| 351. |
- Pigot, Harry, et al.
(författare)
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Ex Vivo Working Porcine Heart Model
- 2024. - 2
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Ingår i: Experimental Models of Cardiovascular Diseases : Methods and Protocols - Methods and Protocols. - 1940-6029 .- 1064-3745. - 9781071638460 - 9781071638484 - 9781071638453 ; 2803, s. 87-107
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Bokkapitel (övrigt vetenskapligt/konstnärligt)abstract
- Ex vivo working porcine heart models allow for the study of a heart’s function and physiology outside the living organism. These models are particularly useful due to the anatomical and physiological similarities between porcine and human hearts, providing an experimental platform to investigate cardiac disease or assess donor heart viability for transplantation. This chapter presents an in-depth discussion of the model’s components, including the perfusate, preload, and afterload. We explore the challenges of emulating cardiac afterload and present a historical perspective on afterload modeling, discussing various methodologies and their respective limitations. An actively controlled afterload device is introduced to enhance the model’s ability to rapidly adjust pressure in the large arteries, thereby providing a more accurate and dynamic experimental model. Finally, we provide a comprehensive experimental protocol for the ex vivo working porcine heart model.
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| 352. |
- Pinto, Ines Fernandes, et al.
(författare)
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Microfluidic Cartridge for Bead-Based Affinity Assays
- 2024
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Ingår i: Methods in Molecular Biology. - : Springer Nature. - 1064-3745 .- 1940-6029. ; 2804, s. 127-138
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Tidskriftsartikel (refereegranskat)abstract
- Within the vast field of medical biotechnology, the biopharmaceutical industry is particularly fast-growing and highly competitive, so reducing time and costs associated to process optimization becomes instrumental to ensure speed to market and, consequently, profitability. The manufacturing of biopharmaceutical products, namely, monoclonal antibodies (mAbs), relies mostly on mammalian cell culture processes, which are highly dynamic and, consequently, difficult to optimize. In this context, there is currently an unmet need of analytical methods that can be integrated at-line in a bioreactor, for systematic monitoring and quantification of key metabolites and proteins. Microfluidic-based assays have been extensively and successfully applied in the field of molecular diagnostics; however, this technology remains largely unexplored for Process Analytical Technology (PAT), despite holding great potential for the at-line measurement of different analytes in bioreactor processes, combining low reagent/molecule consumption with assay sensitivity and rapid turnaround times.Here, the fabrication and handling of a microfluidic cartridge for protein quantification using bead-based affinity assays is described. The device allows geometrical multiplexed immunodetection of specific protein analytes directly from bioreactor samples within 2.5 h and minimal hands-on time. As a proof-of-concept, quantification of Chinese hamster ovary (CHO) host cell proteins (HCP) as key impurities, IgG as product of interest, and lactate dehydrogenase (LDH) as cell viability marker was demonstrated with limits of detection (LoD) in the low ng/mL range. Negligible matrix interference and no cross-reactivity between the different immunoassays on chip were found. The results highlight the potential of the miniaturized analytical method for PAT at reduced cost and complexity in comparison with sophisticated instruments that are currently the state-of-the-art in this context.
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| 353. |
- Poças, Juliana, et al.
(författare)
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Analysis of Extracellular Vesicle-Associated Proteoglycans
- 2023
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Ingår i: Methods in molecular biology (Clifton, N.J.). - New York, NY : Springer US. - 1940-6029. ; 2619, s. 125-139
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Tidskriftsartikel (refereegranskat)abstract
- Extracellular vesicles (EVs) have emerged as a central mechanism of intercellular communication in physiology and disease. EVs participate in paracrine exchange of nucleic acids as well as lipids, proteins, and glycans to elicit a complex biological response in target cells. Proteoglycans (PGs) are widely expressed in EV-producing cells and are sorted to the membrane of secreted EVs to participate in some of the key processes in EV-mediated signaling. Most notably, PGs mainly of the heparan sulfate (HS) type are involved in EV biogenesis and cellular uptake of EVs through endocytic processes. EV-associated PGs may serve as short- and long-range chaperones of signaling molecules with potential implications for intercellular information exchange, most importantly in cancer development. This motivates the development of approaches targeting EV-HSPG interactions as a strategy in cancer treatment. Moreover, the importance of PG remodeling and alterations in their expression in cancer, together with the fact that EVs mimic their cell or tissue of origin, point at an important role of EV-associated PGs as disease biomarkers. Here, we provide methodological insights into the analysis of EV-PGs isolated from cell cultures as well as patient plasma liquid biopsy.
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| 354. |
- Pohl, Carsten, et al.
(författare)
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Genome editing in penicillium chrysogenum using cas9 ribonucleoprotein particles
- 2018
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Ingår i: Methods in Molecular Biology. - New York, NY : Springer New York. - 1940-6029 .- 1064-3745. ; , s. 213-232
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Bokkapitel (övrigt vetenskapligt/konstnärligt)abstract
- Several CRISPR/Cas9 tools have been recently established for precise genome editing in a wide range of filamentous fungi. This genome editing platform offers high flexibility in target selection and the possibility of introducing genetic deletions without the introduction of transgenic sequences. This chapter describes an approach for the transformation of Penicillium chrysogenum protoplasts with preassembled ribonucleoprotein particles (RNPs) consisting of purified Cas9 protein and in vitro transcribed single guide RNA (sgRNA) for the deletion of genome sequences or their replacement with alternative sequences. This method is potentially transferable to all fungal strains where protoplasts can be obtained from.
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| 355. |
- Polisetti, Naresh, et al.
(författare)
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The artificial cornea
- 2013
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Ingår i: Methods in Molecular Biology. - Totowa, NJ : Humana Press. - 1064-3745 .- 1940-6029. ; 1014, s. 45-52
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Tidskriftsartikel (refereegranskat)abstract
- Human corneal transplantation to date suffers from the shortage of good-quality donor tissue, and in some conditions, allografting is contraindicated. A range of artificial replacements to donor allograft corneas have been developed. These range from keratoprostheses (KPro) that replace basic corneal functions of light transmission and protection to regenerative medicine strategies for regenerating one or more layers of the human cornea. This chapter reviews the advances made in developing artificial corneas or more accurately, artificial alternatives to donor allograft corneas for ocular application.
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| 356. |
- Pronk, Kees-Jan, et al.
(författare)
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Flow cytometry-based identification of immature myeloerythroid development.
- 2011
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Ingår i: Methods in Molecular Biology. - Totowa, NJ : Humana Press. - 1940-6029. ; 699, s. 275-293
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Tidskriftsartikel (refereegranskat)abstract
- Precursor cells of the myeloerythroid cell lineages give rise to mature cells of the granulocyte, monocyte, erythroid, and/or thrombocytic lineages. High-resolution profiling of the developmental stages, from hematopoietic stem cells to mature progeny, is important to study and understand the underlying mechanisms that guide various cell fate decisions. In addition, this approach provides greater insights into pathogenic events such as leukemia, diseases that are most often characterized by halted differentiation at defined immature precursor levels. In this chapter, we provide protocols and discuss approaches concerning the analysis and purification of immature myeloerythroid lineages by multiparameter flow cytometry. Although recent data have demonstrated the feasibility of similar approaches also for the human system, we will focus our chapter on C57BL/6 mice, in which immunophenotypic applications have been most widely developed. This should also allow for its application in genetically modified models on this background. For maximal reproducibility, all protocols described have been established using reagents from commercial vendors to be analyzed on a three-laser flow cytometer with factory standard configuration.
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| 357. |
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| 358. |
- Quintana-Hayashi, Macarena P, et al.
(författare)
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Differentiation of Gastrointestinal Cell Lines by Culture in Semi-wet Interface.
- 2018
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Ingår i: Epithelial Cell Culture. Baratta M. (red.) Methods in Molecular Biolog. - New York, NY : Springer. - 1940-6029. - 9781493985999 ; , s. 41-46
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Bokkapitel (refereegranskat)abstract
- Epithelial cells grown in vitro provide opportunities to elucidate cellular mechanisms in response to chemical, viral, or bacterial agents in isolation from the effects of other bodily functions, such as hormonal and immune responses. However, cells that do not form a tight epithelium, polarize or secrete mucins lack some of the important protection mechanisms intrinsic to epithelial cells in vivo, increasing their susceptibility to external agents, and exposing basolateral targets for interactions that may not occur in vivo. Here, we present a method that transforms some epithelial cell lines into mucin secreting polarized epithelial surfaces with high transepithelial resistance: the cells are cultured on semi-permeable membranes in differentiation medium for the first 6days, followed by culture under semi-wet interface with mechanical stimulation for 22days. The procedure can be performed using standard laboratory reagents and equipment. A description on how to fix and paraffin embed these in vitro mucosal membranes for histology purposes is also included.
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| 359. |
- Quist, Ella, et al.
(författare)
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Transcription Factor Programming of Human Pluripotent Stem Cells to Functionally Mature Astrocytes for Monocultures and Cocultures with Neurons
- 2021
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Ingår i: Neural Reprogramming: Methods and Protocols. - New York, NY : Springer US. - 1940-6029 .- 1064-3745. - 9781071616017 - 9781071616000 ; 2352, s. 133-148
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Bokkapitel (refereegranskat)abstract
- Astrocytes are essential cells for normal brain functionality and have recently emerged as key players in many neurological diseases. However, the limited availability of human primary astrocytes for cell culture studies hinders our understanding of their physiology and precise role in disease development and progression. Here, we describe a detailed step-by-step protocol to rapidly and efficiently generate functionally mature induced astrocytes (iAs) from human embryonic and induced pluripotent stem cells (hES/iPSCs). Astrocyte induction is accomplished by ectopic lentiviral expression of two gliogenic transcription factors, Sox9 and Nfib. iAs exhibit morphology features as well as gene and protein expression similar to human mature astrocytes and display important astrocytic functions, such as glutamate uptake, propagation of calcium waves, expression of various cytokines after stimulation, and support of synapse formation and function, making them suitable models for studying the role of astrocytes in health and disease. Moreover, we describe a procedure for cryopreservation of iAs for long-term storage or shipping. Finally, we provide the required information needed to set up cocultures with human induced neurons (iNs, also described in this book), generated from hES/iPSCs, to generate cocultures, allowing studies on astrocyte-neuron interactions and providing new insights in astrocyte-associated disease mechanisms.
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| 360. |
- Ramos, Marta, et al.
(författare)
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Cisterna Magna Injection in Rats to Study Glymphatic Function
- 2019
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Ingår i: Methods in molecular biology (Clifton, N.J.). - New York, NY : Springer New York. - 1940-6029. ; 1938, s. 97-104
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Tidskriftsartikel (refereegranskat)abstract
- The recently discovered glymphatic system, which supports brain-wide clearance of metabolic waste, has become the subject of intense research within the past few years. Its nomenclature arose due to its functionally analogous nature to the lymphatic system in combination with glial cells that are part of its anatomical boundaries. The influx of cerebrospinal fluid (CSF) from perivascular spaces into the brain interstitium acts to clear intraparenchymal solutes. CSF is produced by the choroid plexus and flows from the ventricles to the subarachnoid space via the cisterna magna, and as such the injection of tracer molecules into any one of these spaces could be used for studying CSF movement through the glymphatic system. Of these options, the cisterna magna is most favorable as it offers a route of entry that does not involve craniotomy. Herein we describe the cisterna magna (CM) injection procedure carried out in rats, essential for studying glymphatic influx and efflux dynamics.
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