial chemical stimulus. We confirmed the device functionality as a novel cell-based assay tool. We succeeded in performing localized cell collection using this method, which suggested that the single cell analysis of a cell monolayer that is subjected to a specific chemical stimulus is possible. The method proposed in this paper can contribute significantly to the fields of cell biology and drug development.

A multi-center analysis of adverse events among two thousand, three hundred and seventy two adult patients undergoing adult autologous stem cell therapy for orthopaedic conditions.

INTRODUCTION: The purpose of the present investigation is to report on detailed complications among a much larger group of 2372 orthopaedic patients treated with stem cell injections who were followed in a treatment registry for up to nine years. METHODS: All patients underwent an MSC-based, percutaneous injection treatment of an orthopaedic condition between December 2005 and September 2014 at one of 18 clinical facilities. Treated areas of the body included the knee, hip, ankle/foot, hand/wrist, elbow, shoulder, and spine. The patients were followed prospectively via enrollment in a treatment registry. Patients were followed prospectively at one, three, six and 12 months, and annually thereafter, using an electronic system, ClinCapture software. RESULTS: A total of 3012 procedures were performed on 2372 patients with follow-up period of 2.2 years. A total of 325 adverse events were reported. The majority were pain post-procedure (n = 93, 3.9 % of the study population) and pain due to progressive degenerative joint disease (n = 90, 3.8 % of the study population). Seven cases reported neoplasms, a lower rate than in the general population. The lowest rate of adverse events was observed among patients injected with BMC alone. CONCLUSION: Lowest rate of adverse events was among those patients receiving BMC injections alone, but the higher rate of AEs for BMC plus adipose and cultured cells was readily explained by the nature of the therapy or the longer follow-up. There was no clinical evidence to suggest that treatment with MSCs of any type in this study increased the risk of neoplasm.

Identifying Metabolic Subpopulations from Population Level Mass Spectrometry.

Metabolism underlies many important cellular decisions, such as the decisions to proliferate and differentiate, and defects in metabolic signaling can lead to disease and aging. In addition, metabolic heterogeneity can have biological consequences, such as differences in outcomes and drug susceptibilities in cancer and antibiotic treatments. Many approaches exist for characterizing the metabolic state of a population of cells, but technologies for measuring metabolism at the single cell level are in the preliminary stages and are limited. Here, we describe novel analysis methodologies that can be applied to established experimental methods to measure metabolic variability within a population. We use mass spectrometry to analyze amino acid composition in cells grown in a mixture of (12)C- and (13)C-labeled sugars; these measurements allow us to quantify the variability in sugar usage and thereby infer information about the behavior of cells within the population. The methodologies described here can be applied to a large range of metabolites and macromolecules and therefore have the potential for broad applications.

In vitro model alveoli from photodegradable microsphere templates.

Recreating the 3D cyst-like architecture of the alveolar epithelium in vitro has been challenging to achieve in a controlled fashion with primary lung epithelial cells. Here, we demonstrate model alveoli formed within a tunable synthetic biomaterial platform using photodegradable microspheres as templates to create physiologically relevant, cyst structures. Poly(ethylene glycol) (PEG)-based hydrogels were polymerized in suspension to form microspheres on the order of 120 µm in diameter. The gel chemistry was designed to allow erosion of the microspheres with cytocompatible light doses (≤15 min exposure to 10 mW cm(-2) of 365 nm light) via cleavage of a photolabile nitrobenzyl ether crosslinker. Epithelial cells were incubated with intact microspheres, modified with adhesive peptide sequences to facilitate cellular attachment to and proliferation on the surface. A tumor-derived alveolar epithelial cell line, A549, completely covered the microspheres after only 24 hours, whereas primary mouse alveolar epithelial type II (ATII) cells took ∼3 days. The cell-laden microsphere structures were embedded within a second hydrogel formulation at user defined densities; the microsphere templates were subsequently removed with light to render hollow epithelial cysts that were cultured for an additional 6 days. The resulting primary cysts stained positive for cell-cell junction proteins (ß-catenin and ZO-1), indicating the formation of a functional epithelial layer. Typically, primary ATII cells differentiated in culture to the alveolar epithelial type I (ATI) phenotype; however, each cyst contained ∼1-5 cells that stained positive for an ATII marker (surfactant protein C), which is consistent with ATII cell numbers in native mouse alveoli. This biomaterial-templated alveoli culture system should be useful for future experiments to study lung development and disease progression, and is ideally suited for co-culture experiments where pulmonary fibroblasts or endothelial cells could be presented in the hydrogel surrounding the epithelial cysts.

Semi-quantitative assay for polyketide prymnesins isolated from Prymnesium parvum (Haptophyta) cultures.

A fluorometric assay was developed to semi-quantify co-purified polyketide prymnesins-1 and -2 (PPs) from Prymnesium parvum cultures. Evaluations performed throughout the growth cycle of 5 practical salinity unit (PSU) cultures detected relatively 8-10 × more PPs in the culture medium (exotoxins) than in cells (endotoxins). The [exotoxin] remained stable and relatively low until post-log growth, when they increased significantly. However, on a per-cell basis, [exotoxin] declined throughout log phase and subsequently increased dramatically during late- and post-log phases. The [endotoxin] remained stable until late- and post-log phases, when it achieved its highest level before declining sharply. Shaking cultures of strains from Texas, South Carolina and the United Kingdom displayed dramatically different [exotoxin] during post-log decline. Cultures adapted to 30 PSU had significantly lower [exotoxin] over the course of cultivation than those grown at 5 PSU. Phosphate limitation enhanced [exotoxin] on a per-cell basis, especially in late- and post-log cultures. Media containing streptomycin exhibited a ∼20% increase in [exotoxin] in post-log cultures vs. control treatments, but it had only negligible effects on endotoxin levels. Brefeldin A had minimal effects on [exotoxin], suggesting that the presence of PPs in the medium may be largely derived from cell lysis or some other passive means.

Circulating endometrial cells in peripheral blood.

OBJECTIVES: Endometriosis is a common disorder amongst women of reproductive age. Despite extensive research, no reliable blood tests currently exist for the diagnosis of endometriosis STUDY DESIGN: We report several new approaches enabling study of cell specific characteristic of endometrial cells, introducing enrichment and culturing of viable circulating endometrial cells (CECs) isolated from peripheral blood (PB) and peritoneal endometrial cells (PECs) from peritoneal washing (PW). Size-based enrichment method (MetaCell(®), Czech Republic) has been used for the filtration of PB and PW in patients with diagnosed endometriosis. RESULTS: The PECs were found in the PW in all of the tested patients (n=17), but CECs) only in 23.5% (4/17) cases. Their endometrial origin has been proved by immunohistochemistry. PECs were successfully cultured in vitro directly on the separating membrane (9/17) exhibiting both endometrial cell phenotypes: stromal and glandular within the culture. CECs were successfully cultured in the two of the four positive cases, but in none of them confluence has been reached. The occurrence in CECs in PB is clear and very specific evidence of an active endometrial disease. CONCLUSIONS: We demonstrated efficient, quick and user friendly endometrial cells capture platform based on a cell size. Furthermore, we demonstrated an ability to culture the captured cells, a critical requirement for post-isolation cellular analysis directed to better understanding of endometriosis pathogenesis.

Analysis of polyamines in biological samples by HPLC involving pre-column derivatization with o-phthalaldehyde and N-acetyl-L-cysteine.

Polyamines (putrescine, spermine and spermidine) play a crucial role in the regulation of cell growth, differentiation, death and function. Accurate measurement of these substances is essential for studying their metabolism in cells. This protocol describes detailed procedures for sample preparation and HPLC analysis of polyamines and related molecules (e.g., agmatine and cadaverine) in biological samples. The method is optimized for the deproteinization of samples, including biological fluids (e.g., 10 µl), plant and animal tissues (e.g., 50 mg), and isolated/cultured cells (e.g., 1 × 10(6) cells). The in-line reaction of polyamines with o-phthalaldehyde and N-acetyl-L-cysteine yields fluorescent derivatives which are separated on a reversed-phase C18 column and detected by a fluorometer at an excitation wavelength of 340 nm and an emission wavelength of 450 nm. The total running time for each sample (including column regeneration on the automated system) is 30 min. The detection limit is 0.5 nmol/ml or 0.1 nmol/mg tissue in biological samples. The assays are linear between 1 and 50 µM for each of the polyamines. The accuracy (the nearness of an experimental value to the true value) and precision (agreement between replicate measurement) of the HPLC method are 2.5-4.2 % and 0.5-1.4 %, respectively, for biological samples, depending on polyamine concentrations and sample type. Our HPLC method is highly sensitive, specific, accurate, easily automated, and capable for the analysis of samples with different characteristics and small volume/amount, and provides a useful research tool for studying the biochemistry, physiology, and pharmacology of polyamines and related substances.

Quantum dot-related genotoxicity perturbation can be attenuated by PEG encapsulation.

Nanomaterial-biosystem interaction is emerging as a major concern hindering wide adoption of nanomaterials. Using quantum dots (Qdots) of different sizes (Qdot-440nm and Qdot-680nm) as a model system, we studied the effects of polyethylene glycol (PEG) thin-layer surface modification in attenuating Qdot-related cytotoxicity, genotoxicity perturbation and oxidative stress in a cellular system. We found that uncoated Qdots (U-Qdots) made of core/shell CdSe/ZnS could indeed induce cytotoxic effects, including the inhibition of cell growth. Also, both the neutral comet assay and γH2AX foci formation showed that U-Qdots caused significant DNA damage in a time- and dose-dependent manner. In contrast, results from cytotoxicity analysis and γH2AX generation indicate minimal impact on cells after exposure to PEG-coated Qdots. This lack of observed toxic effects from PEG-coated Qdots may be due to the fact that PEG-coating can inhibit ROS generation induced by U-Qdots. Based on these observations, we conclude that the genotoxicity of Qdots could be significantly decreased following proper surface modification, such as PEG encapsulation. In addition, PEG encapsulation may also serve as a general method to attenuate nanotoxicity for other nanoparticles.

A HaloTag® method for assessing the retrograde axonal transport of the p75 neurotrophin receptor and other proteins in compartmented cultures of rat sympathetic neurons.

We have adapted HaloTag® (HT) technology for use in compartmented cultures of rat sympathetic neurons in order to provide a technique that can be broadly applied to studies of the retrograde transport of molecules that play roles in neurotrophin signaling. Transfected neurons expressing HT protein alone, HT protein fused to the p75 neurotrophin receptor (p75NTR) or HT protein fused to tubulin α-1B were maintained in compartmented cultures in which cell bodies and proximal axons of rat sympathetic neurons reside in proximal compartments and their distal axons extend into distal compartments. HT ligand containing a fluorescent tetramethylrhodamine (TMR) label was applied either in the distal compartments or the proximal compartments, and the transport of labeled proteins was assayed by gel fluorescence imaging and TMR immunoblot. HT protein expressed alone displayed little or no retrograde transport. HT protein fused to either the intracellular C-terminus or the extracellular N-terminus of p75NTR was retrogradely transported. The retrograde transport of p75NTR was augmented when the distal axons were provided with nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) or antibodies to BDNF. The anterograde transport of HT protein fused to the N-terminus of tubulin α-1B was also demonstrated. We conclude that retrograde transport of HT fusion proteins provides a powerful and novel approach in studies of axonal transport.

Biological validation of bio-engineered red blood cell productions.

The generation in vitro of cultured red blood cells (cRBC) could become an alternative to classical transfusion products. However, even when derived from healthy donors, the cRBC generated in vitro from hematopoietic stem cells may display alterations resulting from a poor controlled production process. In this context, we attempted to monitor the quality of the transfusion products arising from new biotechnologies. For that purpose, we developed an in vitro erythrophagocytosis (EP) test with the murine fibroblast cell line MS-5 and human macrophages (reference method). We evaluated 38 batches of cRBC, at the stage of reticulocyte, generated from CD34(+) cells isolated from placental blood or by leukapheresis. We showed that (i) the EP test performed with the MS-5 cell line was sensitive and can replace human macrophages for the evaluation of cultured cells. (ii) The EP tests revealed disparities among the batches of cRBC. (iii) The viability of the cells (determined by calcein-AM test), the expression of CD47 (antiphagocytosis receptor) and the externalization of phosphatidylserine (PS, marker of phagocytosis) were not critical parameters for the validation of the cRBC. (iv) Conversely, the cell deformability determined by ektacytometry was inversely correlated with the intensity of the phagocytic index. Assuming that the culture conditions directly influence the quality of the cell products generated, optimization of the production mode could benefit from the erythrophagocytosis test.

Short term effects of gamma radiation on endothelial barrier function: uncoupling of PECAM-1.

A limiting factor in the treatment of cancer with radiotherapy is the damage to surrounding normal tissue, particularly the vasculature. Vessel pathologies are a major feature of the side effects of radiotherapy and little is known about early events that could initiate subsequent diseases. We tested the hypothesis that gamma radiation has early damaging effects on the human endothelial barrier. Two models were used; Human Brain Microcapillary Endothelial Cells (HBMEC), and Human Umbilical Vein Endothelial Cells (HUVEC). Endpoints included Trans-Endothelial Electrical Resistance (TEER), barrier permeability to 10 kDa and 70 kDa tracer molecules, and the localization of F-actin, and junction proteins and the Platelet Endothelial Cell Adhesion Molecule (PECAM-1). Radiation induced a rapid and transient decrease in TEER at 3 h, with effects also seen at the radiotherapy doses. This dip in resistance correlated to the transient loss of PECAM-1 in discrete areas where cells often detached from the monolayer leaving gaps. Redistribution of PECAM-1 was also seen in 3-D human tissue models. By 6 h, the remaining cells had migrated to reseal the barrier, coincident with TEER returning to control levels. Resealed monolayers contained fewer cells per unit area and their barrier function was weakened as evidenced by an increased permeability over 24 h. This is the first demonstration of a transient and rapid effect of gamma radiation on human endothelial barriers that involves cell detachment and the loss of PECAM-1. Considering the association of cell adhesion molecules with vasculopathies, such an effect has the potential to be clinically relevant to the longer-term effects of radiotherapy.