scholarly journals In Vitro Nasal Tissue Model for the Validation of Nasopharyngeal and Mid-turbinate Swabs for SARS-CoV-2 Testing

2021 ◽  
Author(s):  
Devon R Hartigan ◽  
Miryam Adelfio ◽  
Molly E Shutt ◽  
Stephanie M Jones ◽  
Shreya Patel ◽  
...  
Keyword(s):  
Large Scale ◽  
Rapid Development ◽  
Respiratory Pathogens ◽  
Scale Production ◽  
Upper Airways ◽  
Tissue Model ◽  
Nasal Cavities ◽  
Nasal Tissue ◽  
Scale Population

Large-scale population testing is a key tool to mitigate the spread of respiratory pathogens, as in the current COVID-19 pandemic, where swabs are used to collect samples in the upper airways (e.g. nasopharyngeal and mid-turbinate nasal cavities) for diagnostics. However, the high volume of supplies required to achieve large-scale population testing has posed unprecedented challenges for swab manufacturing and distribution, resulting in a global shortage that has heavily impacted testing capacity world-wide and prompted the development of new swabs suitable for large-scale production. Newly designed swabs require rigorous pre-clinical and clinical validation studies that are costly and time consuming (i.e. months to years long); reducing the risks associated with swab validation is therefore paramount for their rapid deployment. To address these shortages, we developed a 3D-printed tissue model that mimics the nasopharyngeal and mid-turbinate nasal cavities, and we validated its use as a new tool to rapidly test swab performance. In addition to the nasal architecture, the tissue model mimics the soft nasal tissue with a silk-based sponge lining, and the physiological nasal fluid with asymptomatic and symptomatic viscosities of synthetic mucus. We performed several assays comparing standard flocked and injection-molded swabs. We quantified the swab pick-up and release, and determined the effect of viral load and mucus viscosity on swab efficacy by spiking the synthetic mucus with heat-inactivated SARS-CoV-2 virus. By molecular assays, we found that injected molded swabs performed similarly or superiorly in comparison to standard flocked swabs and we underscored a viscosity-dependent difference in cycle threshold values between the asymptomatic and symptomatic mucus for both swabs. To conclude, we developed an in vitro nasal tissue model, that corroborated previous swab performance data from clinical studies, with the potential of providing researchers with a clinically relevant, reproducible, safe, and cost-effective validation tool for the rapid development of newly designed swabs.

Nanofiber-Based Expansion and Differentiation Technology for High-Volume Ex Vivo Production of Reticulocytes for P. Vivax Malaria Research

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2357-2357
Author(s):  
Hong Wang ◽  
Adam M Sorkin ◽  
Ramasamy Sakthivel
Keyword(s):  
Cord Blood ◽  
Large Scale ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Rapid Development ◽  
High Volume ◽  
In Vitro Models ◽  
Scale Production ◽  
Hematopoietic Stem

Abstract Abstract 2357 Infection by Plasmodium Vivax (P. Vivax) is the most common cause of Sleeping Malaria. P. Vivax and other plasmodia have grown increasingly resistant to antimalarial drugs. Introduced by mosquito bite, P. vivax sporozoites enter circulation and preferentially penetrate reticulocytes by attaching to the Fya and Fyb Duffy antigen/chemokine receptor (DARC) via PvRBP-1 and PvRBP-2 proteins located at their apical poles. Once in a reticulocyte, the parasite begins to reproduce asexually, releasing of thousands of merozoites into circulation. At this point, merozoites can also enter the liver and triggering relapses months or years later. The emergence of drug-resistant strains of p. vivax has stimulated development of new vaccines and treatments, but progress has been slowed by the dearth of reliable screening platforms. Many vaccine candidates have been developed to act upon vivax merozoites by preventing binding of PvRBP-1 and 2 to DARC, thereby arresting reproduction. However, there is a distinct lack of in vitro models to evaluate candidates that employ this mechanism. We are addressing this issue with a novel ex vivo expansion and differentiation technology for large-scale production of DARC expressing reticulocytes for in vitro P. vivax infection studies. This technology comprises an expansion system that can produce high yields of hematopoietic precursors (CD133+/CD34+ cells) from a variety of sources (marrow, peripheral blood, and cord blood), and a differentiation system to produce a relatively pure population of enucleated erythrocytes. In this study, we have refined the polyethersulfone (PES) nanofiber-based culturing system containing growth factors and cytokines in a serum-free media, to expand hematopoietic stem and progenitor cells (HSPC) ex vivo. This expansion technology allows rapid 200-fold ex vivo proliferation within 7 days of umbilical cord blood derived CD133+/CD34+ HSPCs from a DARC+ donor. Following expansion, over 50% of these cells retained HSPC phenotype (expression of CD34+). We have subsequently demonstrated that feeder layer free three-step differentiation of nanofiber-expanded cells using cytokines results in a population containing predominately enucleated reticulocyte-like cells. At 21 days of differentiation, cells had expanded 50-fold. Around 41% of cells were enucleated reticulocytes. These cells expressed glycophorin-A, a major sialoglycoprotein present on the human erythrocyte membrane. ∼28% of cells were CD36+, and ∼70% were CD71+ indicating an erythroid lineage. These results suggest that this technology can produce a population of DARC+ reticulocytes that is ∼5,000-fold greater than the starting population of HSPCs. We are partnering with leading malaria vaccine researchers to demonstrate that these reticulocytes can be parasitized by p. vivax. We believe that this will provide a unique platform to jumpstart research of malaria parasites and enable rapid development of effective vaccines. Further development of this technology may also have significant implications for large-scale ex vivo production of erythrocytes for general use. Reticulocyte-like cells and expelled nuclei during differentiation of nanofiber-expanded HSPC. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Lentiviral Vectors for Delivery of Gene-Editing Systems Based on CRISPR/Cas: Current State and Perspectives

Viruses ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 1288
Author(s):  
Wendy Dong ◽  
Boris Kantor
Keyword(s):  
Large Scale ◽  
Lentiviral Vector ◽  
Clinical Applications ◽  
Scale Production ◽  
Base Editing ◽  
Epigenome Editing ◽  
Vector Systems ◽  
Dividing Cells

CRISPR/Cas technology has revolutionized the fields of the genome- and epigenome-editing by supplying unparalleled control over genomic sequences and expression. Lentiviral vector (LV) systems are one of the main delivery vehicles for the CRISPR/Cas systems due to (i) its ability to carry bulky and complex transgenes and (ii) sustain robust and long-term expression in a broad range of dividing and non-dividing cells in vitro and in vivo. It is thus reasonable that substantial effort has been allocated towards the development of the improved and optimized LV systems for effective and accurate gene-to-cell transfer of CRISPR/Cas tools. The main effort on that end has been put towards the improvement and optimization of the vector’s expression, development of integrase-deficient lentiviral vector (IDLV), aiming to minimize the risk of oncogenicity, toxicity, and pathogenicity, and enhancing manufacturing protocols for clinical applications required large-scale production. In this review, we will devote attention to (i) the basic biology of lentiviruses, and (ii) recent advances in the development of safer and more efficient CRISPR/Cas vector systems towards their use in preclinical and clinical applications. In addition, we will discuss in detail the recent progress in the repurposing of CRISPR/Cas systems related to base-editing and prime-editing applications.

Abstract 262: Derivation of Duchenne Muscular Dystrophy Disease Specific Cardiomyocytes From Patient Urine

2013 ◽  
Vol 113 (suppl_1) ◽  
Author(s):  
Xuan Guan ◽  
David L Mack ◽  
Claudia M Moreno ◽  
Fernando Santana ◽  
Charles E Murry ◽  
...  
Keyword(s):  
Stem Cells ◽  
Human Urine ◽  
Large Scale ◽  
Lentiviral Vector ◽  
Cellular Reprogramming ◽  
Urine Samples ◽  
Pcr Analysis ◽  
Scale Production ◽  
Mechanism Study

Introduction: Human somatic cells can be reprogrammed into primitive stem cells, termed induced pluripotent stem cells (iPSCs). These iPSCs can be extensively expanded in vitro and differentiated into multiple functional cell types, enabling faithful preservation of individual’s genotype and large scale production of disease targeted cellular components. These unique cellular reagents thus hold tremendous potential in disease mechanism study, drugs screening and cell replacement therapy. Due to the genetic mutation of the protein dystrophin, many DMD patients develop fatal cardiomyopathy with no effective treatment. The underlying pathogenesis has not been fully elucidated. Hypothesis: We tested the hypothesis that iPSCs could be generated from DMD patients’ urine samples and differentiated into cardiomyocytes, recapitulating the dystrophic phenotype. Methods: iPSCs generation was achieved by introducing a lentiviral vector expressing Oct4, Sox2, c-Myc and Klf4 into cells derived from patient’s (n=1) and healthy volunteers’ (n=3) urine. Cardiomyocytes were derived by sequentially treating iPSCs with GSK3 inhibitor CHIR99021 and Wnt inhibitor IWP4. Differentiated cardiomyocytes were subjected to calcium imaging, electrophysiology recording, Polymerase Chain Reaction (PCR) analysis, and immunostaining. Results: iPSCs were efficiently generated from human urine samples and further forced to differentiate into contracting cardiomyocytes. PCR analysis and immunostaining confirmed the expression of a panel of cardiac markers. Both normal and patient iPSC derived cardiomyocytes exhibited spontaneous and field stimulated calcium transients (up to 2Hz), as well as action potentials with ventricular-like and nodal-like characteristics. Anti-dystrophin antibodies stained normal iPSC-derived cardiomyocyte membranes but did not react against DMD iPSC-derived cardiomyocytes. Conclusions: Cardiomyocytes can be efficiently generated from human urine, through the cellular reprogramming technology. DMD cardiomyocytes retained the patient’s genetic information and manifested a dystrophin-null phenotype. Functional assessments are underway to determine differences that may exist between genotypes.

Optimizing in vitro large scale production of giant reed (Arundo donax L.) by liquid medium culture

2014 ◽  
Vol 69 ◽  
pp. 21-27 ◽  
Author(s):  
Valeria Cavallaro ◽  
Cristina Patanè ◽  
Salvatore L. Cosentino ◽  
Isabella Di Silvestro ◽  
Venera Copani
Keyword(s):  
Liquid Medium ◽  
Large Scale ◽  
Arundo Donax ◽  
Giant Reed ◽  
Scale Production ◽  
Large Scale Production ◽  
Medium Culture ◽  
Arundo Donax L

Scalable Microfluidic Platform for Flexible Configuration of and Experiments with Microtissue Multiorgan Models

2018 ◽  
Vol 24 (1) ◽  
pp. 79-95 ◽  
Author(s):  
Christian Lohasz ◽  
Nassim Rousset ◽  
Kasper Renggli ◽  
Andreas Hierlemann ◽  
Olivier Frey
Keyword(s):  
Large Scale ◽  
Plastic Material ◽  
Microtiter Plate ◽  
Scale Production ◽  
Experimental Conditions ◽  
Precise Control ◽  
Open Design ◽  
Gravity Driven ◽  
Gravity Driven Flow

Microphysiological systems hold the promise to increase the predictive and translational power of in vitro substance testing owing to their faithful recapitulation of human physiology. However, the implementation of academic developments in industrial settings remains challenging. We present an injection-molded microfluidic microtissue (MT) culture chip that features two channels with 10 MT compartments each and that was designed in compliance with microtiter plate standard formats. Polystyrene as a chip material enables reliable, large-scale production and precise control over experimental conditions due to low adsorption or absorption of small, hydrophobic molecules at or into the plastic material in comparison with predecessor chips made of polydimethylsiloxane. The chip is operated by tilting, which actuates gravity-driven flow between reservoirs at both ends of every channel, so that the system does not require external tubing or pumps. The flow rate can be modulated by adjusting the tilting angle on demand. The top-open design of the MT compartment enables efficient MT loading using standard or advanced pipetting equipment, ensures oxygen availability in the chip, and allows for high-resolution imaging. Every channel can be loaded with up to 10 identical or different MTs, as demonstrated by culturing liver and tumor MTs in the same medium channel on the chip.

Large-scale production of polyoma middle T antigen by using genetically engineered tumors

1985 ◽  
Vol 5 (7) ◽  
pp. 1795-1799
Author(s):  
D R Kaplan ◽  
B Bockus ◽  
T M Roberts ◽  
J Bolen ◽  
M Israel ◽  
...  
Keyword(s):  
Nude Mice ◽  
Large Scale ◽  
T Antigen ◽  
Genetically Engineered ◽  
Scale Production ◽  
Large Scale Production ◽  
Metallothionein Promoter ◽  
Polyoma Middle T Antigen ◽  
Nih 3T3

A recombinant plasmid containing a metallothionein promoter-polyoma middle T cDNA fusion was constructed and used to transfect NIH 3T3 cells. Transformed cells expressing middle T were injected into nude mice. Within 3 weeks, each mouse produced tumors containing middle T equivalent to that in 250 to 1,000 100-mm dishes of polyomavirus-infected cells. This middle T, partially purified by immunoaffinity chromatography, retained activity as measured by its ability to be phosphorylated in vitro. The combined approach of fusing strong promoters to genes of interest and utilizing nude mice to grow large quantities of cells expressing the gene provides a quick, inexpensive alternative to other expression systems.

scholarly journals Production of Phytotoxic Metabolite Using Biphasic Fermentation System from Strain C1136 of Lasiodiplodia pseudotheobromae, a Potential Bioherbicidal Agent

2017 ◽  
Vol 9 (3) ◽  
pp. 371-377
Author(s):  
Charles Oluwaseun ADETUNJI ◽  
Julius Kola OLOKE ◽  
Gandham PRASAD ◽  
Moses ABALAKA ◽  
Emenike Onyebum IROKANULO
Keyword(s):  
Large Scale ◽  
Wild Strain ◽  
Fermentation Medium ◽  
Scale Production ◽  
Conventional Farming ◽  
Large Scale Production ◽  
Phytotoxic Metabolite ◽  
Biphasic Fermentation

Formulation of effective and environmental friendly bioherbicides depends on the type of fermentation medium used for the production of phytotoxic metabolites. The effect of biomass, colony forming unit and the phytotoxic metabolite produced from the biphasic fermentation was carried out, while the phytotoxic metabolite was  tested in vivo and in-vitro on Echinochola crus-galli and dicotyledonous Chromolaena odorata. The mutant strain of Lasiodiplodia pseudotheobromae C1136 (Lp90) produced the highest amount of conidia and the largest necrotic area on the two tested weeds when compared to its wild strain in the different biphasic media combinations. The study revealed that the biphasic system containing PDB + rice produced the highest bioherbicidal activities. Therefore, the phytotoxic metabolites from strain C1136 are suggested for large scale production of bioherbicides for the management of weeds in conventional farming to improve yield and enhance food security.

scholarly journals Expansion of Human Pluripotent Stem Cell-derived Early Cardiovascular Progenitor Cells by a Cocktail of Signaling Factors

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Sadaf Vahdat ◽  
Sara Pahlavan ◽  
Elena Mahmoudi ◽  
Maryam Barekat ◽  
Hassan Ansari ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Large Scale ◽  
Culture Medium ◽  
Gene Expression Pattern ◽  
Extended Period ◽  
Scale Production ◽  
Chemical Screening ◽  
Wide Range ◽  
Cell Sources

Abstract Cardiovascular progenitor cells (CPCs) derived from human pluripotent stem cells (hPSCs) are proposed to be invaluable cell sources for experimental and clinical studies. This wide range of applications necessitates large-scale production of CPCs in an in vitro culture system, which enables both expansion and maintenance of these cells. In this study, we aimed to develop a defined and efficient culture medium that uses signaling factors for large-scale expansion of early CPCs, called cardiogenic mesodermal cells (CMCs), which were derived from hPSCs. Chemical screening resulted in a medium that contained a reproducible combination of three factors (A83-01, bFGF, and CHIR99021) that generated 1014 CMCs after 10 passages without the propensity for tumorigenicity. Expanded CMCs retained their gene expression pattern, chromosomal stability, and differentiation tendency through several passages and showed both the safety and possible cardio-protective potentials when transplanted into the infarcted rat myocardium. These CMCs were efficiently cryopreserved for an extended period of time. This culture medium could be used for both adherent and suspension culture conditions, for which the latter is required for large-scale CMC production. Taken together, hPSC-derived CMCs exhibited self-renewal capacity in our simple, reproducible, and defined medium. These cells might ultimately be potential, promising cell sources for cardiovascular studies.

scholarly journals Regeneration Technique of Bamboo Species through Nodal Segments: A Review

2020 ◽  
Vol 8 (1) ◽  
pp. 54-68
Author(s):  
Meena Maiya Suwal ◽  
Janardan Lamichhane ◽  
Dhurva Prasad Gauchan
Keyword(s):  
Large Scale ◽  
Physiological Stress ◽  
Perennial Grass ◽  
Shoot Multiplication ◽  
Nodal Segments ◽  
Individual Species ◽  
Small Scale ◽  
Scale Production ◽  
Bamboo Species

Micropropagation is an alternative technique to propagate at large scale plants to meet global plant demand. Various researchers have worked on the micropropagation technique to regenerate bamboo species by using nodal segments from years. Contamination, browning, necrosis, and acclimatization with physiological stress are the extreme problems of the micropropagation technique. But, many numbers of papers have been published on micropropagation of the bamboo species through nodal segments as explants. The proliferation of the bamboo shoots is dependent on the season of collection, size of explants, the position of explants, diversity of plants, concentration and combination of plant growth regulators, most adequate culture medium, environmental condition of the equipment, handling, and individual species. Bamboo is a monocarpic fast-growing, tall perennial grass and having the high potential to generate economic and social benefits. It helps to maintain land patterns and control soil erosion.  The long life cycle of the bamboo produces a huge amount of seeds but unfortunately, mostly, they are non-viable. So, bamboos are propagated from vegetative by cutting and air layering. However, these methods are only for a small scale and they also tend to destroy large mother plant stocks and difficult to be transported. So, the in vitro propagation technique is useful to obtain large progenies from desired genotypes. Mostly, BAP and TDZ growth hormones are widely used for shoot multiplication and IBA, NAA and IAA are used for root initiation as per developed protocols in tissue culture for large scale production. This review intends to explore an overview of the recent literature reports to summarize the importance of micropropagation by using nodal segments of bamboo species and factors influencing it.

Sign in / Sign up

Export Citation Format

Share Document