Size-selective concentration and label-free characterization of protein aggregates using a Raman active nanofluidic device

Lab on a Chip ◽  
2011 ◽  
Vol 11 (4) ◽  
pp. 632-638 ◽  
Author(s):  
Inhee Choi ◽  
Yun Suk Huh ◽  
David Erickson
Keyword(s):  
Protein Aggregates ◽  
Label Free ◽  
Nanofluidic Device

scholarly journals Characterization of Liposomes Using Quantitative Phase Microscopy (QPM)

Pharmaceutics ◽  
2021 ◽  
Vol 13 (5) ◽  
pp. 590
Author(s):  
Jennifer Cauzzo ◽  
Nikhil Jayakumar ◽  
Balpreet Singh Ahluwalia ◽  
Azeem Ahmad ◽  
Nataša Škalko-Basnet
Keyword(s):  
Rapid Development ◽  
Fluorescent Labeling ◽  
Label Free ◽  
Batch Mode ◽  
Full Field ◽  
Quantitative Phase ◽  
Phase Microscopy ◽  
Quantitative Phase Microscopy ◽  
Tracking Ability

The rapid development of nanomedicine and drug delivery systems calls for new and effective characterization techniques that can accurately characterize both the properties and the behavior of nanosystems. Standard methods such as dynamic light scattering (DLS) and fluorescent-based assays present challenges in terms of system’s instability, machine sensitivity, and loss of tracking ability, among others. In this study, we explore some of the downsides of batch-mode analyses and fluorescent labeling, while introducing quantitative phase microscopy (QPM) as a label-free complimentary characterization technique. Liposomes were used as a model nanocarrier for their therapeutic relevance and structural versatility. A successful immobilization of liposomes in a non-dried setup allowed for static imaging conditions in an off-axis phase microscope. Image reconstruction was then performed with a phase-shifting algorithm providing high spatial resolution. Our results show the potential of QPM to localize subdiffraction-limited liposomes, estimate their size, and track their integrity over time. Moreover, QPM full-field-of-view images enable the estimation of a single-particle-based size distribution, providing an alternative to the batch mode approach. QPM thus overcomes some of the drawbacks of the conventional methods, serving as a relevant complimentary technique in the characterization of nanosystems.

scholarly journals Microfluidic Tools for Enhanced Characterization of Therapeutic Stem Cells and Prediction of Their Potential Antimicrobial Secretome

Antibiotics ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 750
Author(s):  
Pasquale Marrazzo ◽  
Valeria Pizzuti ◽  
Silvia Zia ◽  
Azzurra Sargenti ◽  
Daniele Gazzola ◽  
...  
Keyword(s):  
Stem Cells ◽  
Defense Mechanisms ◽  
Live Cells ◽  
Label Free ◽  
Bacterial Diseases ◽  
Prospective Application ◽  
Therapy Strategies ◽  
Stromal Stem Cells ◽  
Different Sources

Antibiotic resistance is creating enormous attention on the development of new antibiotic-free therapy strategies for bacterial diseases. Mesenchymal stromal stem cells (MSCs) are the most promising candidates in current clinical trials and included in several cell-therapy protocols. Together with the well-known immunomodulatory and regenerative potential of the MSC secretome, these cells have shown direct and indirect anti-bacterial effects. However, the low reproducibility and standardization of MSCs from different sources are the current limitations prior to the purification of cell-free secreted antimicrobial peptides and exosomes. In order to improve MSC characterization, novel label-free functional tests, evaluating the biophysical properties of the cells, will be advantageous for their cell profiling, population sorting, and quality control. We discuss the potential of emerging microfluidic technologies providing new insights into density, shape, and size of live cells, starting from heterogeneous or 3D cultured samples. The prospective application of these technologies to studying MSC populations may contribute to developing new biopharmaceutical strategies with a view to naturally overcoming bacterial defense mechanisms.

scholarly journals Characterization of Recombinant Adeno-Associated Viruses (rAAVs) for Gene Therapy Using Orthogonal Techniques

Pharmaceutics ◽  
2021 ◽  
Vol 13 (4) ◽  
pp. 586
Author(s):  
Liam Cole ◽  
Diogo Fernandes ◽  
Maryam T. Hussain ◽  
Michael Kaszuba ◽  
John Stenson ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Light Scattering ◽  
Dynamic Light Scattering ◽  
Viral Vector ◽  
Structural Characteristics ◽  
Genetic Material ◽  
Label Free ◽  
Gene Therapies ◽  
Multiple Challenges

Viruses are increasingly used as vectors for delivery of genetic material for gene therapy and vaccine applications. Recombinant adeno-associated viruses (rAAVs) are a class of viral vector that is being investigated intensively in the development of gene therapies. To develop efficient rAAV therapies produced through controlled and economical manufacturing processes, multiple challenges need to be addressed starting from viral capsid design through identification of optimal process and formulation conditions to comprehensive quality control. Addressing these challenges requires fit-for-purpose analytics for extensive characterization of rAAV samples including measurements of capsid or particle titer, percentage of full rAAV particles, particle size, aggregate formation, thermal stability, genome release, and capsid charge, all of which may impact critical quality attributes of the final product. Importantly, there is a need for rapid analytical solutions not relying on the use of dedicated reagents and costly reference standards. In this study, we evaluate the capabilities of dynamic light scattering, multiangle dynamic light scattering, and SEC–MALS for analyses of rAAV5 samples in a broad range of viral concentrations (titers) at different levels of genome loading, sample heterogeneity, and sample conditions. The study shows that DLS and MADLS® can be used to determine the size of full and empty rAAV5 (27 ± 0.3 and 33 ± 0.4 nm, respectively). A linear range for rAAV5 size and titer determination with MADLS was established to be 4.4 × 1011–8.7 × 1013 cp/mL for the nominally full rAAV5 samples and 3.4 × 1011–7 × 1013 cp/mL for the nominally empty rAAV5 samples with 3–8% and 10–37% CV for the full and empty rAAV5 samples, respectively. The structural stability and viral load release were also inferred from a combination of DLS, SEC–MALS, and DSC. The structural characteristics of the rAAV5 start to change from 40 °C onward, with increasing aggregation observed. With this study, we explored and demonstrated the applicability and value of orthogonal and complementary label-free technologies for enhanced serotype-independent characterization of key properties and stability profiles of rAAV5 samples.

scholarly journals Immortalization and Characterization of Rat Lingual Keratinocytes in a High-Calcium and Feeder-Free Culture System Using ROCK Inhibitor Y-27632

2021 ◽  
Vol 22 (13) ◽  
pp. 6782
Author(s):  
Zixing Chen ◽  
Wenmeng He ◽  
Thomas Chun Ning Leung ◽  
Hau Yin Chung
Keyword(s):  
Calcium Imaging ◽  
Cultured Cells ◽  
Rho Kinase ◽  
Culture Method ◽  
Taste Bud ◽  
Label Free ◽  
Sprague Dawley ◽  
Rock Inhibitor ◽  
High Calcium

Cultured keratinocytes are desirable models for biological and medical studies. However, primary keratinocytes are difficult to maintain, and there has been little research on lingual keratinocyte culture. Here, we investigated the effect of Y-27632, a Rho kinase (ROCK) inhibitor, on the immortalization and characterization of cultured rat lingual keratinocyte (RLKs). Three Y-27632–supplemented media were screened for the cultivation of RLKs isolated from Sprague–Dawley rats. Phalloidin staining and TUNEL assay were applied to visualize cytoskeleton dynamics and cell apoptosis following Y-27632 removal. Label-free proteomics, RT-PCR, calcium imaging, and cytogenetic studies were conducted to characterize the cultured cells. Results showed that RLKs could be conditionally immortalized in a high-calcium medium in the absence of feeder cells, although they did not exhibit normal karyotypes. The removal of Y-27632 from the culture medium led to reversible cytoskeletal reorganization and nuclear enlargement without triggering apoptosis, and a total of 239 differentially expressed proteins were identified by proteomic analysis. Notably, RLKs derived from the non-taste epithelium expressed some molecular markers characteristic of taste bud cells, yet calcium imaging revealed that they rarely responded to tastants. Collectively, we established a high-calcium and feeder-free culture method for the long-term maintenance of RLKs. Our results shed some new light on the immortalization and differentiation of lingual keratinocytes.

Label-Free Characterization of Collagen Crosslinking in Bone-Engineered Materials Using Nonlinear Optical Microscopy

2021 ◽  
pp. 1-11
Author(s):  
Chao-Wei Hung ◽  
Nirmal Mazumder ◽  
Dan-Jae Lin ◽  
Wei-Liang Chen ◽  
Shih-Ting Lin ◽  
...  
Keyword(s):  
Optical Microscopy ◽  
Nonlinear Optical ◽  
Label Free ◽  
Nonlinear Optical Microscopy ◽  
Collagen Crosslinking ◽  
Engineered Materials

Abstract

Label‐free 3D characterization of cardiac fibrosis in muscular dystrophy using SHG imaging of cleared tissue

2021 ◽  
Author(s):  
Julien Pichon ◽  
Mireille Ledevin ◽  
Thibaut Larcher ◽  
Frédéric Jamme ◽  
Karl Rouger ◽  
...  
Keyword(s):  
Muscular Dystrophy ◽  
Cardiac Fibrosis ◽  
Label Free

Automatic Counting of Intra-Cellular Ribonucleo-Protein Aggregates in Saccharomyces cerevisiae Using a Textural Approach

2019 ◽  
Vol 25 (1) ◽  
pp. 164-179
Author(s):  
Ambroise Marin ◽  
Emmanuel Denimal ◽  
Lucie Bertheau ◽  
Stéphane Guyot ◽  
Ludovic Journaux ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Fluorescent Protein ◽  
Protein Aggregates ◽  
Point Of View ◽  
Zernike Moment ◽  
Two Photon ◽  
Haralick Features ◽  
Green Fluorescent ◽  
Two Photon Fluorescence

AbstractIn the context of microbiology, recent studies show the importance of ribonucleo-protein aggregates (RNPs) for the understanding of mechanisms involved in cell responses to specific environmental conditions. The assembly and disassembly of aggregates is a dynamic process, the characterization of the stage of their evolution can be performed by the evaluation of their number. The aim of this study is to propose a method to automatically determine the count of RNPs. We show that the determination of a precise count is an issue by itself and hence, we propose three textural approaches: a classical point of view using Haralick features, a frequency point of view with generalized Fourier descriptors, and a structural point of view with Zernike moment descriptors (ZMD). These parameters are then used as inputs for a supervised classification in order to determine the most relevant. An experiment using a specific Saccharomyces cerevisiae strain presenting a fusion between a protein found in RNPs (PAB1) and the green fluorescent protein was performed to benchmark this approach. The fluorescence was observed with two-photon fluorescence microscopy. Results show that the textural approach, by mixing ZMD with Haralick features, allows for the characterization of the number of RNPs.

scholarly journals Label-Free Prostate Cancer Detection by Characterization of Extracellular Vesicles Using Raman Spectroscopy

2018 ◽  
Vol 90 (19) ◽  
pp. 11290-11296 ◽  
Author(s):  
Wooje Lee ◽  
Afroditi Nanou ◽  
Linda Rikkert ◽  
Frank A. W. Coumans ◽  
Cees Otto ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Raman Spectroscopy ◽  
Extracellular Vesicles ◽  
Cancer Detection ◽  
Label Free ◽  
Prostate Cancer Detection

scholarly journals PATH-43. DIAGNOSIS OF GLIOMAS USING CIRCULATING GLIAL CELLS

2019 ◽  
Vol 21 (Supplement_6) ◽  
pp. vi153-vi153
Author(s):  
Darshana Patil ◽  
Dadasaheb Akolkar ◽  
Stefan Schuster ◽  
H B Govardhan ◽  
Vineet Datta ◽  
...  
Keyword(s):  
Glial Cells ◽  
High Morbidity ◽  
Label Free ◽  
Circulating Biomarkers ◽  
Formidable Challenge ◽  
Selective Elimination ◽  
Technological Challenge ◽  
Cns Malignancies ◽  
Responsive Cell

Abstract Invasive procedures for diagnosis of CNS malignancies carry inherent risks of high morbidity and mortality. Although circulating biomarkers such as cell free DNA (cfDNA) and microvesicle (MV) borne nucleic acids have been proposed as potential diagnostic aids, their stand-alone utility has inherent limitations. However, Circulating Glial Cells (CGCs) combined with cfDNA could offer a viable alternative to invasive biopsies for diagnosis of CNS malignancies; yet the technological challenge in the detection of CGCs in glioma patients presents a formidable challenge. In this study, we evaluated the feasibility of harvesting CGCs from suspected cases of Glioma. From a cohort of 23 suspected cases of CNS malignancies, we used 15ml of peripheral blood and used the CellWizard™ process and related protocol for isolation of CGCs. CellWizard™ is an epigenetically active media with paradoxical chemo-toxicity that selectively induces lethality in normal cells which have a functionally responsive cell death (apoptosis) mechanism, while simultaneously conferring survival privilege on apoptosis resistant cells typically released from a malignant tumor. This paradoxical cytotoxicity of the medium leads to selective elimination of most leukocytes thus facilitating a label free negative enrichment of CGCs, which can be harvested and further characterized. Patients included 11 Glioblastoma, 3 Anaplastic astrocytoma, 2 Medulloblastoma, 5 Oligodendroglioma, 1 Gliosarcoma and 1 meningioma patient. Characterization of CGCs was performed using GFAP, S100 and CD45 markers. CGCs were detected in 16 out of 23 (69.6 %) patients and could be stained positively for both GFAP and S100 and negatively for CD45. Detection of viable CGCs in cases of CNS malignancies can be used for characterization of markers related to the diagnosis.

Sign in / Sign up

Export Citation Format

Share Document