1,721,015 research outputs found
Engineering biomaterials to tailor the microenvironment for macrophage–endothelium interactions
No full textMacrophages and endothelial cells (ECs) have essential roles in physiological and pathological conditions by regulating inflammation, vascularization and tissue remodelling. Although the interactions between macrophages and ECs in tissue homeostasis and disease progression have been extensively studied in the past few decades, the role of the extracellular matrix in this intercellular process is less known. Here, we review the current knowledge on how microenvironmental cues, biophysical and biochemical, dictate macrophage–endothelium crosstalk in the pathology of different diseases. We summarize studies using biomaterials as extracellular matrix with tenable properties to manipulate macrophage–EC fate to regulate innate and adaptive immunity, angiogenesis and regeneration. Finally, we discuss the potential and challenges of developing novel therapeutic strategies to tailor macrophage–EC niches to restore homeostasis in various diseases. © 2023, Springer Nature Limited
An Improved Method for Assessing the Biocidal Activity of Silver-Bearing Wound Dressings Against Staphylococcus Aureus
Full text linkThis investigation evaluated the biocidal efficacy of silver-bearing wound dressings against Staphylococcus aureus, a pervasive wound pathogen. Using the FDA-recognized AATCC-100 cell-viability assay, we found that the silver-bearing wound dressings may no longer be biocidal after 1 day of use, exhibiting only 1 log reduction after 7 days (p < 0.05). * As an alternative to this cell-viability assay, we discuss the advantages of qRT-PCR, including better precision, accuracy, reproducibility, and time-efficiency. We show that the standard deviation in log reduction is on average 13x higher for the plate-based than it is for the qRT-PCR method. While both qRT-PCR and the plate-based method show bacterial persistence after 1 day, the plate-based method may overestimate log reductions when compared to qRT-PCR. We present a standard curve for real time quantification of cell concentration in wound dressing extracts. We test rhodopsin (rho), gyrase (gyr), and universal bacteria 16s rRNA primers and find that 16s rRNA results in 100% reaction efficiency and an optimal quantification limit (~1 x 103 CFU/mL). We also discuss qRT-PCR’s potential for monitoring Staphylococcus aureus persistence and virulence, using assays targeting the associated biomarkers (Staphylococcus protein A, mecA, gamma-hemolysin, beta-toxin, leukotoxin-F). Our preliminary qRT-PCR assay results show that the day 1 wound dressing extracts are negative for the methicillin-resistance gene (mecA), in accordance with the genome sequence for Staphylococcus aureus ATTCC-3556. Using the delta-delta Ct method for these day 1 samples, the relative abundance of the housekeeping gene rhodopsin (rho) is 0.15. The relative abundance of the virulence factor gene Staphylococcus protein A (spA) is 0.0042 and 0.087 for day 1 and day 3 samples, respectively
Bioreactor Controls for High Oxygen Uptake Rate (OUR) Demand Processes
Full text linkIn the pharmaceutical industry, development of novel methodologies to increase the bioreactor capabilities is crucial to meet the ever-growing demand. With the introduction of high oxygen uptake rate (OUR) demand processes, production facilities may find current equipment and control settings inadequate to handle the processes safely and efficiently. Hence, the objective of the project was to develop a new set of control parameters and automatization that utilize OUR measurements to improve bioreactor performance with minimal capital investment and disruption in the plant workflow. To meet newly introduced high OUR demand, four control loops were chosen: antifoam, dissolved oxygen (DO), pH, and glucose.
Statistical tools were used for antifoam control to predict foam accumulation and dosage from OUR measurements. Arduino was used to handshake with the bioreactor for the delivery. For both DO and pH controls, the gain-scheduling method was applied with the Direct Synthesis-disturbance rejection method to determine controller tuning parameters. For glucose control, OUR measurement was used to calculate the glucose concentration and modified the controller output, acting as a feed forward controller.
The results demonstrated promising potentials for the proposed approaches. The antifoam controller successfully managed foam without any maintenance, albeit further characterization may be necessary for scale-up. The DO controller controlled DO without trial and error at different scales, while maintaining significant disturbance rejection; it eliminated the typical 10~15% decrease in DO during feed events. However, more in-depth stability analysis is necessary for implementation. The pH controller achieved tight control without the deadband, but it has to be tested at various scales. The handshake between the hardware and the software was established for the glucose controller. The controller output also functioned as predicted, but further stability analysis is recommended. These findings elucidate the capability of the OUR measurements and provide a solid theoretical and experimental basis for future application and characterization of bioreactors
Developing and Establishing a Platform for the Automation of Monoclonal Antibody Drug Analysis
Full text linkThe use of automation has been standardized for generations in many industries, ranging from car manufacturing to oil refinery, and continues to advance at a rapid pace. While the power of automation has been harnessed in many other industries, the biotechnology sector has lagged behind, largely due to the complexity of working with biological matter as well as the complexity and the ever-changing nature of the processes involved.1 In recent years, the automation trend has gained in popularity in the field of biotechnology and many companies are seeking to utilize the new instruments and technology available to increase efficiency as well as optimize many other characteristics of their processes.
Automation has the potential to significantly increase the overall productivity of a biopharmaceutical company by strategically implementing it where workflow traditionally slows due to bottlenecks, such as in analytical departments. Yet, in addition to improving efficiency, automation offers the unique ability of improving reproducibility and decreasing variation by transferring repetitive tasks to a robot. The elimination of human error and the ability to track all robotic movements well suits the ultimate goal of producing a high-quality product, which is of utmost importance in a good manufacturing practices (GMP) compliant environment.
Currently, the standard for analyzing developmental drug samples is either by manually performed assays or on instruments dedicated to a particular assay. Therefore, we developed a customizable and versatile automated platform to replace a range of existing manual assays that test varying aspects of a monoclonal antibody drug sample.
In this study, we used the Hamilton Microlab STAR robot to automate two relative drug potency assays and one residual Chinese Hamster Ovarian (CHO) host cell protein (HCP) assay. For all assays, all liquid handling steps are automated and performed by the robot. In this essay, we demonstrate that our automated assays can achieve equal or superior accuracy and precision compared to their manual counterparts, have a greater maximum efficiency, and track and record all robotic movements performed during the assay. Furthermore, our work establishes a precedent upon which future assays can be more easily translated into an automated environment
Fibronectin Network Influences Endothelial Cell Migration During Vascular Morphogenesis in a Breast Cancer Model
Full text linkAngiogenesis, the formation of new blood vessels from pre-existing vessels, is a major research topic in the biomedical field due to its role in regenerative medicine, tissue engineering, and cancer development, growth, and metastasis. This process is driven by the demand for oxygen and nutrients from the surrounding tissues, and is heavily regulated by the extracellular matrix (ECM). The ECM is a non-cellular, complex network made up of fibrous proteins, growth factors, and signaling molecules. Many researchers have studied the individual constituents of this protein rich matrix and the specific roles they play during angiogenesis hoping to find a potential target for cancer treatments.
Previous research conducted in our lab has established fibroblast and breast cancer cell-derived scaffolds as a platform to investigate tumor angiogenesis. This thesis focuses on the influence of the fibronectin network in the cell-derived scaffolds on endothelial cell (EC) migration. We used pUR4B, a fibronectin polymerization inhibitor, and III-11C, the control peptide that has no effect on fibronectin polymerization to manipulate matrix deposition. We also established a line of green fluorescent protein (GFP) tagged EC’s using lentiviral GFP. After seeding the GFP ECs on the de-cellularized scaffolds, we tracked the migration of the cells and quantified several migration parameters. Our results showed that the polymerized fibronectin restricts EC migration during angiogenesis. Its function to support the surrounding matrix components and adhesive properties are essential for endothelial cell migration. These findings shed new light on the role of polymerized fibronectin in endothelial cell migration during angiogenesis, and further prove its potential as a target for future cancer treatment
Optimization of Ex Vivo Rat Pulmonary Artery Contractility Measurement by Wire Myography
Full text linkPulmonary arterial hypertension (PAH) is an acutely lethal disease with 3-year survival rates ranging from 55 to 73%.1 Despite its detrimental effects and the inevitable eventual progression to right ventricular failure, the pathogenesis and biology of pulmonary arterial hypertension are not yet completely understood. The underlying causes of pulmonary arterial hypertension are a complex combination of endothelial cell dysfunction, platelet activation, vasoconstriction, loss of relaxing factors, hypertrophy, and inflammation. Currently, PAH treatments are generally aimed to correct the imbalance between vasoconstriction and vasodilation in the pulmonary arteries, and thus improve oxygenation, functional class, exercise capacity, and quality of life.2
Wire myography is an effective in-vitro technique to investigate the vasomotor functions of blood vessels ex-vivo. With multiple wire myograph units, the technique allows researchers to perform comparative analysis of not only vasomotor functions of pulmonary vessels isolated from different pathological states of pulmonary arterial hypertensions, but also their vasomotor functions of under the influence of various pharmaceutical agents.
Wire myograph experiment protocols have been well-established for different vessel types of the systemic circulation, from large compliance vessels such as the aorta to small resistance vessels such as the mesenteric artery. However, there is a lack of consensus on the experimental protocol for one of the most widely used models in wire myograph analysis, rat pulmonary arteries from animals subjected to experimental PAH.
In present study, we identified the initial tension of pulmonary arteries as a key element in determining the result of rat pulmonary artery wire myography experiments. By subjecting segments of rat pulmonary arteries to a wide range of initial tensions (100 – 3000 mg-Force) and performing wire myography with various pharmaceutical agents such as phenylephrine, acetylcholine and sodium nitroprusside, we determined that a moderate initial tension range of 1500 – 2000 mg-Force to be optimal for rat pulmonary artery wire myography experiments involving a variety of vasoconstrictors and vasodilators. Furthermore, we found the optimum equilibration time of rat pulmonary artery vessel segments in the presence of 2M KCl to range between 45 and 50 minutes
RECOMBINANT AAV6 GENE THERAPY VECTORS: PROCESS OPTIMIZATION AND SCALE UP INTO THE iCELLis NANO BIOREACTOR
No full textABSTRACT
Adeno-associated viral vector gene therapy is a treatment for genetic diseases that involves the introduction of a healthy copy of a mutated gene through the use of adeno-associated viral vectors. There has been a growing level of interest in gene therapy with as many as 2600 clinical trials reported as either completed, ongoing, or having approval as of 2018. With 56.8% of all trials in Phase I and 17.1% in Phase II, batch requirements that contain a good yield and are of high-purity are important. Industry has developed scalable processes for manufacture of large batches of highly-pure AAV product. These systems, while seemingly robust, have many challenges yet to overcome. One limitation to production are vector serotypes having lower yields compared to their counterparts. The AAV6 serotype, used to treat diseases such as cystic fibrosis and muscular dystrophy, have been observed to have a thirty-fold decrease in production compared to AAV9 serotype. To date, AAV6 production at Paragon has been confined to serum-free, suspension cell culture. By increasing the yield, we can affectively meet batch requirements for clinical trials while lowering the amount of resources and cost of goods. This study represents the first production of AAV6 at Paragon Bioservices using an adherent cell platform. Scale down studies were undertaken to increase production by optimizing transfection conditions such as the total DNA concentration during transfection, the transfection reagent-DNA ratio, the harvest days post transfection, seeding density, and plasmid ratio. The optimized conditions were tested in the iCELLis® Nano bioreactor and successful production of AAV6 was achieved at titers equivalent or better to those achieved in suspension culture
ENDOTHELIAL PROGENITOR CELL RECRUITMENT IN A WOUND HEALING MICROFLUIDIC VASCULAR MODEL
Full text linkDuring wound healing, endothelial progenitor cells (EPCs) are recruited from the bone marrow and directed to the site of injury. At the site of injury, hypoxic conditions promote TNF-α, which up regulates intercellular adhesion molecule-1 (ICAM-1). EPCs use ICAM-1 to attach to endothelial cells (ECs) lining blood vessels. Here, we design, develop and test a three-dimensional microbioreactor system (3-D MBR) with precise control and monitoring of oxygen and media flow rate. We first analyze the transport of oxygen in the proposed device. Following fabrication of the 3-D MBR, we next utilized a step-wise seeding technique, which resulted in confluency of human umbilical vein endothelial cells (HUVECs) on all four sides of the device. We next examine endothelial colony forming cell (ECFC) attachment and retention onto HUVECs using conventional 2-D cultures. HUVECs are pre-stimulated with one of four conditions: 21% oxygen (atmospheric), atmospheric with TNF-α-supplemented media, 1% oxygen (hypoxia), and lastly hypoxia with TNF-α-supplemented media. We show the highest attachment and retention of ECFCs on HUVECs pre-treated with TNF-α and 1% oxygen, which correlated with the highest expression levels of ICAM-1. Using the new 3-D MBR system we next demonstrate that TNF-α and hypoxia, when used in conjunction, significantly increase EPC attachment on ECs under pathologically relevant flow conditions. The 3-D MBR system allows us to mimic the oxygen and shear stress environment in the vasculature, thus providing a step between traditional in-vitro and in-vivo experimentation to model a variety of vascular-related disorders, especially wound healing
Anticoagulant drug attachment to small-diameter vascular graft
Full text linkCurrent tissue engineered vascular grafts (TEVGs) fail to demonstrate patency due to high rates of thrombus formation for small diameters in coronary artery bypass grafting (CABG). To address this problem, we developed grafts with conjugated anticoagulants. Initially, an anticoagulant surface coated graft was tested, but did not demonstrate consistent anticoagulant attachment to the graft. We progressed to a TEVG with an anticoagulant embedded throughout the graft. The anticoagulant fibrinogen synthesis process was optimized to ensure maximal anticoagulant conjugation as well as purification of the synthesized biomaterial. The initial anticoagulant-modified fibrin TEVGs proved to reduce platelet adhesion in both 0.6 mm and 5 mm inner diameter grafts compared to control grafts. A two-dimensional (2D) fibrin sheet was developed to accelerate testing of the synthesized anticoagulant-fibrinogen material. We then modified the platelet adhesion assay to be more rigorous with the addition of thrombin to the platelet rich plasma (PRP) to further activate the platelets. A concentration curve for platelet adhesion was created to determine the optimal combination of fibrinogen with anticoagulant modified fibrinogen for electrospinning scaffolds. The 2D sheets with 30% - 40% anticoagulant modified fibrinogen concentrations demonstrated significantly lower platelet adhesion. This established that the synthesized material may reduce TEVG thrombogenicity when used for future in vivo studies
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