scholarly journals Neurosurgical Approaches to Brain Tissue Harvesting for the Establishment of Cell Cultures in Neural Experimental Cell Models

Materials ◽  
2021 ◽  
Vol 14 (22) ◽  
pp. 6857
Author(s):  
Lidija Gradišnik ◽  
Roman Bošnjak ◽  
Gorazd Bunc ◽  
Janez Ravnik ◽  
Tina Maver ◽  
...  
Keyword(s):  
Brain Tissue ◽  
Cell Biology ◽  
Cell Isolation ◽  
Neurosurgical Procedures ◽  
Cell Models ◽  
Tissue Samples ◽  
Testing Procedures ◽  
Experimental Cell ◽  
Wide Range

In recent decades, cell biology has made rapid progress. Cell isolation and cultivation techniques, supported by modern laboratory procedures and experimental capabilities, provide a wide range of opportunities for in vitro research to study physiological and pathophysiological processes in health and disease. They can also be used very efficiently for the analysis of biomaterials. Before a new biomaterial is ready for implantation into tissues and widespread use in clinical practice, it must be extensively tested. Experimental cell models, which are a suitable testing ground and the first line of empirical exploration of new biomaterials, must contain suitable cells that form the basis of biomaterial testing. To isolate a stable and suitable cell culture, many steps are required. The first and one of the most important steps is the collection of donor tissue, usually during a surgical procedure. Thus, the collection is the foundation for the success of cell isolation. This article explains the sources and neurosurgical procedures for obtaining brain tissue samples for cell isolation techniques, which are essential for biomaterial testing procedures.

A System for 256-Channel in Vitro Recording of the Electrophysiological Activity of Brain Tissue

2013 ◽  
Vol 20 (3) ◽  
pp. 371-384
Author(s):  
Miroslaw Zoladz
Keyword(s):  
Brain Tissue ◽  
Support System ◽  
Microelectrode Array ◽  
Life Support ◽  
Hippocampal Formation ◽  
Measurement Data ◽  
Life Support System ◽  
Tissue Samples ◽  
Electrophysiological Activity

Abstract A measurement system for 256-channel in vitro recordings of brain tissue electrophysiological activity is presented in the paper. The system consists of the brain tissue life support system, Microelectrode Array (MEA), conditioning Application Specific Integrated Circuits (ASIC’s) for signals conditioning, Digitizer and PC application for measurement data presentation and storage. The life support system keeps brain tissue samples in appropriately saturated artificial cerebrospinal fluid at a very stable temperature. The MEA consists of two hundred and fifty-six 40 μm diameter tip-shaped electrodes. The ASIC’s performs amplification and filtering of the 256-field and action potential signals. The Digitizer performs simultaneous data acquisition from 256 channels 14 kS/s sample rate and 12-bit resolution. The resulting byte stream is transmitted to the PC via USB (Universal Serial Bus). Preliminary tests confirm that the system is capable of keeping the extracted brain tissue active (hippocampal formation slices) and simultaneously to record action potentials, as well as local theta field potentials with very small amplitudes from multiple neurons

scholarly journals In VivoExpression of Streptococcus pyogenes Immunogenic Proteins during Tibial Foreign Body Infection

2014 ◽  
Vol 82 (9) ◽  
pp. 3891-3899 ◽  
Author(s):  
Jeffrey A. Freiberg ◽  
Kevin S. McIver ◽  
Mark E. Shirtliff
Keyword(s):  
Group A Streptococcus ◽  
Rabbit Model ◽  
Growth Conditions ◽  
Rabbit Serum ◽  
Tissue Samples ◽  
Wide Range ◽  
Group A ◽  
Immunogenic Proteins

ABSTRACTGroup A streptococcus (GAS) is an important human pathogen that causes a number of diseases with a wide range of severities. While all known strains of GAS are still sensitive to penicillin, there have been reports of antibiotic treatment failure in as many as 20% to 40% of cases. Biofilm formation has been implicated as a possible cause for these failures. A biofilm is a microbially derived, sessile community where cells grow attached to a surface or as a bacterial conglomerate and surrounded by a complex extracellular matrix. While the ability of group A streptococcus to form biofilms in the laboratory has been shown, there is a lack of understanding of the role of GAS biofilms during an infection. We hypothesized that during infections, GAS exhibits a biofilm phenotype, complete with unique protein expression. To test this hypothesis, a rabbit model of GAS osteomyelitis was developed. A rabbit was inoculated with GAS using an infected indwelling device. Following the infection, blood and tissue samples were collected. Histological samples of the infected tibia were prepared, and the formation of a biofilmin vivowas visualized using peptide nucleic acid fluorescentin situhybridization (PNA-FISH) and confocal microscopy. In addition, Western blotting with convalescent rabbit serum detected cell wall proteins expressedin vitrounder biofilm and planktonic growth conditions. Immunogenic proteins were then identified using matrix-assisted laser desorption ionization–time of flight tandem mass spectrometry (MALDI-TOF/TOF MS). These identities, along with thein vivoresults, support the hypothesis that GAS forms biofilms during an infection. This unique phenotype should be taken into consideration when designing a vaccine or any other treatment for group A streptococcus infections.

scholarly journals BrainPhys neuronal medium optimized for imaging and optogenetics in vitro

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Michael Zabolocki ◽  
Kasandra McCormack ◽  
Mark van den Hurk ◽  
Bridget Milky ◽  
Andrew P. Shoubridge ◽  
...  
Keyword(s):  
Cell Biology ◽  
Culture Conditions ◽  
Cellular Reprogramming ◽  
Synaptic Activity ◽  
Neuronal Cultures ◽  
Neuronal Viability ◽  
Human Neurons ◽  
Wide Range ◽  
And Function

AbstractThe capabilities of imaging technologies, fluorescent sensors, and optogenetics tools for cell biology are advancing. In parallel, cellular reprogramming and organoid engineering are expanding the use of human neuronal models in vitro. This creates an increasing need for tissue culture conditions better adapted to live-cell imaging. Here, we identify multiple caveats of traditional media when used for live imaging and functional assays on neuronal cultures (i.e., suboptimal fluorescence signals, phototoxicity, and unphysiological neuronal activity). To overcome these issues, we develop a neuromedium called BrainPhys™ Imaging (BPI) in which we optimize the concentrations of fluorescent and phototoxic compounds. BPI is based on the formulation of the original BrainPhys medium. We benchmark available neuronal media and show that BPI enhances fluorescence signals, reduces phototoxicity and optimally supports the electrical and synaptic activity of neurons in culture. We also show the superior capacity of BPI for optogenetics and calcium imaging of human neurons. Altogether, our study shows that BPI improves the quality of a wide range of fluorescence imaging applications with live neurons in vitro while supporting optimal neuronal viability and function.

scholarly journals Impact of isolation method on cellular activation and presence of specific tendon cell subpopulations during in vitro culture

2021 ◽  
Author(s):  
Anne E.C. Nichols ◽  
Sarah E. Miller ◽  
Luke J. Green ◽  
Michael S. Richards ◽  
Alayna E. Loiselle
Keyword(s):  
Cell Biology ◽  
Tendon Healing ◽  
Marker Genes ◽  
Cell Models ◽  
Isolation Method ◽  
Tendon Cell ◽  
Tendon Cells ◽  
Tissue Systems

AbstractTendon injuries are common and heal poorly, due in part to a lack of understanding of fundamental tendon cell biology. A major impediment to the study of tendon cells is the absence of robust, well-characterized in vitro models. Unlike other tissue systems, current tendon cell models do not account for how differences in isolation methodology may affect the activation state of tendon cells or the presence of various tendon cell sub-populations. The objective of this study was to characterize how common isolation methods affect the behavior, fate, and lineage composition of tendon cell cultures. Tendon cells isolated by explant exhibited reduced proliferative capacity, decreased expression of tendon marker genes, and increased expression of genes associated with fibroblast activation compared to digested cells. Consistently, explanted cells also displayed an increased propensity to differentiate to myofibroblasts compared to digested cells. Explanted cultures from multiple different tendons were substantially enriched for the presence of scleraxis-lineage (Scx-lin+) cells compared to digested cultures, while the overall percentage of S100a4-lineage (S100a4-lin+) cells was dependent on both isolation method and tendon of origin. Neither isolation method preserved the ratios of Scx-lin+ or S100a4-lin+ to non-lineage cells seen in tendons in vivo. Combined, these data indicate that further refinement of in vitro cultures models is required in order to more accurately understand the effects of various stimuli on tendon cell behavior.Statement of clinical significanceThe development of informed in vitro tendon cell models will facilitate enhanced screening of potential therapeutic candidates to improve tendon healing.

scholarly journals BrainPhys neuronal medium optimized for imaging and optogenetics in vitro

2020 ◽  
Author(s):  
Michael Zabolocki ◽  
Kasandra McCormack ◽  
Mark van den Hurk ◽  
Bridget Milky ◽  
Andrew Shoubridge ◽  
...  
Keyword(s):  
Neuronal Activity ◽  
Cortical Neurons ◽  
Cell Biology ◽  
Cellular Reprogramming ◽  
Synaptic Activity ◽  
Neuronal Cultures ◽  
Light Spectrum ◽  
Human Neurons ◽  
Wide Range

AbstractThe capabilities of imaging technologies, fluorescent sensors, and optogenetics tools for cell biology have improved exponentially in the last ten years. At the same time, advances in cellular reprogramming and organoid engineering have quickly expanded the use of human neuronal models in vitro. Altogether this creates an increasing need for tissue culture conditions better adapted to live-cell imaging. Here, we identified multiple caveats of traditional media when used for live imaging and functional assays on neuronal cultures (e.g., phototoxicity, suboptimal fluorescence signals, and unphysiological neuronal activity). To overcome these issues, we developed a new neuromedium, “BrainPhys™ Imaging”, in which we adjusted fluorescent and phototoxic compounds. The new medium is based on the formulation of the original BrainPhys medium, which we designed to better support the neuronal activity of human neurons in vitro1. We tested the new imaging-optimized formulation on human neurons cultured in monolayers or organoids, and rat primary neurons. BrainPhys Imaging enhanced fluorescence signals and reduced phototoxicity throughout the entire light spectrum. Importantly, consistent with standard BrainPhys, we showed that the new imaging medium optimally supports the electrical and synaptic activity of midbrain and human cortical neurons in culture. We also benchmarked the capacity of the new medium for functional calcium imaging and optogenetic control of human neurons. Altogether, our study shows that the new BrainPhys Imaging improves the quality of a wide range of fluorescence imaging applications with live neurons in vitro while supporting cell viability and neuronal functions.

scholarly journals A Living Cell Repository of the Cranio-/Orofacial Region to Advance Research and Promote Personalized Medicine

2021 ◽  
Vol 9 ◽  
Author(s):  
Ludovica Parisi ◽  
Patrick O. Knapp ◽  
Eleftheria Girousi ◽  
Silvia Rihs ◽  
Giorgio C. La Scala ◽  
...  
Keyword(s):  
Living Cell ◽  
Specific Cell ◽  
Cell Models ◽  
Cell Bank ◽  
Tissue Samples ◽  
Genetic Characteristics ◽  
Societal Pressure ◽  
Orofacial Region ◽  
Quality Checks

The prevalence of congenital anomalies in newborns is estimated to be as high as 6%, many of which involving the cranio-/orofacial region. Such malformations, including several syndromes, are usually identified prenatally, at birth, or rarely later in life. The lack of clinically relevant human cell models of these often very rare conditions, the societal pressure to avoid the use of animal models and the fact that the biological mechanisms between rodents and human are not necessarily identical, makes studying cranio-/orofacial anomalies challenging. To overcome these limitations, we are developing a living cell repository of healthy and diseased cells derived from the cranio-/orofacial region. Ultimately, we aim to make patient-derived cells, which retain the molecular and genetic characteristics of the original anomaly or disease in vitro, available for the scientific community. We report our efforts in establishing a human living cell bank derived from the cranio-/orofacial region of otherwise discarded tissue samples, detail our strategy, processes and quality checks. Such specific cell models have a great potential for discovery and translational research and might lead to a better understanding and management of craniofacial anomalies for the benefit of all affected individuals.

scholarly journals ATM Knockdown renders Glioma resistant to Temozolomide by Inhibiting IFIT1 Expression

2021 ◽  
Author(s):  
Lijuan Yang ◽  
Jinfeng Zhang ◽  
Shengmei Weng ◽  
Zhixiong Lin
Keyword(s):  
Cell Biology ◽  
Dna Methyltransferase ◽  
Chemotherapy Resistance ◽  
Tumor Model ◽  
Standard Of Care ◽  
Tissue Samples ◽  
Mgmt Expression ◽  
Whole Genome Expression

Abstract Background: Temozolomide (TMZ) chemotherapy has been a standard of care in treating malignant glioma. Although TMZ chemotherapy can extend patient’s survival, resistance to TMZ is observed in most cases. The drug resistance is reported to be mainly mediated by O6- methyl guanine-DNA methyltransferase (MGMT) expression.Methods: By means of molecular biology, cell biology, construction of nude mouse xenograft tumor model and analysis of clinical specimens, a new molecular mechanism for the regulation of TMZ chemotherapy resistance of glioma was proposed.Result: The present study indicates a pathway of TMZ resistance in glioma via suppressing ataxia-telangiectasia mutated (ATM) gene. Whole genome expression profile of glioma cells and tissue samples revealed a positive correlation between the ATM and IF1T1, the decreased expression of which might be the underlying cause of the ATM knockdown induced TMZ resistance.Conclusions: Our study revealed that ATM silencing induced TMZ chemotherapy resistance of glioma in vitro and in vivo by inhibite IFIT1 expression, the p-ATM, IFIT1 and MGMT expression acts as a prognostic marker of glioma chemotherapy. And combination of IFIT1 and MGMT seems to be a more significant molecular marker to predict the prognosis in glioma patients.

scholarly journals Role of M Cells in Human Mucosal Immunity to Mycobacterium tuberculosis

2017 ◽  
Vol 4 (suppl_1) ◽  
pp. S48-S48
Author(s):  
Vidhya Nair ◽  
Haaris Khan ◽  
Ron Mitchell ◽  
Michael U Shiloh
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Virulence Factor ◽  
Mucosal Immunity ◽  
Cell Biology ◽  
Infection Model ◽  
M Cells ◽  
Dependent Manner ◽  
M Cell ◽  
Wide Range

Abstract Background Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), is a bacterial pathogen that infects roughly one-third of the worldÕs population and causes 1–2 million deaths per year. The current paradigm is that phagocytosis of Mtb by patrolling alveolar macrophages initiates Mtb infection. While this model can account for pulmonary TB, it does not adequately explain the occurrence of extrapulmonary forms of TB that manifest in the absence of obvious lung involvement, such as tuberculous cervical lymphadenitis, also known as scrofula. We hypothesized that specialized epithelial cells called microfold cells (M cells) may be an alternate portal of entry for Mtb. Previously we demonstrated that Mtb is able to transcytose across an epithelial barrier in an M cell dependent manner and that M cell mediated transcytosis is vital for Mtb pathogenesis in a mouse model of tuberculosis. Methods We used an in vitro M-cell mediated translocation assay and a Mtb mutant lacking a key virulence factor, ESAT6. We used biochemistry and genetics to identify a novel receptor for ESAT6. We also developed a novel explanted human adenoid Mtb infection model to study mucosal immunity. Results We now demonstrate that the Mtb virulence factor ESAT6 is necessary and sufficient to mediate binding and transcytosis by M cells in vitro and in vivo, and that uptake of Mtb by M cells requires a unique cell surface ESAT6 receptor. We developed a novel explanted human adenoid model of M cell biology and demonstrate rapid Mtb transcytosis by primary human tissue within 60–120 minutes. Using flow cytometry we find that Mtb is first ingested by M cells and then after transcytosis, by tissue resident antigen-presenting cells. Explanted adenoids from 10 independent donors display a wide range of Mtb uptake. Conclusion We conclude that Mtb ESAT6 is necessary for Mtb uptake by M-cells and that binding and transcytosis require a host receptor. Because explanted adenoids display a wide range of Mtb uptake, M cell mediated transcytosis may confer differential susceptibility to scrofula and disseminated disease. These findings are significant as M cells could potentially serve as the basis for novel therapeutic targets against primary Mtb infection. Disclosures All authors: No reported disclosures.

scholarly journals In vitro bioavailability and cellular bioactivity studies of flavonoids and flavonoid-rich plant extracts: questions, considerations and future perspectives

2016 ◽  
Vol 76 (3) ◽  
pp. 175-181 ◽  
Author(s):  
Gerard Bryan Gonzales
Keyword(s):  
Plant Extracts ◽  
Flavonoid Glycosides ◽  
Cell Models ◽  
Future Perspectives ◽  
Oral Ingestion ◽  
Detailed Mechanism ◽  
Flavonoid Aglycones ◽  
Wide Range ◽  
Biochemical Mechanisms

In vitro techniques are essential in elucidating biochemical mechanisms and for screening a wide range of possible bioactive candidates. The number of papers published reporting in vitro bioavailability and bioactivity of flavonoids and flavonoid-rich plant extracts is numerous and still increasing. However, even with the present knowledge on the bioavailability and metabolism of flavonoids after oral ingestion, certain inaccuracies still persist in the literature, such as the use of plant extracts to study bioactivity towards vascular cells. There is therefore a need to revisit, even question, these approaches in terms of their biological relevance. In this review, the bioavailability of flavonoid glycosides, the use of cell models for intestinal absorption and the use of flavonoid aglycones and flavonoid-rich plant extracts in in vitro bioactivity studies will be discussed. Here, we focus on the limitations of current in vitro systems and revisit the validity of some in vitro approaches, and not on the detailed mechanism of flavonoid absorption and bioactivity. Based on the results in the review, there is an apparent need for stricter guidelines on publishing data on in vitro data relating to the bioavailability and bioactivity of flavonoids and flavonoid-rich plant extracts.

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