Number Of Adherent Cells Research Articles

Prostaglandin E2 activates complement protein CD55 to enhance cell adhesion in endometriosis

Endometriosis is a leading cause of pelvic pain and infertility. Prostaglandin E2 (PGE2) is widely regarded to be central to its pathogenesis.1 A promising novel target of PGE2 signaling is decay accelerating factor (CD55) which has increased RNA expression in endometriotic stromal cells.2 CD55 has a well described role in the complement pathway, but emerging evidence indicates other functions in cell proliferation and activation of cellular signaling. The objective of this work was to determine whether CD55 protein is expressed in ectopic endometrium, whether PGE2 is a regulator of CD55 expression, and whether CD55 is necessary and sufficient for endometriosis pathogenesis via a non-canonical pathway. Laboratory investigation. To study the principal cellular compartments of endometriotic lesions in vitro, immortalized endometrial stromal (T-HESC) and endometriotic epithelial (12Z) cell lines were studied. Immunohistochemistry was performed to localize the presence of CD55 in a random sample of eight human pathology specimens of eutopic and ectopic endometrium. CD55 expression analysis was performed on cell lines (RNA expression by quantitative real-time PCR; protein expression by immunoblotting and flow cytometry) treated first with 0, 1, 3, 10, 30, and 100 uM of PGE2 then with 10uM PGE2 for 0, 1, 2, 6, and 24 hours. Overexpression of CD55 in 12Z cells was achieved using lentiviral transduction with an empty vector control. Cell adhesion was determined by plating cells on cell culture plates coated with fibronectin, collagen I, or bovine serum albumin at different time points, washing the plates, then quantifying remaining adherent cells using a luminescent ATP detection assay. Intra- and inter-experimental variation was addressed by multiple wells per condition and ≥2 replicates per experiment. Both 12Z and T-HESC lines demonstrated CD55 expression at baseline, as did histologic specimens of eutopic and ectopic endometrium. Treatment with PGE2 of 12Z and T-HESC lines resulted in increased CD55 expression in a dose dependent fashion until maximal treatment effect (3-fold) was achieved at 10 uM. In time course experiments, CD55 RNA expression was maximal at 6 hours and protein expression at 18 hours. 12Z cells overexpressing CD55 demonstrated more than 2-fold number of adherent cells as compared to wildtype and empty vector controls. These preliminary cellular studies indicate that PGE2 induction of CD55 can lead to promotion of adhesion of endometriotic cells, a necessary step in the pathogenesis of endometriosis. Future in vivo studies using CD55-/- mice will investigate CD55 function in adhesion in mouse models of endometriosis. Understanding this downstream pathway of PGE2 signaling may identify points of fragility for future translational therapies.

Well-defined monolith morphology regulates cell adhesion and its functions

Hydrophilic epoxy resin-based monoliths were employed as cell culture substrates. The monoliths were made of a porous material with a bicontinuous structure that consisted of a porous channel and a resin skeleton. Monolith disks were prepared with a skinless surface through polymerization-induced spinodal decomposition-type phase separation. The pore sizes, which were well controlled by the polymerization temperature, ranged from 70 to 380 nm. The quantity of protein adsorbed per unit area and the early-stage adhesion of HepG2 cells on the monolith substrates were independent of pore size, meaning they were not affected by surface topology. Long-term cell adhesion, as indicated by adherent cell number and shape, as well as liver-specific gene expression were significantly affected by pore size. In terms of cell shape, number, and gene expression, pores of approximately 200 nm were most suitable for HepG2 cell growth. These results highlight the importance of monolith morphology for use as a cell culture substrate. The well-controlled morphology demonstrated in this work indicates monoliths are capable of supporting growth for various types of cells in a range of applications.

Differential Sensitivity of Myeloid and Lymphoid Cell Populations to Apoptosis in Peritoneal Cavity of Mice with Model Larval Mesocestoides Vogae Infection.

SummaryThe metacestode stage of the tapeworm Mesocestoides vogae (M. vogae) has the ability of asexual growth in the peritoneal cavity of rodents and other intermediate hosts without restriction. Early immunological events have decisive role in the establishment of infection. In the present study we investigated the kinetic of myeloid and lymphoid cell populations and the proportions of cells undergoing apoptosis in peritoneal cavities of mice within the first month after oral infection with M. vogae larvae. Proportions of cell phenotypes and apoptotic cells were examined by flow cytometry and by microscopical analysis of cells following May/Grünwald staining and fluorescent stain Hoechst 33234, respectively. Total numbers of peritoneal cells increased and their distribution changed towards accumulation of myelo-monocytic cell lineage in the account of reduced proportions of lymphoid cells. CD4+ T cell subpopulations were more abundant than CD8+ and their proportions elevated within two weeks post infection (p.i.) which was followed by a significant decline. Expression level of CD11c marker on myelo-monocytic cells revealed phenotype heterogeneity and proportions of cells with low and medium expression elevated from day 14 p.i. along with concurrent very low presence of CD11chigh phenotype. Lymphoid cell population was highly resistant to apoptosis but elevated proportions of myeloid cells were in early/late stage of apoptosis. Apoptosis was detected in a higher number of adherent cells from day 14 p.i. onwards as evidenced by nuclear fluorescent staining. By contrast, cells adherent to larvae, mostly macrophages and eosinophils, did not have fragmented nuclei. Our data demonstrated that apoptosis did not account for diminished population of peritoneal lymphoid cells and substantial proportions of myeloid cells seem to be more susceptible to apoptotic turnover in peritoneal cavity of mice with ongoing M. vogae infection, suggesting their important role in the host-parasite interactions.

Fabrication of thermoresponsive degradable hydrogel made by radical polymerization of 2-methylene-1,3-dioxepane: Unique thermal coacervation in hydrogel

Thermoresponsive degradable hydrogels have attracted attention because their properties allow for their use as smart drug carriers for drug delivery systems and cell scaffolds in the living body. In this study, we synthesized thermoresponsive degradable hydrogels via radical copolymerization of 2-methylene-1,3-dioxepane and 2-hydroxyethylacrylate in the presence of a crosslinker at various feed ratios. The synthesized hydrogels showed a temperature-dependent swelling/shrinking behavior, which was also observed in the corresponding linear polymers. During shrinking, coacervate droplets were observed within the hydrogel. Under mild alkaline conditions, the hydrogels were degraded and converted into hydrophilic oligomers. Moreover, cell adhesion to the hydrogel surfaces was observed using poly (d-lysine)-modified hydrogels. The number of adhered cells increased on the hydrogels that showed shrinking behavior at 37 °C. The prepared hydrogels are expected to be applicable for use in functional cell scaffolding instead of as non-degradation materials.

BMSC affinity peptide-functionalized \u03b2-tricalcium phosphate scaffolds promoting repair of osteonecrosis of the femoral head

BackgroundOsteonecrosis of the femoral head (ONFH) is a disabling disease. Early treatment is crucial to the prognosis of the disease. Core decompression (CD) is one of the most commonly used methods for the treatment of early ONFH. But it could not prevent the collapse of the necrotic femoral head. How to improve the therapeutic effect of early ONFH on the basis of CD has become an area of focused research.MethodsFunctional β-tricalcium phosphate (β-TCP) scaffolds modified by DPIYALSWSGMA (DPI) peptide, a bone marrow-derived mesenchymal stem cell (BMSC) affinity peptide, were constructed using an adsorption/freeze-drying strategy. The affinity of DPI peptide towards rabbit BMSCs was investigated using flow cytometry and fluorescence cytochemistry. In vitro cell adhesion assay was performed to study the adherent ability of rabbit BMSCs on functional β-TCP scaffolds. After the rabbit model of early ONFH was established, DPI peptide-modified and pure β-TCP scaffolds were transplanted into the remaining cavity after CD. Meanwhile, rabbits treated with pure CD were used as blank control. Twelve weeks after surgery, histological analysis was performed to show the therapeutic effect of three methods on early ONFH.ResultsThe result of ImageXpress Micro Confocal indicated that fabricated DPI peptide-modified functional β-TCP scaffolds exhibited green fluorescence. In flow cytometry, the average fluorescence intensity for rabbit BMSCs incubated with FITC-DPI was significantly higher than that of FITC-LSP (P = 2.733 × 10−8). In fluorescence cytochemistry, strong fluorescent signals were observed in rabbit BMSCs incubated with FITC-DPI and FITC-RGD, whereas no fluorescent signals in cells incubated with FITC-LSP. In cell adhesion assay, the number of adherent cells to β-TCP-DPI scaffolds was more than that of pure β-TCP scaffolds (P = 0.033). The CD + β-TCP-DPI group expressed the lowest vacant bone lacunae percentage compared to CD group (P = 2.350 × 10−4) and CD + β-TCP group (P = 0.020). The expression content of COL1 in CD + β-TCP-DPI group was much higher than CD group (P = 1.262 × 10−7) and CD + β-TCP group (P = 1.666 × 10−7) according to the integrated optical density (IOD) analyses.ConclusionFunctional β-TCP scaffolds modified by DPI peptide were successfully synthesized using an adsorption/freeze-drying strategy. DPI peptide has good affinity towards rabbit BMSCs. The adhesion of rabbit BMSCs on DPI peptide-modified β-TCP scaffolds was apparently enhanced. CD followed by implantation of DPI peptide-modified β-TCP scaffolds can apparently improve the treatment of early ONFH compared with pure CD and CD followed by implantation of unmodified β-TCP scaffolds. Our current study provides an improved method for the treatment of early ONFH.

Multi-parametric Cell-Based Inflammation Assays for Cytokines and Cell Surface Antigens

Abstract Macrophages and vascular cells upregulate expression of cytokines and adhesion molecules in response to inflammation stimuli. Monitoring these molecules can provide a physiological read-out for inflammation. We present results from a multiparametric assays that used a combination of low-volume ELISA for secreted cytokines and automated cell imaging for cell surface markers to evaluate the effect of different mediators on inflammatory response with two cell models. First, human monocytic THP-1 cells were differentiated to macrophages and activated by LPS. Phenotypic changes in cell morphology were quantified by cell area and number of adherent cells using TL imaging. Secretion of IL-8, IL-1β and TNF-α was quantified by an automated low-volume ELISA (PuMA System). Second, primary human umbilical vein endothelial cells (HUVEC) were stimulated with inflammation cytokines (TNF-α, IFN-γ, and IL-1β), then PuMA System was used to quantify amount of MCP-1, IL-8, and IL-6 in cell supernatants. Expression of adhesion molecules VCAM and HLADR was quantified by measurement of total fluorescence from antibody-stained cells using an ImageXpress Pico system. Concentration dependent inhibition of inflammation by the compounds AG126 and MG132, SB202190 and PDTC were measured and EC50s calculated. Clear differences in cytokine expression were seen between the compounds consistent with reported mechanism of actions of the kinase inhibitors. The combination of imaging and microfluidic-based assays provides an efficient multiparametric assay system that can test the efficacy of anti-inflammatory compounds versus toxicity and provide insight into their MoA by selective inhibition of markers triggered by different signaling pathways.

Beyond Gut Instinct: Metabolic Short-Chain Fatty Acids Moderate the Pathogenesis of Alphaherpesviruses.

Short-chain fatty acids (SCFA), such as sodium butyrate (SB), sodium propionate (SPr), and sodium acetate (SAc), are metabolic end-products of the fermentation of dietary fibers. They are linked with multiple beneficial effects on the general mammalian health, based on the sophisticated interplay with the host immune response. Equine herpesvirus 1 (EHV1) is a major pathogen, which primarily replicates in the respiratory epithelium, and disseminates through the body via a cell-associated viremia in leukocytes, even in the presence of neutralizing antibodies. Infected monocytic CD172a+ cells and T-lymphocytes transmit EHV1 to the endothelium of the endometrium or central nervous system (CNS), causing reproductive or neurological disorders. Here, we questioned whether SCFA have a potential role in shaping the pathogenesis of EHV1 during the primary replication in the URT, during the cell-associated viremia, or at the level of the endothelium of the pregnant uterus and/or CNS. First, we demonstrated the expression of SCFA receptors, FFA2 and FFA3, within the epithelium of the equine respiratory tract, at the cell surface of immune cells, and equine endothelium. Subsequently, EHV1 replication was evaluated in the URT, in the presence or absence of SB, SPr, or SAc. In general, we demonstrated that SCFA do not affect the number of viral plaques or virus titer upon primary viral replication. Only SB and SPr were able to reduce the plaque latitudes. Similarly, pretreatment of monocytic CD172a+ cells and T-lymphocytes with different concentrations of SCFA did not alter the number of infected cells. When endothelial cells were treated with SB, SPr, or SAc, prior to the co-cultivation with EHV1-inoculated mononuclear cells, we observed a reduced number of adherent immune cells to the target endothelium. This was associated with a downregulation of endothelial adhesion molecules ICAM-1 and VCAM-1 in the presence of SCFA, which ultimately lead to a significant reduction of the EHV1 endothelial plaques. These results indicate that physiological concentrations of SCFA may affect the pathogenesis of EHV1, mainly at the target endothelium, in favor of the fitness of the horse. Our findings may have significant implications to develop innovative therapies, to prevent the devastating clinical outcome of EHV1 infections.

The ability of usage various RGD configurations for the biodegradable vascular graft modification

The results of modifying non-woven matrices based on polyhydroxybutyrate / valerate and polycaprolactone with various RGD peptides (peptides: RGDK, AhRGD, c[RGDFK]) attached to the surface of matrices using hexamethylenediamine were studied. The structure of the surface of the matrices before and after the modification was not significantly changed. After 7 days of cultivation, the largest number of adherent and viable human umbilical vein endothelial cells of a person were detected on the surface of matrices with AhRGD or c[RGDFK]. However, cell proliferation was practically absent on the matrices with AhRGD. Thus, the modification of the surface of matrices with the AhRGD peptide is most suitable for products requiring rapid endothelization.

L-cysteine improves blood fluidity impaired by acetaldehyde: In vitro evaluation.

Blood fluidity is reportedly influenced by the volume and function of blood cells and plasma and is a predictor of primary cardiovascular events in patients with traditional cardiovascular risk factors. Heavy alcohol consumption was shown to be associated with a higher risk for cardiovascular diseases. Acetaldehyde (ACD), an oxidizing substance formed from ethanol, reportedly stimulates monocyte adhesion, causes abnormalities in the red blood cell (RBC) membrane, and decreases RBC deformability. In addition, it was reported that blood ACD levels are reduced in mice pretreated with L-cysteine. However, there are no studies on the effect of ACD and/or L-cysteine on blood fluidity. In the present study, we evaluated whether ACD impairs blood fluidity. In addition, the effect of L-cysteine on blood fluidity impaired by ACD was examined. Blood samples were obtained from 10 healthy, non-smoking, male volunteers (age: 23.4 ± 1.2 years, body mass index: 21.8 ± 2.6 kg/m2). ACD or ACD and L-cysteine were added to the blood samples before each experiment. We measured the passage time of 100 μL blood and RBC suspension using Kikuchi’s microchannel method. Percentage of microchannel obstruction and the number of adherent white blood cells (WBCs) on microchannel terrace were counted. The blood passage time, percentage of microchannel obstruction, and numbers of adherent WBCs on the microchannel terrace increased after adding ACD in a concentration-dependent manner, whereas they decreased after adding ACD and L-cysteine in a L-cysteine concentration-dependent manner. No significant effects were observed in passage time for 100 μL RBC suspension after adding ACD and L-cysteine. This study suggested that blood fluidity impaired by ACD might improve after adding L-cysteine.

Deleterious Effects of Methylene Blue on Rat Nucleus Pulposus Cell in Vitro: Changes in Cell Viability and Secretory Phenotype in Exposed Cells.

To investigate the effects of methylene blue (MB) on the viability and secretory phenotype of rat nucleus pulposus (NP) cells in vitro. Rat NP cells were isolated, cultured, and treated with different MB concentrations (0-6.25 ng/mL) for different lengths of time. We evaluated the changes in cell morphology and cell viability. We also examined the cells for expression of collagen II, aggrecan, matrix metalloproteinase (MMP)-3, MMP-9, and inducible nitric oxide synthase (iNOS). After 2.5 to 6.25 ng/mL MB induced for 6 hours, numerous NP cells were dyed blue and rounded up. The adherent cell number was reduced by MB treatment. The viability of rat NP cells was significantly inhibited by MB in a dose- and time-dependent manner. Treatment with a very low dose of MB (1.5625 ng/mL) resulted in lower expression of collagen II and aggrecan and higher expression of MMP-3, MMP-9, and iNOS in rat NP cells. Rat NP cells exposed in vitro to MB significantly reduced their viability. Moreover, MB upregulated catabolism gene expression and downregulated anabolism gene expression in rat NP cells. These results suggest MB may be harmful to NP cells. The dose of intradiskal injected MB should be as low as possible to prevent or limit the damage to intervertebral disks.

Combinatorial effects of inorganic ions on adhesion and proliferation of osteoblast-like cells.

Combinatorial effects of three ions, namely silicate (Si), calcium (Ca), and magnesium (Mg) ions, on the adhesion and proliferation of MC3T3-E1 mouse osteoblast-like cells were evaluated. The cells were cultured in single-, dual-, or triple-ion-conditioned culture media with systematically changed ion concentrations. The ranges of Si, Ca, and Mg ion concentrations were set as 10-70, 80-400, and 25-500 ppm, respectively. The numbers of adherent live cells were measured after culturing for 3 h and for 1, 3, and 5 days to examine cell adhesion and proliferation, respectively. Mg ions predominantly enhanced cell adhesion in both the dual-ion (xSi-zMg and yCa-zMg) and triple-ion (xSi-yCa-zMg) systems but had no effect when they acted individually in the single-ion system. Conversely, Si ions predominantly enhanced cell proliferation in most single- and triple-ion-conditioned media. Evaluation of the combinatorial effects of the three ions on cell adhesion and proliferation revealed that the dual- and triple-ion-conditioned media mainly conferred synergistic effects on adhesion but antagonistic effects on proliferation. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 1042-1051, 2019.

Rapid quantification of cellular proliferation and migration using ImageJ.

Cellular proliferation and migration are crucial during development, regeneration and disease. Methods to quantify these processes are available; however, many are time consuming and require specialized equipment and costly reagents. Simple cell counts (proliferation analysis) and the scratch assay (migration analysis) are favorable methods due to their simplicity and cost-effectiveness; however, they rely on subjective and labor-intensive manual analysis, resulting in low throughput. We have developed optimized protocols to rapidly and accurately quantify adherent cell number and wound area using ImageJ, an open-source image processing program. Notably, these adaptable protocols facilitate quantification with significantly greater accuracy than manual identification.

Equilin in conjugated equine estrogen increases monocyte-endothelial adhesion via NF-κB signaling.

The adhesion of monocytes to endothelial cells, which is mediated by adhesion molecules, plays a crucial role in the onset of atherosclerosis. Conjugated equine estrogen, which is widely used for estrogen-replacement therapy, contains both estrone sulfate and various nonhuman estrogens, including equilin. To investigate the association between various estrogen types and atherosclerosis risk, we examined their effect on adhesion-molecule expression in human umbilical vein endothelial cells (HUVECs). In estrogen-treated HUVECs, the mRNA and protein expression levels of adhesion molecules were quantified by real-time polymerase chain reaction and enzyme immunoassay. Additionally, a flow-chamber system was used to assess the effects of estrogens on the adherence of U937 monocytoid cells to HUVECs. Equilin, but not 17β-estradiol (E2) or other types of estrogen, significantly increased the mRNA (P < 0.01) and protein (P < 0.05) expression of the adhesion molecules E-selectin and intercellular adhesion molecule-1 as compared with levels in controls. Equilin treatment increased the adherence of U937 monocytoid cells to HUVECs relative to the that in the control (P < 0.05), decreased estrogen receptor (ER)β expression, and increased the expression of proteins involved in nuclear factor kappa-B (NF-κB) activation relative to levels in controls. Furthermore, the accumulation of NF-κB subunit p65 in HUVEC nuclei was promoted by equilin treatment. By contrast, E2 treatment neither increased the number of adhered monocytoid cells to HUVECs nor altered the expression of ERβ or NF-κB-activating proteins. Our findings suggest that in terms of the adhesion of monocytes at the onset of atherosclerosis, E2 may be preferable for estrogen-replacement therapy. Further studies comparing equilin treatment with that of E2 are needed to investigate their differential impacts on atherosclerosis.

"Solvent/non-solvent" treatment as a method for non-covalent immobilization of gelatin on the surface of poly(l-lactic acid) electrospun scaffolds.

In the present study, we report a simple and efficient method of gelatin immobilization on the surface of PLA electrospun fibers using pre-treatment with a mixture of toluene and ethanol allowing to form swelled surface layer followed by gelatin adsorption from its solution in PBS. Our results demonstrate that gelatin immobilization leads to a decrease in the water contact angle from 120° to 0°, enhances scaffold strength up to 50%, and doubles the number of adhered cells and their average area. We observed that the maximum amount of gelatin (0.07 ± 0.01 mg per cm3 of the scaffold) was immobilized during the first five minutes of exposure to the gelatin solution. Modified scaffolds demonstrated increased strength.

Nitrogen-Doped Titanium Dioxide Thin Films Formation on the Surface of PLLA Electrospun Microfibers Scaffold by Reactive Magnetron Sputtering Method

Nitrogen-doped thin titanium dioxide films formed by the reactive magnetron sputtering method on the surface of PLLA electrospun microfibers scaffold were investigated. It was shown that the chemical composition of the films is shifting from titanium dioxide (TiO2) composites saturated with C–NH, C=N, N–C=N and HN–C=O compounds to solid solutions of titanium oxides (TixOy) and titanium oxynitrides (TiOxNy) with the increased time of the treatment. An empirical model describing changes in the chemical composition of the surface due to the treatment was proposed. It was shown that the modification of the PLLA microfibers scaffolds surface improves cell-scaffold and cell–cell interactions with the highest number of viable adherent cells observed on the scaffold treated for 4 min.

Collagen type-IV Langmuir and Langmuir–Schäfer layers as model biointerfaces to direct stem cell adhesion

In biomaterial development, the design of material surfaces that mimic the extra-cellular matrix (ECM) in order to achieve favorable cellular instruction is rather challenging. Collagen-type IV (Col-IV), the major scaffolding component of Basement Membranes (BM), a specialized ECM with multiple biological functions, has the propensity to form networks by self-assembly and supports adhesion of cells such as endothelial cells or stem cells. The preparation of biomimetic Col-IV network-like layers to direct cell responses is difficult. We hypothesize that the morphology of the layer, and especially the density of the available adhesion sites, regulates the cellular adhesion to the layer. The Langmuir monolayer technique allows for preparation of thin layers with precisely controlled packing density at the air–water (A–W) interface. Transferring these layers onto cell culture substrates using the Langmuir–Schäfer (LS) technique should therefore provide a pathway for preparation of BM mimicking layers with controlled cell adherence properties. In situ characterization using ellipsometry and polarization modulation-infrared reflection absorption spectroscopy of Col-IV layer during compression at the A–W interface reveal that there is linear increase of surface molecule concentration with negligible orientational changes up to a surface pressure of 25 mN m−1. Smooth and homogeneous Col-IV network-like layers are successfully transferred by LS method at 15 mN m−1 onto poly(ethylene terephthalate) (PET), which is a common substrate for cell culture. In contrast, the organization of Col-IV on PET prepared by the traditionally employed solution deposition method results in rather inhomogeneous layers with the appearance of aggregates and multilayers. Progressive increase in the number of early adherent mesenchymal stem cells (MSCs) after 24 h by controlling the areal Col-IV density by LS transfer at 10, 15 and 20 mN m−1 on PET is shown. The LS method offers the possibility to control protein characteristics on biomaterial surfaces such as molecular density and thereby, modulate cell responses.

Spontaneously contracting cell aggregates derived from grass carp heart as a promising tool in in vitro heart research.

Regarding challenges in isolation and maintenance of cultured heart cells, introduction of new in vitro heart model that is stable and exhibits long-term spontaneously contracting cell aggregates (SCCs), whose electrophysiological properties are comparable to the human heart, is required. This research aimed to establish cardiac primary cells and to evaluate the effects of culture conditions. Also the effect of fish age on beating SCC and cardiac cell morphology were studied. Twelve healthy grass carps (Ctenopharyngodon idella) were divided into four groups based on their age. Non-enzymatic explant culture was used and heart explants were incubated at 21-31°C for 60days. After proliferation of the cardiac primary cells, changes in their morphology and their beatings were recorded. The results showed that the explants derived from different age of grass carp fish are fully viable and proliferative with formation of SCC for a long period of time. By increasing the number of adhered cells, the cardiac primary cells became more flat and elongated. Increasing the medium temperature and fetal bovine serum concentration in culture medium led to decline and enhancement in beat frequencies of heart cell aggregates, respectively. Also, grass carp heart explant had high potential in regeneration (especially in young fish) and thus high feasibility to generate stable long-term cultures. In general, it seems that explant culture of heart from grass carp can be considered as a promising tool in heart research area.

Assessment of a photobioreactor-coupled modified Robbins device to compare the adhesion of Nannochloropsis gaditana on different materials

The prevention of biofouling in the inner walls of closed photobioreactors (PBRs) becomes a critical step in improving the performance of photosynthetic microalgae bioprocesses. Selection of antifouling materials implies approaches at laboratory scale. This work reports the use of a flow cell of the modified Robbins device (MRD) type coupled to a PBR, operated in both batch and continuous modes with the model marine microalgae Nannochloropsis gaditana to study the biofouling formation on diverse materials. The fluid-dynamics within the MRD was studied via CFD-aided simulations. At separation distances lower than the cells' diffusion layer thickness, a diffusion-controlled transport of the cells to the material surface was postulated. Results suggested that the flow density of cells in the MRD (Jz), governed by cell concentration gradients, is a significant factor in the adhesion intensity (B) when the PBR is operated in batch mode; not in the continuous mode where the differences observed in B between materials were mainly attributed to the type of material. Polyvinylchloride (PVC) was clearly the best anti-biofouling material compared to polycarbonate, polystyrene, borosilicate glass and stainless steel. The B maximum occurred at the end of the stationary phase in batch culture mode. Continuous culture operation seemed to be preferable since once steady state is achieved, the B value remained low and constant, indicating equilibrium between the number of adhered cells per surface unit and the cell concentration in the culture broth – this was because the adhered cells did not grow on the surface due to phosphate limitation. The PBR-coupled MRD has demonstrated to be well-suited for the screening of antifouling materials under fluid-dynamic conditions relevant in PBRs.

Heavy Metal Ion Stress on Halobacterium salinarum R1 Planktonic Cells and Biofilms

Halobacterium salinarum R1 is an extremely halophilic archaeon, able to attach to the surface and to form characteristic biofilm structures under physiological conditions. However, the effect of environmental stress factors like heavy metals on biofilms was still unknown. Here, we report on the first insights into H. salinarum biofilm formation when exposed to copper, nickel and zinc and describe the effects of metal ions on the architecture of mature biofilms. We also studied the effects on gene expression in planktonic cells. Investigation of planktonic growth and cell adhesion in the presence of sub-lethal metal concentrations yielded an up to 60% reduced adhesion in case of copper and a significantly enhanced adhesion in case of zinc, whereas nickel treatment had no effect on adhesion. A PMA-qPCR assay was developed to quantify live/dead cells in planktonic cultures and mature biofilms, enabling the investigation of cell vitality after metal exposure. An increased resistance was observed in biofilms with up to 80% in case of copper- and up to 50% in case of zinc exposure compared to planktonic cells. However, nickel-treated biofilms showed no significant increase of cell survival. Microscopic investigation of the architecture of mature biofilms exposed to lethal metal concentrations demonstrated an increased detachment and the formation of large microcolonies after copper treatment, whereas the number of adherent cells increased strongly in nickel-exposed biofilms. In contrast, zinc exposed-biofilms showed no differences compared to the control. Analysis of the expression of genes encoding putative metal transporters by qRT-PCR revealed specific changes upon treatment of the cells with heavy metals. Our results demonstrate diverse effects of heavy metal ions on H. salinarum and imply a metal-specific protective response of cells in biofilms.

α-Chymotrypsin Immobilized on a Low-Density Polyethylene Surface Successfully Weakens Escherichia coli Biofilm Formation.

The protease α-chymotrypsin (α-CT) was covalently immobilized on a low-density polyethylene (LDPE) surface, providing a new non-leaching material (LDPE-α-CT) able to preserve surfaces from biofilm growth over a long working timescale. The immobilized enzyme showed a transesterification activity of 1.24 nmol/h, confirming that the immobilization protocol did not negatively affect α-CT activity. Plate count viability assays, as well as confocal laser scanner microscopy (CLSM) analysis, showed that LDPE-α-CT significantly impacts Escherichia coli biofilm formation by (i) reducing the number of adhered cells (−70.7 ± 5.0%); (ii) significantly affecting biofilm thickness (−81.8 ± 16.7%), roughness (−13.8 ± 2.8%), substratum coverage (−63.1 ± 1.8%), and surface to bio-volume ratio (+7.1 ± 0.2-fold); and (iii) decreasing the matrix polysaccharide bio-volume (80.2 ± 23.2%). Additionally, CLSM images showed a destabilized biofilm with many cells dispersing from it. Notably, biofilm stained for live and dead cells confirmed that the reduction in the biomass was achieved by a mechanism that did not affect bacterial viability, reducing the chances for the evolution of resistant strains.