A modular microfluidic system based on a multilayered configuration to generate large-scale perfusable microvascular networks

AbstractThe vascular network of the circulatory system plays a vital role in maintaining homeostasis in the human body. In this paper, a novel modular microfluidic system with a vertical two-layered configuration is developed to generate large-scale perfused microvascular networks in vitro. The two-layer polydimethylsiloxane (PDMS) configuration allows the tissue chambers and medium channels not only to be designed and fabricated independently but also to be aligned and bonded accordingly. This method can produce a modular microfluidic system that has high flexibility and scalability to design an integrated platform with multiple perfused vascularized tissues with high densities. The medium channel was designed with a rhombic shape and fabricated to be semiclosed to form a capillary burst valve in the vertical direction, serving as the interface between the medium channels and tissue chambers. Angiogenesis and anastomosis at the vertical interface were successfully achieved by using different combinations of tissue chambers and medium channels. Various large-scale microvascular networks were generated and quantified in terms of vessel length and density. Minimal leakage of the perfused 70-kDa FITC-dextran confirmed the lumenization of the microvascular networks and the formation of tight vertical interconnections between the microvascular networks and medium channels in different structural layers. This platform enables the culturing of interconnected, large-scale perfused vascularized tissue networks with high density and scalability for a wide range of multiorgan-on-a-chip applications, including basic biological studies and drug screening.

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Adaptation of a microfluidic qPCR system for enzyme kinetics studies

10.1101/2020.09.18.303248 ◽

2020 ◽

Author(s):

Elzbieta Rembeza ◽

Martin KM Engqvist

Keyword(s):

Enzyme Activity ◽

Enzyme Kinetics ◽

Large Scale ◽

Microfluidic System ◽

Kinetic Properties ◽

Kinetics Studies ◽

Biological Studies ◽

Wide Range ◽

Hands On ◽

Drastic Increase

AbstractMicrofluidic platforms offer a drastic increase in throughput while minimizing sample usage and hands-on time, which makes them important tools for large-scale biological studies. A range of such systems have been developed for enzyme activity studies, although their complexity largely hinders their application by a wider scientific community. Here we present adaptation of an easy-to-use commercial microfluidic qPCR system for performing enzyme kinetics studies. We demonstrate functionality of the Fluidigm Biomark HD system (the Fluidigm system) by determining kinetic properties of three oxidases in a resorufin-based fluorescent assay. The results obtained in the microfluidic system proved reproducible and comparable to the ones obtained in a standard microplate-based assay. With a wide range of easy-to-use, off-the-shelf components, the microfluidic system presents itself as a simple and customizable platform for high-throughput enzyme activity studies.Graphical abstract

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The cardiac nanoenvironment: form and function at the nanoscale

Biophysical Reviews ◽

10.1007/s12551-021-00834-5 ◽

2021 ◽

Author(s):

Jashan P. Singh ◽

Jennifer L. Young

Keyword(s):

Large Scale ◽

New Materials ◽

Length Scales ◽

Form And Function ◽

Molecular Features ◽

Wide Range ◽

Physiological Homeostasis ◽

Organ Level ◽

And Function

AbstractMechanical forces in the cardiovascular system occur over a wide range of length scales. At the whole organ level, large scale forces drive the beating heart as a synergistic unit. On the microscale, individual cells and their surrounding extracellular matrix (ECM) exhibit dynamic reciprocity, with mechanical feedback moving bidirectionally. Finally, in the nanometer regime, molecular features of cells and the ECM show remarkable sensitivity to mechanical cues. While small, these nanoscale properties are in many cases directly responsible for the mechanosensitive signaling processes that elicit cellular outcomes. Given the inherent challenges in observing, quantifying, and reconstituting this nanoscale environment, it is not surprising that this landscape has been understudied compared to larger length scales. Here, we aim to shine light upon the cardiac nanoenvironment, which plays a crucial role in maintaining physiological homeostasis while also underlying pathological processes. Thus, we will highlight strategies aimed at (1) elucidating the nanoscale components of the cardiac matrix, and (2) designing new materials and biosystems capable of mimicking these features in vitro.

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

Scientific Reports ◽

10.1038/s41598-019-52516-8 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 3

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.

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Ecological Structuring of Temperate Bacteriophages in the Inflammatory Bowel Disease-Affected Gut

Microorganisms ◽

10.3390/microorganisms8111663 ◽

2020 ◽

Vol 8 (11) ◽

pp. 1663

Author(s):

Hiroki Nishiyama ◽

Hisashi Endo ◽

Romain Blanc-Mathieu ◽

Hiroyuki Ogata

Keyword(s):

Inflammatory Bowel Disease ◽

Bowel Disease ◽

Large Scale ◽

Sequence Data ◽

Vital Role ◽

Temperate Bacteriophage ◽

Human Gut ◽

Potential Association ◽

Wide Range ◽

Inflammatory Bowel

The aim of this study was to elucidate the ecological structure of the human gut temperate bacteriophage community and its role in inflammatory bowel disease (IBD). Temperate bacteriophages make up a large proportion of the human gut microbiota and are likely to play a role in IBD pathogenesis. However, many of these bacteriophages await characterization in reference databases. Therefore, we conducted a large-scale reconstruction of temperate bacteriophage and bacterial genomes from the whole-metagenome sequence data generated by the IBD Multi’omics Database project. By associating phages with their hosts via genome comparisons, we found that temperate bacteriophages infect a phylogenetically wide range of bacteria. The majority of variance in bacteriophage community composition was explained by variation among individuals, but differences in the abundance of temperate bacteriophages were identified between IBD and non-IBD patients. Of note, in active ulcerative colitis patients, temperate bacteriophages infecting Bacteroides uniformis and Bacteroides thetaiotaomicron—two species experimentally proven to be beneficial to gut homeostasis—were over-represented, whereas their hosts were under-represented in comparison with non-IBD patients. Supporting the mounting evidence that gut viral community plays a vital role in IBD, our results show potential association between temperate bacteriophages and IBD pathogenesis.

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Long-term monitoring in a microfluidic system to study tumour spheroid response to chronic and cycling hypoxia

Scientific Reports ◽

10.1038/s41598-019-54001-8 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 2

Author(s):

Samantha M. Grist ◽

S. Soroush Nasseri ◽

Loïc Laplatine ◽

Jonathan C. Schmok ◽

Dickson Yao ◽

...

Keyword(s):

Cancer Progression ◽

3D Culture ◽

Microfluidic System ◽

Cancer Diagnostics ◽

Hypoxic Exposure ◽

Oxygen Control ◽

Tumour Spheroid ◽

Wide Range

AbstractWe demonstrate the application of a microfluidic platform combining spatiotemporal oxygen control and long-term microscopy monitoring to observe tumour spheroid response to hypoxia. The platform is capable of recreating physiologically-relevant low and cycling oxygen levels not attainable in traditional cell culture environments, while image-based monitoring visualizes cell response to these physiologically-relevant conditions. Monitoring spheroid cultures during hypoxic exposure allows us to observe, for the first time, that spheroids swell and shrink in response to time-varying oxygen profiles switching between 0% and 10% O2; this swelling-shrinkage behaviour appears to be driven by swelling of individual cells within the spheroids. We also apply the system to monitoring tumour models during anticancer treatment under varying oxygen conditions. We observe higher uptake of the anticancer agent doxorubicin under a cycling hypoxia profile than under either chronic hypoxia or in vitro normoxia, and the two-photon microscopy monitoring facilitated by our system also allows us to observe heterogeneity in doxorubicin uptake within spheroids at the single-cell level. Combining optical sectioning microscopy with precise spatiotemporal oxygen control and 3D culture opens the door for a wide range of future studies on microenvironmental mechanisms driving cancer progression and resistance to anticancer therapy. These types of studies could facilitate future improvements in cancer diagnostics and treatment.

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Cyclodextrin–Drug Inclusion Complexes: In Vivo and In Vitro Approaches

International Journal of Molecular Sciences ◽

10.3390/ijms20030642 ◽

2019 ◽

Vol 20 (3) ◽

pp. 642 ◽

Cited By ~ 50

Author(s):

Simone Carneiro ◽

Fernanda Costa Duarte ◽

Luana Heimfarth ◽

Jullyana Siqueira Quintans ◽

Lucindo Quintans-Júnior ◽

...

Keyword(s):

Inclusion Complexes ◽

Biological Properties ◽

Pharmaceutical Products ◽

Vital Role ◽

Performance Tests ◽

Biological Studies ◽

Therapeutic Properties ◽

Biological Performance

This review aims to provide a critical review of the biological performance of natural and synthetic substances complexed with cyclodextrins, highlighting: (i) inclusion complexes with cyclodextrins and their biological studies in vitro and in vivo; (ii) Evaluation and comparison of the bioactive efficacy of complexed and non-complexed substances; (iii) Chemical and biological performance tests of inclusion complexes, aimed at the development of new pharmaceutical products. Based on the evidence presented in the review, it is clear that cyclodextrins play a vital role in the development of inclusion complexes which promote improvements in the chemical and biological properties of the complexed active principles, as well as providing improved solubility and aqueous stability. Although the literature shows the importance of their ability to help produce innovative biotechnological substances, we still need more studies to develop and expand their therapeutic properties. It is, therefore, very important to gather together evidence of the effectiveness of inclusion complexes with cyclodextrins in order to facilitate a better understanding of research on this topic and encourage further studies.

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Significant Interference with Porcine Epidemic Diarrhea Virus Pandemic and Classical Strain Replication in Small-Intestine Epithelial Cells Using an shRNA Expression Vector

Vaccines ◽

10.3390/vaccines7040173 ◽

2019 ◽

Vol 7 (4) ◽

pp. 173

Author(s):

Da Shi ◽

Xiaobo Wang ◽

Hongyan Shi ◽

Jiyu Zhang ◽

Yuru Han ◽

...

Keyword(s):

Epithelial Cells ◽

Porcine Epidemic Diarrhea Virus ◽

Large Scale ◽

N Gene ◽

Shrna Expression ◽

Diarrhea Virus ◽

Shrna Expression Vector ◽

Wide Range ◽

Porcine Epidemic Diarrhea

Porcine epidemic diarrhea (PED) re-emerged in China in 2010 and is now widespread. Evidence indicates that highly virulent porcine epidemic diarrhea virus (PEDV) strains belonging to genotype G2 caused a large-scale outbreak of diarrhea. Currently, vaccines derived from PEDV classical strains do not effectively prevent infection by virulent PEDV strains, and no specific drug is available to treat the disease. RNA interference (RNAi) is a novel and effective way to cure a wide range of viruses. We constructed three short hairpin RNA (shRNA)-expressing plasmids (shR-N307, shR-N463, and shR-N1071) directed against nucleocapsid (N) and determined their antiviral activities in intestine epithelial cells infected with a classical CV777 strain and LNCT2. We verified that shR-N307, shR-N463, and shR-N1071 effectively inhibited the expression of the transfected N gene in vitro, comparable to the control shRNA. We further demonstrated the shRNAs markedly reduced PEDV CV777 and LNCT2 replication upon downregulation of N production. Therefore, this study provides a new strategy for the design of antiviral methods against coronaviruses by targeting their processivity factors.

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Synthetic Studies towards Fungal glycosides: An Overview

Current Organic Chemistry ◽

10.2174/1385272824999201105160034 ◽

2020 ◽

Vol 24 (24) ◽

pp. 2865-2901

Author(s):

Hidayat Hussain ◽

Iftikhar Ali ◽

Elizbit ◽

Wahid Hussain ◽

Nilufar Z. Mamadalieva ◽

...

Keyword(s):

Natural Product ◽

Large Scale ◽

Chemical Diversity ◽

Tetramic Acid ◽

Total Syntheses ◽

Biological Studies ◽

Wide Range ◽

Therapeutic Molecules ◽

Synthetic Studies ◽

Pharmacologically Active

: Fungi have provided intriguing chemical diversity and have additionally proven to be a tremendous source for a great variety of therapeutic molecules. Various fungal glycosides have been reported from fungi and the majority of these metabolites possess cytotoxic and antimicrobial effects. Although natural products are obtained in most cases in small amounts from the specific natural source, total syntheses of these valuable commodities remain one of the most important ways of obtaining them on a large scale for more detailed and comprehensive biological studies. In addition, the total synthesis of secondary metabolites is a useful tool, not only for the disclosure of novel complex pharmacologically active molecules but also for the establishment of cutting-edge methodologies in synthetic chemistry. Numerous fungal glycosides have been synthesized in the last four decades regarding the following natural product classes viz., tetramic acid glycosides (epicoccamides A and D), polyketide glycosides (TMC-151C), 2-pyrone glycosides (epipyrone A), diterpene glycosides (sordarin), depside glycosides (CRM646-A and –B, KS-501 and KS- 502), caloporosides (caloporoside A), glycolipids (emmyguyacins A and B, acremomannolipin A), and cerebrosides (cerebroside B, Asperamide B, phalluside-1, Sch II). The current literature review about fungal glycoside synthetic studies is, therefore, of interest for a wide range of scientists and researchers in the field of organic, natural product, and medicinal chemists as it outlines key strategies of fungal glycosides and, in particular, glycosylation, the known biological and pharmacological effects of these natural compounds have afforded a new dimension of exposure.

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Organogenic Nodule Formation in Hop: A Tool to Study Morphogenesis in Plants with Biotechnological and Medicinal Applications

Journal of Biomedicine and Biotechnology ◽

10.1155/2010/583691 ◽

2010 ◽

Vol 2010 ◽

pp. 1-16 ◽

Cited By ~ 5

Author(s):

Ana M. Fortes ◽

Filipa Santos ◽

Maria S. Pais

Keyword(s):

Tissue Culture ◽

Large Scale ◽

Root Nodules ◽

Molecular Networks ◽

Nodule Formation ◽

Culture Methods ◽

Culture Techniques ◽

Wide Range ◽

Morphogenic Process

The usage ofHumulus lupulusfor brewing increased the demand for high-quality plant material. Simultaneously, hop has been used in traditional medicine and recently recognized with anticancer and anti-infective properties. Tissue culture techniques have been reported for a wide range of species, and open the prospect for propagation of disease-free, genetically uniform and massive amounts of plantsin vitro.Moreover, the development of large-scale culture methods using bioreactors enables the industrial production of secondary metabolites. Reliable and efficient tissue culture protocol for shoot regeneration through organogenic nodule formation was established for hop. The present review describes the histological, and biochemical changes occurring during this morphogenic process, together with an analysis of transcriptional and metabolic profiles. We also discuss the existence of common molecular factors among three different morphogenic processes: organogenic nodules and somatic embryogenesis, which strictly speaking depend exclusively on intrinsic developmental reprogramming, and legume nitrogen-fixing root nodules, which arises in response to symbiosis. The review of the key factors that participate in hop nodule organogenesis and the comparison with other morphogenic processes may have merit as a study presenting recent advances in complex molecular networks occurring during morphogenesis and together, these provide a rich framework for biotechnology applications.

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Addressing Challenges to Enhance the Bioactives ofWithania somniferathrough Organ, Tissue, and Cell Culture Based Approaches

BioMed Research International ◽

10.1155/2017/3278494 ◽

2017 ◽

Vol 2017 ◽

pp. 1-15 ◽

Cited By ~ 4

Author(s):

Pritika Singh ◽

Rupam Guleri ◽

Amrita Angurala ◽

Kuldeep Kaur ◽

Kulwinder Kaur ◽

...

Keyword(s):

Cell Culture ◽

Secondary Metabolites ◽

Culture System ◽

Large Scale ◽

Distinct Advantage ◽

Scale Production ◽

Genetic Base ◽

Wide Range ◽

Organ Tissue

Withania somniferais a highly valued medicinal plant in traditional home medicine and is known for a wide range of bioactivities. Its commercial cultivation is adversely affected by poor seed viability and germination. Infestation by various pests and pathogens, survival under unfavourable environmental conditions, narrow genetic base, and meager information regarding biosynthesis of secondary metabolites are some of the other existing challenges in the crop. Biotechnological interventions through organ, tissue, and cell culture provide promising options for addressing some of these issues.In vitropropagation facilitates conservation and sustainable utilization of the existing germplasms and broadening the genetic base. It would also provide means for efficient and rapid mass propagation of elite chemotypes and generating uniform plant material round the year for experimentation and industrial applications. The potential ofin vitrocell/organ cultures for the production of therapeutically valuable compounds and their large-scale production in bioreactors has received significant attention in recent years.In vitroculture system further provides distinct advantage for studying various cellular and molecular processes leading to secondary metabolite accumulation and their regulation. Engineering plants through genetic transformation and development of hairy root culture system are powerful strategies for modulation of secondary metabolites. The present review highlights the developments and sketches current scenario in this field.

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