Mixed Cell Suspensions Research Articles

Biofunctionalized self-assembly of peptide amphiphile induces the differentiation of bone marrow mesenchymal stem cells into neural cells.

Bone marrow mesenchymal stem cells (BMSCs) are multipotential differentiation cells which can differentiate into different cell types such as osteoblasts, chondrocytes, adipocytes, cardiomyocytes, hepatocytes, endothelial cells, and neuronal cells. Such multipotential differentiation makes them attractive for stem cell-based therapy aimed at treating previously incurable disorders. In the present work, we encapsulated BMSCs into a hydrogel with a three-dimensional (3D) network of nanofibers, formed from self-assembling of peptide amphiphile. The self-assembling of peptide amphiphile into hydrogel was triggered by mixing cell suspensions with dilute aqueous solutions of amphipathic peptide. Moreover, this hydrogel was designed to present cells the neurite-promoting laminin epitope IKVAV at nearly van der Waals density, which induced the successful differentiation of BMSCs into neural cells.

Isolation of Dendritic Cells from the Human Female Reproductive Tract for Phenotypical and Functional Studies.

The characterization of the human dendritic cells (DCs) resident in mucosal tissues is challenging due to the difficulty in obtaining samples, and the low numbers of DCs present per tissue. Yet, as the phenotype and function of DCs is modified by the tissue environment, it is necessary to analyze tissue resident DC populations, since blood derived DCs incompletely reflect the complexities of DCs in tissues. Here we present a protocol to isolate DCs from the human female reproductive tract (FRT) using hysterectomy specimens that allows both phenotypical and functional analyses. The protocol consists of tissue digestion to generate a single cell mixed cell suspension, followed by positive magnetic bead selection. Our tissue digestion protocol does not cleave surface markers, which allows phenotypical and functional analysis of DCs in the steady state, without overnight incubation or cell activation. This protocol can be adapted for the isolation of other immune cell types or isolation of DCs from other tissues.

Selective targeting of tumor associated macrophages in different tumor models.

Tumor progression largely depends on the presence of alternatively polarized (M2) tumor-associated macrophages (TAMs), whereas the classical M1-polarized macrophages can promote anti-tumorigenic immune responses. Thus, selective inhibition of M2-TAMs is a desirable anti-cancer approach in highly resistant tumor entities such as hepatocellular carcinoma (HCC) or breast cancer. We here examined whether a peptide that selectively binds to and is internalized by in vitro-differentiated murine M2 macrophages as compared to M1 macrophages, termed M2pep, could be used to selectively target TAMs in HCC and breast carcinoma. We confirmed selectivity of M2pep for in vitro M2 polarized macrophages. Upon incubation of suspended mixed 4T1 tumor cells with M2pep, high amounts of the TAMs were found to be associated with M2pep, whereas in mixed tumor cell suspensions from two HCC mouse models, M2pep showed only low-degree binding to TAMs. M2pep also showed low-degree targeting of liver macrophages. This indicates that the TAMs in different tumor entities show different targeting of M2pep and that M2pep is a very promising approach to develop selective M2-TAM-targeting in tumor entities containing M2-TAMs with significant amounts of the so far elusive M2pep receptor(s).

Minimally invasive and targeted therapeutic cell delivery to the skin using microneedle devices.

Translation of cell therapies to the clinic is accompanied by numerous challenges, including controlled and targeted delivery of the cells to their site of action, without compromising cell viability and functionality. To explore the use of hollow microneedle devices (to date only used for the delivery of drugs and vaccines into the skin and for the extraction of biological fluids) to deliver cells into skin in a minimally invasive, user-friendly and targeted fashion. Melanocyte, keratinocyte and mixed epidermal cell suspensions were passed through various types of microneedles and subsequently delivered into the skin. Cell viability and functionality are maintained after injection through hollow microneedles with a bore size ≥75μm. Healthy cells are delivered into the skin at clinically relevant depths. Hollow microneedles provide an innovative and minimally invasive method for delivering functional cells into the skin. Microneedle cell delivery represents a potential new treatment option for cell therapy approaches including skin repigmentation, wound repair, scar and burn remodelling, immune therapies and cancer vaccines.

Droplet Array-Based 3D Coculture System for High-Throughput Tumor Angiogenesis Assay.

Angiogenesis is critical for tumor progression and metastasis, and it progresses through orchestral multicellular interactions. Thus, there is urgent demand for high-throughput tumor angiogenesis assays for concurrent examination of multiple factors. For investigating tumor angiogenesis, we developed a microfluidic droplet array-based cell-coculture system comprising a two-layer polydimethylsiloxane chip featuring 6 × 9 paired-well arrays and an automated droplet-manipulation device. In each droplet-pair unit, tumor cells were cultured in 3D in one droplet by mixing cell suspensions with Matrigel, and in the other droplet, human umbilical vein endothelial cells (HUVECs) were cultured in 2D. Droplets were fused by a newly developed fusion method, and tumor angiogenesis was assayed by coculturing tumor cells and HUVECs in the fused droplet units. The 3D-cultured tumor cells formed aggregates harboring a hypoxic center-as observed in vivo-and secreted more vascular endothelial growth factor (VEGF) and more strongly induced HUVEC tubule formation than did 2D-cultured tumor cells. Our single array supported 54 assays in parallel. The angiogenic potentials of distinct tumor cells and their differential responses to antiangiogenesis agent, Fingolimod, could be investigated without mutual interference in a single array. Our droplet-based assay is convenient to evaluate multicellular interaction in high throughput in the context of tumor sprouting angiogenesis, and we envision that the assay can be extensively implementable for studying other cell-cell interactions.

Electro-Optical Assays for Immunoindication of Microbial Cells

Background: Last years the development of various rapid methods for detection and identification of microorganisms is one of the most intensively growing research trends because they are available and in use in the industrial microbiological market today not only for microbiological diagnostics but for controlling food quality, pharmaceutical industry products and environmental control also. These methods tend to be nongrowth-based on various technology platforms. The more common technologies among them include nucleic-acid-based detection, which uses DNA or RNA targets; methods; enzymatic detection such as adenosine triphosphate (ATP) methods; flow-cytometry-based biochemical and various types of biosensors methods. The possibility of using the electro-optical method of analysis for the detection of microbial cells is very relevant and will be described in this paper. Methods: We undertook a structured search of bibliographic databases for peer-reviewed research literature using a focused review question and inclusion/exclusion criteria. The quality of retrieved papers was appraised using standard tools. The characteristics of our results were descriebed and were applied to analyse the findings of included studies using a conceptual framework. Results: In the article are described the results of the long-year research of our group devoted to developing of the real-time immunoindication (rapid detection) method basing on the registering of interactions of bacterial cells with polyclonal or phage display antibodies (the antibody-based detection) by the electro-optical analysis with the specially designed electro-optical sensor and to studying electro-optical characteristics of corresponding biological and physical processes occurring in the bacterial suspensions in the presence of various biological reagents. It was shown that the electro-optical analysis provides comparatively high sensitivity for cell detection and can be used for characterization of bacteria interactions with specific monoclonal antibodies, polyclonal antibodies or mini-antibodies. Also the theoretical fundamentals (basic principles) of the electro-optical analysis of microbial cells were described. Conclusion: The suggested electro-optical method can be recommended for analyzing of mixed cell suspensions (detection of bacterial cells in liquid phase) by using specific antibodies with further registration of their interactions measuring changes of electro-optical parameters, but without immobilization of antibodies on the solid surface (various kinds of plates). Keywords: Immunoindication, electro-optical analysis, electro-optical signal, electric-field orientation, Azospirillum brasilense, polyclonal antibodies, phage display technology, H-antigen, O-antigen, flagellin.

Mesenchymal Stem Cells Promote Tumor Progression via Inducing Stroma Remodeling on Rabbit VX2 Bladder Tumor Model.

Background and aim: Mesenchymal stem cells (MSCs) are capable of impacting tumor progression but its role in tumor stroma remodeling still remains unclear. This present study was aimed to evaluate the potential function of MSCs on tumor stroma remodeling using rabbits VX2 bladder tumor model.Methods: The VX2 bladder tumor models were established by injecting mixed cell suspensions (106 of VX2 tumor cells and 0/106/107 of autologous MSCs in group A, B, C, respectively) into the bladder mucosa using thirty male New Zealand white rabbits. The tumor volume was measured by ultrasound at the time points of 1st, 2nd, 3rd and 4th week after inoculation. At the end of the fourth week, the tumor tissue expressions of basic fibroblast growth factor (bFGF), transforming growth factor beta 1 (TGFβ-1), hepatocyte growth factor (HGF), matrix metalloproteinase 2 (MMP2) and matrix metalloproteinase 9 (MMP9) were determined using Real-time quantitative PCR and immunohistochemistry. Masson trichrome staining and Cy3-FITC double-labelled immunofluorescence staining were used to determine the MSCs distribution in tumor tissue in another two rabbits implanted with a cell suspension of 106 VX2 tumor cells and 106 autologous MSCs.Results: MSCs were homogeneously distributed in tumor tissues after 7 days of inoculation, which were not consistent with the distribution of tumor stroma. After 21 days of inoculation, MSCs have been integrated into tumor interstitial tissue and mainly distributed in the mesenchyma around the tumor nest. At the 1st, 2nd, 3rd and 4th week time point, tumor volume in group A < group B < group C, and the difference has statistical significance (all p<0.001).The relative mRNA and protein levels of bFGF, TGFβ-1 and HGF were significantly higher in group B and C compared with group A (all p<0.05), as well as the mRNA levels of bFGF, HGF were higher in group C than group B (p<0.05), and the protein levels of bFGF, TGFβ-1 were higher in group C than group B (p<0.05). The mRNA and protein levels of MMP2 were significantly higher in group B, C than group A (p<0.05). MMP9 was increasingly over expressed along with the growing amount of MSCs inoculated within tumor, both at the level of mRNA and protein (all p<0.05).Conclusion: MSCs participate in tumor stroma remodeling via inducing overexpression of some important growth factors and MMPs.

New in vivo avatars of diffuse intrinsic pontine gliomas (DIPG) from stereotactic biopsies performed at diagnosis.

Diffuse Instrinsic Pontine Glioma is the most aggressive form of High Grade Gliomas in children. The lack of biological material and the absence of relevant models have hampered the development of new therapeutics. Their extensive infiltration of the brainstem renders any surgical resection impossible and until recently biopsies were considered not informative enough and therefore not recommended. Thus, most models were derived from autopsy material. We aimed to develop relevant in vivo DIPG models that mimic this specific disease and its molecular diversity from tumor material obtained at diagnosis. Eight patient-derived orthotopic xenograft models were obtained after direct stereotactic injection of a mixed cell suspension containing tumor cells and stromal cells in the brainstem or thalamus of nude mice and serially passaged thereafter. In parallel, we developed 6 cell-derived xenograft models after orthotopic injection of tumor-initiating cells cultured from stereotactic biopsies. Cells were modified to express luciferase to enable longitudinal tumor growth monitoring, and fluorescent reporter proteins to trace the tumor cells in the brain.These models do not form a tumor mass, they are invasive, show the H3K27 trimethylation loss in vivo and the tumor type diversity observed in patients in terms of histone H3 mutations and lineage markers. Histological and MRI features at 11.7 Tesla show similarities with treatment naïve human DIPG, and in this respect, both direct and indirect orthotopic xenograft looked alike. These DIPG models will therefore constitute valuable tools for evaluating new therapeutic approaches in this devastating disease.

Streamline-based purification of bacterial samples from liquefied sputum utilizing microfluidics.

The separation or purification of bacterial samples from a mixed cell suspension is critical in a variety of biomedical applications, such as sputum diagnostics and cell biology studies. We propose a streamline-based microfluidic filtration device for highly efficient purification of bacterial samples from a mixed cell suspension. The device is composed of tens of repeated streamline-based separation units that continuously filter the solution. By injecting a liquid sample such as liquefied human sputum solution through the device, approximately 50% of the injected sample solution can be collected from the filtration collection channels, which filter approximately 99.9% of the mammalian cells but retain approximately 60% to 90% of the bacteria. Different injection rates (0.2 ml h-1 to 30 ml h-1), different sample viscosities, and different initial bacterial densities were tested and confirmed that our separation method was robust. The easy operation, robustness and high efficiency indicate that our method may be useful for the separation or purification of bacterial samples from a mixed cell suspension, such as bacterial samples for sputum diagnostics.

The Effects of Co-transplantation of Olfactory Ensheathing Cells and Schwann Cells on Local Inflammation Environment in the Contused Spinal Cord of Rats.

Inflammatory response following spinal cord injury (SCI) is important in regulation of the repair process. Olfactory ensheathing cells (OECs) and Schwann cells (SCs) are important donor cells for repairing SCI in different animal models. However, synergistic or complementary effects of co-transplantation of both cells for this purpose have not been extensively investigated. In the present study, we investigated the effects of co-transplantation of OECs and SCs on expression of pro- or anti-inflammatory factor and polarization of macrophages in the injured spinal cord of rats. Mixed cell suspensions containing OECs and SCs were transplanted into the injured site at 7days after contusion at the vertebral T10 level. Compared with the DMEM, SC, or OEC group, the co-transplantation group had a more extensive distribution of the grafted cells and significantly reduced number of astrocytes, microglia/macrophage infiltration, and expression of chemokines (CCL2 and CCL3) at the injured site. The co-transplantation group also significantly increased arginase+/CD206+ macrophages (IL-4) and decreased iNOS+/CD16/32+ macrophages (IFN-γ), which was followed by higher IL-10 and IL-13 and lower IL-6 and TNF-α in their expression levels, a smaller cystic cavity area, and improved motor functions. These results indicate that OEC and SC co-transplantation could promote the shift of the macrophage phenotype from M(IFN-γ) to M(IL-4), reduce inflammatory cell infiltration in the injured site, and regulate inflammatory factors and chemokine expression, which provide a better immune environment for SCI repair.

Serially Ordered Magnetization of Nanoclusters via Control of Various Transition Metal Dopants for the Multifractionation of Cells in Microfluidic Magnetophoresis Devices.

A novel method (i.e., continuous magnetic cell separation in a microfluidic channel) is demonstrated to be capable of inducing multifractionation of mixed cell suspensions into multiple outlet fractions. Here, multicomponent cell separation is performed with three different distinguishable magnetic nanoclusters (MnFe2O4, Fe3O4, and CoFe2O4), which are tagged on A431 cells. Because of their mass magnetizations, which can be ideally altered by doping with magnetic atom compositions (Mn, Fe, and Co), the trajectories of cells with each magnetic nanocluster in a flow are shown to be distinct when dragged under the same external magnetic field; the rest of the magnetic characteristics of the nanoclusters are identically fixed. This proof of concept study, which utilizes the magnetization-controlled nanoclusters (NCs), suggests that precise and effective multifractionation is achievable with high-throughput and systematic accuracy for dynamic cell separation.

Multi-analyte profiling in human carotid atherosclerosis uncovers pro-inflammatory macrophage programming in plaques.

Molecular characterisation of vulnerable atherosclerosis is necessary for targeting functional imaging and plaque-stabilising therapeutics. Inflammation has been linked to atherogenesis and the development of high-risk plaques. We set to quantify cytokine, chemokine and matrix metalloproteinase (MMP) protein production in cells derived from carotid plaques to map the inflammatory milieu responsible for instability. Carotid endarterectomies from carefully characterised symptomatic (n=35) and asymptomatic (n=32) patients were enzymatically dissociated producing mixed cell type atheroma cell suspensions which were cultured for 24 hours. Supernatants were interrogated for 45 analytes using the Luminex 100 platform. Twenty-nine of the 45 analytes were reproducibly detectable in the majority of donors. The in vitro production of a specific network of mediators was found to be significantly higher in symptomatic than asymptomatic plaques, including: tumour necrosis factor α, interleukin (IL) 1β, IL-6, granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF), CCL5, CCL20, CXCL9, matrix metalloproteinase (MMP)-3 and MMP-9. Ingenuity pathway analysis of differentially expressed analytes between symptomatic and asymptomatic patients identified a number of key biological pathways (p< 10(-25)). In conclusion, the carotid artery plaque culprit of ischaemic neurological symptoms is characterised by an inflammatory milieu favouring inflammatory cell recruitment and pro-inflammatory macrophage polarisation.

Detecting Secreted Analytes from Immune Cells: An Overview of Technologies.

The tumor microenvironment is largely shaped by secreted factors and infiltrating immune cells and the nature of this environment can profoundly influence tumor growth and progression. As such, there is an increasing need to identify and quantify secreted factors by tumor cells, tumor-associated cells, and infiltrating immune cells. To meet this need, the dynamic range of immunoassays such as ELISAs and ELISpots have been improved and the scope of reagents commercially available has been expanded. In addition, new bead-based and membrane-based screening arrays have been developed to allow for the simultaneous detection of multiple analytes in one sample. Similarly, the optimization of intracellular staining for flow cytometry now allows for the quantitation of multiple cytokines from either a purified cell population or a complex mixed cell suspension. Herein, we review the rapidly evolving technologies that are currently available to detect secreted analytes. Emphasis is placed on discussing the advantages and disadvantages of these assays and their applications.

Hematopoietic Stem and Progenitor Cell Expansion in Contact with Mesenchymal Stromal Cells in a Hanging Drop Model Uncovers Disadvantages of 3D Culture.

Efficient ex vivo expansion of hematopoietic stem cells with a concomitant preservation of stemness and self-renewal potential is still an unresolved ambition. Increased numbers of methods approaching this issue using three-dimensional (3D) cultures were reported. Here, we describe a simplified 3D hanging drop model for the coculture of cord blood-derived CD34+ hematopoietic stem and progenitor cells (HSPCs) with bone marrow-derived mesenchymal stromal cells (MSCs). When seeded as a mixed cell suspension, MSCs segregated into tight spheroids. Despite the high expression of niche-specific extracellular matrix components by spheroid-forming MSCs, HSPCs did not migrate into the spheroids in the initial phase of coculture, indicating strong homotypic interactions of MSCs. After one week, however, HSPC attachment increased considerably, leading to spheroid collapse as demonstrated by electron microscopy and immunofluorescence staining. In terms of HSPC proliferation, the conventional 2D coculture system was superior to the hanging drop model. Furthermore, expansion of primitive hematopoietic progenitors was more favored in 2D than in 3D, as analyzed in colony-forming assays. Conclusively, our data demonstrate that MSCs, when arranged with a spread (monolayer) shape, exhibit better HSPC supportive qualities than spheroid-forming MSCs. Therefore, 3D systems are not necessarily superior to traditional 2D culture in this regard.

High-dose immunosuppressant alters the immunological status of New Zealand white rabbits following skin transplantation.

The aim of this study was to investigate the effect of an immunosuppressant on the immunological status of New Zealand white rabbits after skin grafting, and to evaluate a method for monitoring the immunological status of subjects with skin transplants. The rabbits were randomly divided into allograft rejection, autograft tolerance, nontransplant, allograft low-dose immunosuppressant and allograft high-dose immunosuppressant groups. The rabbits in the low- and high-dose immunosuppressant groups were treated with cyclosporine A intravenously 8 h prior to skin transplantation and once daily following transplantation at doses of 2 and 25 mg/kg, respectively. At 12 days after skin transplantation, the spleens of donor (female) rabbits and recipient (male) rabbits were harvested for the preparation of single-cell suspensions. The splenocytes from recipient and donor rabbits were labeled with 0.3 or 6 µM carboxy fluorescein diacetate succinimidyl ester, respectively, and a mixed cell suspension was prepared. The final preparation was intravenously injected into recipient New Zealand white rabbits. The ratio of the two fluorescently labeled cell populations in the peripheral blood was measured using flow cytometry at 1, 2, 4 and 8 h after the injection, and the cell death rate was calculated. Histological analysis was also performed on samples collected at the time of splenectomy. The cell death rates of the allograft rejection and low-dose immunosuppressant groups reached their highest levels 8 h after the injection of spleen cell suspension. Allogeneic spleen cells from donor male rabbits were almost completely removed within 8 h of injection. The cell death rate increased slowly in the nontransplant, autograft and high-dose immunosuppressant groups without specificity. This study provides a specific method for the in vivo monitoring of the immunological status of patients after skin grafting. This method can quickly and accurately detect the immunological status of recipients following the injection of a mixed splenocyte suspension, thereby indicating the strength of immune rejection by the immune systems of the recipients.

Immobilization of &lt;i&gt;Lactobacillus plantarum&lt;/i&gt; B134 Cells using Sodium Alginate for Lactose Hydrolysis in UHT Milk

Hydrolysis of lactose in milk by β-galactosidase from immobilized bacterial cells has the potential to alleviate the problem of lactose intolerance. The present study was aimed to immobilize cells of L. plantarum strain B134 and evaluate their efficiency in hydrolyzing lactose in ultra high temperature (UHT) milk. Immobilized cells were generated by mixing cell suspensions with solutions of sodium alginate and calcium chloride. The β-galactosidase activity of the immobilized cells was tested by determining their ability in hydrolyzing lactose in UHT milk (whole milk and skimmed milk). Results showed that cells of L. plantarum strain B134 were entrapped optimally using a combination of 1 % sodium alginate, 100 mM calcium chloride and 12 % w/v cell suspension. The highest β-galactosidase activity was achieved at pH 6.5 and a temperature of 45 ºC for 5 minutes incubation time. The immobilization efficiency achieved was 28.95 %. The immobilized cells could reduce lactose by up to 85.45 % in UHT whole milk and 91.26 % in UHT skimmed milk. The times required for that reduction of lactose in UHT whole milk and UHT skimmed milk were 12 hours and 9 hours respectively. The immobilized cells could be re-used up to 4 times for efficient lactose hydrolysis for both types of milk. Therefore, immobilized cells of L. plantarum B134 have the potential to be used for lactose hydrolysis in UHT milk.

From the rat to the beta cell: a fast and effective technique of separation of Langerhans islets and direct purification of pancreatic beta cells

Isolated Langerhans islets represent a useful model for the study of the endocrine pancreas. The possibility to purify pancreatic beta cells from a mixed Langerhans islet cell population may lead towards a dedicated focus on beta cell research. We describe an effective and rapid immunomagnetic technique for the direct purification of beta cells from isolated Langerhans islets of rat. After the sacrifice of the rat, the Langerhans islets were separated by ductal injection of the pancreas with collagenase, altered to a mixed Langerhans islet cell population and incubated with conditioned immunomagnetic beads targeted to the beta cell surface. The beads were previously coated with a specific antibody against the surface of the beta cell, namely K14D10. The suspension of mixed Langerhans islet cells and immunomagnetic K14D10-conditioned beads was pelleted by a magnetic particle concentrator to isolate the bead-bound cells, which were finally suspended in a culture medium. The purified cells were immunoreactive for insulin and no glucagon-positive cells were detected at immunocytochemistry. Real Time PCR confirmed the purification of the pancreatic beta cells. This immunomagnetic technique allows a rapid, effective and consistent purification of beta cells from isolated Langerhans islets in a direct manner by conditioning the immunomagnetic beads only. This technique is easy, fast and reproducible. It promises to be a reliable method for providing purified beta cells for in vitro research.

Domoic acid toxicokinetics in Dungeness crabs: New insights into mechanisms that regulate bioaccumulation

Domoic acid (DA) is an excitatory neurotoxic amino acid produced by several marine algal species and is the causative agent of amnesic shellfish poisoning. Profound differences in the toxicokinetics of DA have been identified in a wide variety of shellfish. We characterized the toxicokinetics of DA in Dungeness crabs (Metacarcinus magister) after oral and intravascular dosing (IV) using a variety of doses ranging from 0.1 to 20mg/kg. After a 1mg/kg oral dose, DA disappeared from the foregut within 2h and largely accumulated in the hepatopancreas, with hemolymph and other tissues having 100–1000 times lower concentrations. After IV dosing, hemolymph concentrations of DA were unexpectedly high and toxicokinetic analysis indicated the steady-state volume of distribution (Vss) was 123–197ml/kg, which is well below the hemolymph volume of 350ml/kg for crabs. This indicated only limited extravascular distribution of DA was occurring after IV injection, which is surprising considering the capacity of the hepatopancreas to sequester DA after oral dosing. Additional studies measured the partitioning of DA in hepatopancreas cellular and subcellular fractions. The subcellular distribution of DA was primarily associated with the S8 fraction and could be filtered through a 30,000MW cut-off filter, indicating DA was not appreciably bound to macromolecules. Interestingly, very little (<0.4%) of the total hepatopancreas DA tissue content was associated with the cellular fraction isolated after dissociation and separation from tissue fragments. The in vivo and in vitro results led us to hypothesize that DA uptake and distribution is regulated by crustacean orthologs of ATP-binding cassette (ABC) type transporters. We tested this hypothesis by co-exposing crabs to DA and known inhibitors of ABC transporters (verapamil, cyclosporine A and MK-571) and through in vitro studies using isolated hepatopancreas tissue and mixed cell suspensions prepared from hepatopancreas tissue. The in vivo results were inconclusive in that the toxicokinetics of DA was not consistently altered by co-administration of the inhibitors. Two exceptions were MK-571, which significantly increased the total body clearance of DA and co-administration of verapamil, which significantly increased the hepatopancreas tissue content of DA 24h after IV injection. Isolated pieces of hepatopancreas tissue were able to readily absorb DA from incubation media, but mixed cell suspensions did not. The absorption of DA or lack thereof was largely unaffected by co-incubation with verapamil although cell suspensions appeared to accumulate small quantities of DA in the presence of verapamil. Collectively, the results of this study suggest DA accumulates in the extracellular spaces of the hepatopancreas, such as the tubular lumen. Under natural circumstances, crabs feeding on contaminated shellfish would be expected to readily absorb DA, which is then stored and slowly eliminated in urine. If the DA exposure level exceeds the storage capacity of the tissue (as occurred with the 20mg/kg dose), breakthrough occurs resulting in much higher systemic exposure and potential for DA toxicity.

BAS/BSCR38 Cytokine profiling in culture reveals a predominance of M1 macrophage polarisation in symptomatic carotid plaques

RationaleMacrophages in atherosclerotic plaques are a heterogeneous population. To uncover signatures of classical (M1) or alternative (M2) macrophage polarisation during plaque instability, we compared cytokine and chemokine production via Luminex...

Enhancement of specific cell-capture efficiency using a reversible dielectrophoresis field

This work proposes using a periodic and reversible DEP (dielectrophoresis) field to improve the immuno-capture efficiency of the rare specific cells in a mixed cell suspension. The forward DEP field gently guides all the cells to interact with the bioprobes that are immobilized on a PVDF substrate in the reactor, and the reverse DEP field redistributes the un-bonded cells into the suspension for next reaction chance. The fluorescence visibility of the specific T-cells was also improved via quantum dots/tetramer complex staining. Accordingly, the specifically stained and captured cells could be imaged and counted. The experimental results indicated that the forward DEP can double the cell-capture efficiency compared with that without DEP enhancement, and the reversible DEP scheme can improve the efficiency about 25% further. This assay is also successively used to detect the population of rare cytotoxic Tc cells in a human blood sample; and the Tc cell has specific receptors on the membrane surface to identify the Epstein–Barr virus (EBV).