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Methods in Molecular Biology (Clifton,... 2018Large-scale transient transfection of mammalian cell suspension cultures enables the production of biological products in sufficient quantity and under stringent quality...
Large-scale transient transfection of mammalian cell suspension cultures enables the production of biological products in sufficient quantity and under stringent quality attributes to perform accelerated in vitro evaluations and has the potential to support preclinical or even clinical studies. Here we describe the methodology to produce VLPs in a 3L bioreactor, using suspension HEK 293 cells and PEIPro as a transfection reagent. Cells are grown in the bioreactor to 1 × 10 cells/mL and transfected with a plasmid DNA-PEI complex at a ratio of 1:2. Dissolved oxygen and pH are controlled and are online monitored during the production phase and cell growth and viability can be measured off line taking samples from the bioreactor. If the product is labeled with a fluorescent marker, transfection efficiency can be also assessed using flow cytometry analysis. Typically, the production phase lasts between 48 and 96 h until the product is harvested.
Topics: Animals; Biological Products; Bioreactors; Cell Culture Techniques; Cell Line; Cell Proliferation; Cell Survival; HEK293 Cells; Humans; Hydrogen-Ion Concentration; Mammals; Oxygen; Plasmids; Suspensions; Transfection
PubMed: 28921433
DOI: 10.1007/978-1-4939-7312-5_10 -
Chemosphere Apr 2015The preparation of a stable nanoparticle stock suspension is the first step in nanotoxicological studies, but how different preparation methods influence the...
Interactions between suspension characteristics and physicochemical properties of silver and copper oxide nanoparticles: a case study for optimizing nanoparticle stock suspensions using a central composite design.
The preparation of a stable nanoparticle stock suspension is the first step in nanotoxicological studies, but how different preparation methods influence the physicochemical properties of nanoparticles in a solution, even in Milli-Q water, is often under-appreciated. In this study, a systematic approach using a central composite design (CCD) was employed to investigate the effects of sonication time and suspension concentration on the physicochemical properties (i.e. hydrodynamic diameter, zeta potential and ion dissolution) of silver (Ag) and copper oxide (CuO) nanoparticles (NPs) and to identify optimal conditions for suspension preparation in Milli-Q water; defined as giving the smallest particle sizes, highest suspension stability and lowest ion dissolution. Indeed, all the physicochemical properties of AgNPs and CuONPs varied dramatically depending on how the stock suspensions were prepared and differed profoundly between nanoparticle types, indicating the importance of suspension preparation. Moreover, the physicochemical properties of AgNPs and CuONPs, at least in simple media (Milli-Q water), behaved in predictable ways as a function of sonication time and suspension concentration, confirming the validity of our models. Overall, the approach allows systematic assessment of the influence of various factors on key properties of nanoparticle suspensions, which will facilitate optimization of the preparation of nanoparticle stock suspensions and improve the reproducibility of nanotoxicological results. We recommend that further attention be given to details of stock suspension preparation before conducting nanotoxicological studies as these can have an important influence on the behavior and subsequent toxicity of nanoparticles.
Topics: Copper; Hydrodynamics; Ions; Metal Nanoparticles; Silver; Sonication; Suspensions; Toxicity Tests; Water Pollutants, Chemical
PubMed: 25550107
DOI: 10.1016/j.chemosphere.2014.12.005 -
Journal of Oncology Pharmacy Practice :... Mar 2022Imatinib is a protein-tyrosine kinase inhibitor which is currently only commercially available as a tablet dosage form in the strength of 100mg and 400mg. The...
BACKGROUND
Imatinib is a protein-tyrosine kinase inhibitor which is currently only commercially available as a tablet dosage form in the strength of 100mg and 400mg. The elaboration of new oral liquid formulations is suitable in pediatrics and for patients who have difficulties to swallow, notably in the absence of commercial forms. This enables the adaptation of dosage and secure the administration.
OBJECTIVES
The formulation of an oral pediatric solution of imatinib at a concentration of 30 mg/mL and the evaluation of its stability for the treatment of pediatric patients with Philadelphia chromosome positive chronic myeloid leukemia.
METHODS
The physicochemical stability parameters: appearance, pH, osmolality, and drug content of formulation were evaluated for 30 days when stored at 2-8°C. Concentration of solution was measured with a validated method using high performance liquid chromatography (HPLC) coupled with an absorbance UV detector. Equally, microbiological stability was performed.
RESULTS
The remaining imatinib concentration was at least 95% of the initial concentration after 30 days stored in fridge temperature. No changes were observed regarding the physical properties of the formulation during the study period.
CONCLUSIONS
The stability study showed that the imatinib oral solution at a concentration of 30 mg/mL provides an alternative option at the commercial tablet dosage forms for pediatric patients and patients who have difficulties to swallow.
Topics: Administration, Oral; Child; Drug Stability; Humans; Imatinib Mesylate; Pediatrics; Suspensions; Tablets
PubMed: 33522386
DOI: 10.1177/1078155221991200 -
International Journal of Pharmaceutics Jan 2023It has long been accepted that suspension pressurized metered-dose inhalers (pMDIs) must be shaken if a correct dose is to be delivered, if not, it will usually be...
It has long been accepted that suspension pressurized metered-dose inhalers (pMDIs) must be shaken if a correct dose is to be delivered, if not, it will usually be higher than the label claim. The purpose of this work was to investigate the influence of the device being unshaken, shaken and after a period of delay in pMDI actuation on the Fine Particle Mass (<5 µm), Extra Fine Particle Mass (<2 µm) and MMAD. Solution and suspension commercial pMDIs containing one, two or three components were used in the study. Most of the suspension pMDIs produced variable amounts of respirable size drug following the shake-fire delays tested in terms of the label claim dose. The effect was even more critical if the inhaler was not shaken and the FPM was found to be between -82 % for Symbicort and 363 % for Ventolin compared with the control values. In the case of MMAD measurements, Seretide and Serzyl inhalers showed the largest change from around 3 µm to 4.2-5.1 µm when not shaken. Conversely, the FPM and MMAD for the solution aerosols remained unchanged whether or not they were shaken or when a progressive increase in the delay in actuation after shaking was employed.
Topics: Bronchodilator Agents; Administration, Inhalation; Metered Dose Inhalers; Aerosols; Albuterol; Suspensions; Particle Size; Equipment Design
PubMed: 36535456
DOI: 10.1016/j.ijpharm.2022.122478 -
Journal of Visualized Experiments : JoVE Dec 2023Tear fluid is one of the easily accessible biofluids that can be collected non-invasively. Tear proteomics has the potential to discover biomarkers for several ocular...
Tear fluid is one of the easily accessible biofluids that can be collected non-invasively. Tear proteomics has the potential to discover biomarkers for several ocular diseases and conditions. The suspension trapping column has been reported to be an efficient and user-friendly sample preparation workflow for the broad application of downstream proteomic analysis. Yet, this strategy has not been well-studied in the analysis of human tear proteome. The present protocol describes an integrated workflow from clinical human tear samples to purified peptides for non-invasive tear protein biomarker research using mass spectrometry, which provides insights into disease biomarkers and monitoring when combined with bioinformatics analysis. A protein suspension trapping sample preparation was applied and demonstrated the discovery of tear proteome with fast, reproducible, and user-friendly procedures, as a universal, optimized sample preparation for human tear fluid analysis. In particular, the suspension trapping procedure outperformed in-solution sample preparation in terms of peptide recovery, protein identification, and shorter sample preparation time.
Topics: Humans; Proteome; Proteomics; Tandem Mass Spectrometry; Lacerations; Chromatography, Liquid; Suspensions; Biomarkers
PubMed: 38108381
DOI: 10.3791/64617 -
Nature Protocols Dec 2020sBLISS (in-suspension breaks labeling in situ and sequencing) is a versatile and widely applicable method for identification of endogenous and induced DNA double-strand...
sBLISS (in-suspension breaks labeling in situ and sequencing) is a versatile and widely applicable method for identification of endogenous and induced DNA double-strand breaks (DSBs) in any cell type that can be brought into suspension. sBLISS provides genome-wide profiles of the most consequential DNA lesion implicated in a variety of pathological, but also physiological, processes. In sBLISS, after in situ labeling, DSB ends are linearly amplified, followed by next-generation sequencing and DSB landscape analysis. Here, we present a step-by-step experimental protocol for sBLISS, as well as a basic computational analysis. The main advantages of sBLISS are (i) the suspension setup, which renders the protocol user-friendly and easily scalable; (ii) the possibility of adapting it to a high-throughput or single-cell workflow; and (iii) its flexibility and its applicability to virtually every cell type, including patient-derived cells, organoids, and isolated nuclei. The wet-lab protocol can be completed in 1.5 weeks and is suitable for researchers with intermediate expertise in molecular biology and genomics. For the computational analyses, basic-to-intermediate bioinformatics expertise is required.
Topics: Base Sequence; Cell Line; DNA Breaks, Double-Stranded; Genomics; Suspensions
PubMed: 33139954
DOI: 10.1038/s41596-020-0397-2 -
Aerospace Medicine and Human Performance Mar 2023The purpose of this study was to report the temporary loss of medical license and pilot incapacitations in the United Arab Emirates from 2018-2021. The General Civil...
The purpose of this study was to report the temporary loss of medical license and pilot incapacitations in the United Arab Emirates from 2018-2021. The General Civil Aviation Authority database was searched for all reported temporary suspensions of license between 2018-2021 and the ICD-10 codes were extracted. A total of 1233 incapacitations was reported with a mean license suspension of 148.4 ± 276.8 d. The mean days of suspension for the various medical specialties were 115.2 ± 188.4 for musculoskeletal conditions ( = 392), 189.3 ± 324.8 for medicine ( = 335), 101.6 ± 231.4 for surgery, 109.4 ± 223.5 for urology ( = 93), 90.3 ± 128.7 for ophthalmology ( = 68), 385.6 ± 594.3 for psychiatry ( = 61), 150.4+285.9 for ENT ( = 59), 419.4 ± 382.6 for obstetrics and gynecology ( = 30), and 44.9+39 for dermatology ( = 21). Permanent suspensions were as follows: total = 100 (8.1%), musculoskeletal = 13 (3.3%), medicine = 37 (11%), surgery = 10 (5.7%), urology = 10 (10.7%), ophthalmology = 2 (2.9%), psychiatry = 20 (32.8%), ENT = 1 (1.7%), obstetrics and gynecology = 4 (13.1%), and dermatology = 3 (14.3%). Musculoskeletal conditions are the most common reason for temporary loss of medical license followed by medical and surgical conditions. The least common reason was dermatological conditions. The longest period of incapacitation was associated with psychiatric conditions followed by medical and ENT conditions. The annual calculated temporary incapacitation rate was 2.8% and the permanent suspension rate was 0.25%.
Topics: Female; Pregnancy; Humans; Pilots; Aerospace Medicine; Suspensions; Aviation; Musculoskeletal Diseases
PubMed: 36829285
DOI: 10.3357/AMHP.6071.2023 -
European Journal of Pharmaceutics and... Jul 2022Limited research has been performed on the downstream processing of nano-co-crystal suspensions into solid oral dosage forms. The objectives of this study were to...
Limited research has been performed on the downstream processing of nano-co-crystal suspensions into solid oral dosage forms. The objectives of this study were to evaluate the impact of three downstream processes (wet granulation, spray granulation and bead layering) on the performance of itraconazole-succinic acid (ITZ-SUC) nano-co-crystal suspension. An optimized ITZ-SUC nano-co-crystal suspension mixed with HPMC E5 was utilized for the downstream processing. The suspension was converted in the solid state either by wet or spray granulation (with microcrystalline cellulose or lactose as substrates) or by layering onto microcrystalline cellulose and sugar beads. The multiparticulate solid dosage forms were characterized by optical microscopy, differential scanning calorimeter (DSC), X-ray powder diffraction (XRPD) and in situ dissolution studies. Spray granulation and bead layering resulted in less particle aggregation, a faster dissolution rate, and higher kinetic solubility when compared to wet granulation. ITZ-SUC nano-co-crystals spray granulated with lactose resulted in higher kinetic solubility profiles compared to microcrystalline cellulose granules. The type of bead core had no impact on the dissolution behavior. A slower dissolution and decreased kinetic solubility were observed with increasing drug loading for sprayed granules when microcrystalline cellulose was used as substrate. All dosage forms were stable under accelerated storage conditions (40 °C/75% RH) when blistered. Nano-co-crystals incorporated in granules were less stable than layered beads under non-blistered condition. Nano-co-crystals layered sugar beads are an interesting alternative to amorphous solid dispersion; a comparable kinetic solubility but a faster drug release were achieved. This study identified bead layering as a superior downstream process approach for incorporating ITZ-SUC nano-co-crystals into an oral solid dosage form without compromising drug dissolution.
Topics: Drug Compounding; Itraconazole; Lactose; Nanoparticles; Particle Size; Solubility; Suspensions
PubMed: 35598769
DOI: 10.1016/j.ejpb.2022.05.011 -
Electrophoresis Mar 2018This paper aims to summarize the series of investigations on coupling suspension yield stress and DLVO (Derjaguin-Landau-Verwey-Overbeek) forces, i.e. van der Waals and... (Review)
Review
This paper aims to summarize the series of investigations on coupling suspension yield stress and DLVO (Derjaguin-Landau-Verwey-Overbeek) forces, i.e. van der Waals and electrical double layer forces. This summary provides a better understanding of the basic phenomena associated, historical development and current status of this useful coupling, and also discusses the applicability and limitations/variations of such coupling applied to different types of concentrated aqueous particle suspensions. Aqueous suspensions discussed are composed of charged inorganic fine particles, including metal oxide colloidal particles, mineral fine particles, and clays. The research gaps are identified and specific future perspectives are discussed to further enhance the use of this unique and useful coupling, and to aim for the transition from the modelling of similar particle suspension systems to its dissimilar/mix particle suspension systems that fit more with the current and future industry needs in particle processing.
Topics: Aluminum Silicates; Clay; Colloids; Hydrogen-Ion Concentration; Ions; Metals; Minerals; Models, Theoretical; Oxides; Particle Size; Physical Phenomena; Surface Properties; Suspensions; Water
PubMed: 29330873
DOI: 10.1002/elps.201700314 -
Journal of Visualized Experiments : JoVE Aug 2023In basic experimental acupuncture research, rats are commonly used as laboratory animals. However, it is difficult for them to maintain a fixed posture. During...
In basic experimental acupuncture research, rats are commonly used as laboratory animals. However, it is difficult for them to maintain a fixed posture. During electroacupuncture procedures, proper immobilization of rats is essential. Various methods of rat fixation are currently used, including anesthesia fixation, high-platform fixation, binding fixation, and fixation with a self-made rat coat. However, these methods have their limitations, which may affect the efficiency and operability of the experiment to some extent. This protocol introduces a method of suspending and fixing rats using rat clothes. Firstly, rats are clothed with rat jackets that match their body shape, taking advantage of their preference for darkness and burrowing. The needling operation can then be carried out after the rats have worn rat clothes. When suspended, the rats are relatively still, as their limbs cannot move. This fixation method offers not only economical and user-friendly benefits but also ensures a stable and reliable fixation of the rats in a comfortably relaxed position. It also effectively minimizes time consumption, experimental space, and manpower resources. Additionally, this method allows for the exposure of most acupoints used for acupuncture in rats. This article primarily concentrates on the device's composition, encompassing a specially designed rat jacket, an elevated fixation rack, and their connecting structures. Additionally, an illustrative example will be presented to demonstrate the application of the rat clothing-based suspension fixation method in rat electroacupuncture procedures.
Topics: Animals; Rats; Acupuncture Therapy; Anesthesia; Acupuncture Points; Extremities; Histological Techniques; Suspensions
PubMed: 37607089
DOI: 10.3791/65652