A needle-like optofluidic probe enables targeted intracellular delivery of molecular cargo into single-cells and tissue ex-vivo (Conference Presentation)

2019 ◽  
Author(s):  
Andrew Doppenberg ◽  
Michel Meunier ◽  
Christos Boutopoulos
Keyword(s):  
Ex Vivo ◽  
Single Cells ◽  
Intracellular Delivery

scholarly journals A small molecule screen identified N-Acetyl-L-cysteine for promoting ex vivo growth of single circulating tumor cells

2020 ◽  
Author(s):  
Teng Teng ◽  
Mohamed Kamal ◽  
Oihana Iriondo ◽  
Yonatan Amzaleg ◽  
Chunqiao Luo ◽  
...  
Keyword(s):  
Tumor Cells ◽  
Circulating Tumor Cells ◽  
Small Molecule ◽  
Ex Vivo ◽  
Single Cells ◽  
Small Subset ◽  
Blood Draw ◽  
Small Molecule Screen ◽  
Functional Analyses ◽  
Single Ctcs

AbstractCirculating tumor cells (CTCs) can be isolated via a minimally invasive blood draw and are considered a “liquid biopsy” of their originating solid tumors. CTCs contain a small subset of metastatic precursors that can form metastases in secondary organs, and provide a resource to identify mechanisms underlying metastasis-initiating properties. Despite technological advancements that allow for highly sensitive approaches of detection and isolation, CTCs are very rare and often present as single cells, posing an extreme challenge for ex vivo expansion after isolation. Here, using previously established patient-derived CTC lines, we performed a small molecule drug screening to identify compounds that can improve ex vivo culture efficiency for single CTCs. We found that N-acetylcysteine (NAC) and other antioxidants can promote ex vivo expansion of single CTCs, by reducing oxidative and other stress particularly at the initial stage of single cell expansion. RNA-seq analysis of growing clones and non-growing clones confirmed the effect by NAC, but also indicate that NAC-induced decrease in oxidative stress is insufficient for promoting proliferation of a subset of cells with heterogeneous quiescent and senescent features. Despite the challenge in expanding all CTCs, NAC treatment lead to establishment of single CTC clones that have similar tumorigenic features, which will facilitate future functional analyses.

scholarly journals On-Demand Intracellular Delivery of Single Particles in Single Cells by 3D Hollow Nanoelectrodes

Nano Letters ◽  
2019 ◽  
Vol 19 (2) ◽  
pp. 722-731 ◽  
Author(s):  
Jian-An Huang ◽  
Valeria Caprettini ◽  
Yingqi Zhao ◽  
Giovanni Melle ◽  
Nicolò Maccaferri ◽  
...  
Keyword(s):  
Single Cells ◽  
Intracellular Delivery ◽  
Single Particles ◽  
On Demand

scholarly journals Gonadotrope Plasticity at Cellular and Population Levels

Endocrinology ◽  
2012 ◽  
Vol 153 (10) ◽  
pp. 4729-4739 ◽  
Author(s):  
Zahara Alim ◽  
Cheryl Hartshorn ◽  
Oliver Mai ◽  
Iain Stitt ◽  
Colin Clay ◽  
...  
Keyword(s):  
Pituitary Gland ◽  
Anterior Pituitary ◽  
Ex Vivo ◽  
Single Cells ◽  
Anterior Pituitary Gland ◽  
Female Mice ◽  
Model Animal ◽  
Pituitary Glands ◽  
Acute Changes

Abstract Hormone-secreting cells within the anterior pituitary gland may form organized and interdigitated networks that adapt to changing endocrine conditions in different physiological contexts. For gonadotropes, this might reflect a strategy to cope with acute changes throughout different female reproductive stages. The current study examined gonadotropes in female mice at characteristically different hormonal stages: prepubertal, postpubertal, and lactating. Gonadotrope plasticity was examined at the level of the whole population and single cells at different stages by imaging both fixed and live pituitary slices. The use of a model animal providing for the identification of selectively fluorescent gonadotropes allowed the particular advantage of defining cellular plasticity specifically for gonadotropes. In vivo analyses of gonadotropes relative to vasculature showed significantly different gonadotrope distributions across physiological states. Video microscopy studies using live slices ex vivo demonstrated pituitary cell plasticity in the form of movements and protrusions in response to GnRH. As positive feedback from rising estradiol levels is important for priming the anterior pituitary gland for the LH surge, experiments provide evidence of estradiol effects on GnRH signaling in gonadotropes. The experiments presented herein provide new insight into potential plasticity of gonadotropes within the anterior pituitary glands of female mice.

scholarly journals Two-Dimensional Modeling of Nanomechanical Strains in Healthy and Diseased Single-Cells During Microfluidic Stress Applications

2010 ◽  
Vol 1 (2) ◽  
Author(s):  
Zachary D. Wilson ◽  
Sean S. Kohles
Keyword(s):  
Ex Vivo ◽  
Single Cells ◽  
Extensional Flow ◽  
Molecular Engineering ◽  
Creeping Flow ◽  
Recent Analysis ◽  
Two Dimensional ◽  
Monitoring And Control ◽  
The Impact

Investigations in cellular and molecular engineering have explored the impact of nanotechnology and the potential for monitoring and control of human diseases. In a recent analysis, the dynamic fluid-induced stresses were characterized during microfluidic applications of an instrument with nanometer and picoNewton resolution as developed for single-cell biomechanics (Kohles, S. S., Nève, N., Zimmerman, J. D., and Tretheway, D. C., 2009, “Stress Analysis of Microfluidic Environments Designed for Isolated Biological Cell Investigations,” ASME J. Biomech. Eng., 131(12), p. 121006). The results described the limited stress levels available in laminar, creeping-flow environments, as well as the qualitative cellular strain response to such stress applications. In this study, we present a two-dimensional computational model exploring the physical application of normal and shear stress profiles (with 0.1, 1.0, and 10.0 Pa peak amplitudes) potentially available within uniform and extensional flow states. The corresponding cellular strains and strain patterns were determined within cells modeled with healthy and diseased mechanical properties (5.0–0.1 kPa moduli, respectively). Strain energy density results integrated over the volume of the planar section indicated a strong mechanical sensitivity involving cells with disease-like properties. In addition, ex vivo microfluidic environments creating in vivo stress states would require freestream flow velocities of 2–7 mm/s. Knowledge of the nanomechanical stresses-strains necessary to illicit a biologic response in the cytoskeleton and cellular membrane will ultimately lead to refined mechanotransduction relationships.

scholarly journals From cells to tissue: How cell scale heterogeneity impacts glioblastoma growth and treatment response

2019 ◽  
Author(s):  
Jill A. Gallaher ◽  
Susan C. Massey ◽  
Andrea Hawkins-Daarud ◽  
Sonal S. Noticewala ◽  
Russell C. Rockne ◽  
...  
Keyword(s):  
Growth Factor ◽  
Cell Tracking ◽  
Ex Vivo ◽  
Wide Spectrum ◽  
Single Cells ◽  
Intratumor Heterogeneity ◽  
Model Parameters ◽  
Mathematical Framework ◽  
Migration Behavior ◽  
Wide Range

AbstractGlioblastomas are aggressive primary brain tumors known for their inter- and intratumor heterogeneity. This disease is uniformly fatal, with intratumor heterogeneity the major reason for treatment failure and recurrence. Just like the nature vs nurture debate, heterogeneity can arise from heritable or environmental influences. Whilst it is impossible to clinically separate observed behavior of cells from their environmental context, using a mathematical framework combined with multiscale data gives us insight into the relative roles of variation from inherited and environmental sources.To better understand the implications of intratumor heterogeneity on therapeutic outcomes, we created a hybrid agent-based mathematical model that captures both the overall tumor kinetics and the individual cellular behavior. We track single cells as agents, cell density on a coarser scale, and growth factor diffusion and dynamics on a finer scale over time and space. Our model parameters were fit utilizing serial MRI imaging and cell tracking data from ex vivo tissue slices acquired from a growth-factor driven glioblastoma murine model.When fitting our model to serial imaging only, there was a spectrum of equally-good parameter fits corresponding to a wide range of phenotypic behaviors. This wide spectrum of in silico tumors also had a wide variety of responses to an application of an antiproliferative treatment. Recurrent tumors were generally less proliferative than pre-treatment tumors as measured via the model simulations and validated from human GBM patient histology. When fitting our model using imaging and cell scale data, we determined that heritable heterogeneity is required to capture the observed migration behavior. Further, we found that all tumors increased in size after an anti-migratory treatment, and some tumors were larger after a combination treatment than with an anti-proliferative treatment alone. Together our results emphasize the need to understand the underlying phenotypes and tumor heterogeneity in designing therapeutic regimens.

scholarly journals Live Imaging, Identifying, and Tracking Single Cells in Complex Populations In Vivo and Ex Vivo

2013 ◽  
pp. 109-123 ◽  
Author(s):  
Minjung Kang ◽  
Panagiotis Xenopoulos ◽  
Silvia Muñoz-Descalzo ◽  
Xinghua Lou ◽  
Anna-Katerina Hadjantonakis
Keyword(s):  
Ex Vivo ◽  
Single Cells ◽  
Live Imaging

scholarly journals Biomechanics of Ex Vivo-Generated Red Blood Cells Investigated by Optical Tweezers and Digital Holographic Microscopy

Cells ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 552
Author(s):  
Claudia Bernecker ◽  
Maria Augusta R. B. F. Lima ◽  
Catalin D. Ciubotaru ◽  
Peter Schlenke ◽  
Isabel Dorn ◽  
...  
Keyword(s):  
Red Blood Cells ◽  
Optical Tweezers ◽  
Blood Cells ◽  
Ex Vivo ◽  
Single Cells ◽  
Membrane Lipid ◽  
Membrane Properties ◽  
Digital Holographic Microscopy ◽  
Holographic Microscopy

Ex vivo-generated red blood cells are a promising resource for future safe blood products, manufactured independently of voluntary blood donations. The physiological process of terminal maturation from spheroid reticulocytes to biconcave erythrocytes has not been accomplished yet. A better biomechanical characterization of cultured red blood cells (cRBCs) will be of utmost interest for manufacturer approval and therapeutic application. Here, we introduce a novel optical tweezer (OT) approach to measure the deformation and elasticity of single cells trapped away from the coverslip. To investigate membrane properties dependent on membrane lipid content, two culture conditions of cRBCs were investigated, cRBCPlasma with plasma and cRBCHPL supplemented with human platelet lysate. Biomechanical characterization of cells under optical forces proves the similar features of native RBCs and cRBCHPL, and different characteristics for cRBCPlasma. To confirm these results, we also applied a second technique, digital holographic microscopy (DHM), for cells laid on the surface. OT and DHM provided related results in terms of cell deformation and membrane fluctuations, allowing a reliable discrimination between cultured and native red blood cells. The two techniques are compared and discussed in terms of application and complementarity.

scholarly journals An event-based paradigm for analyzing fluorescent astrocyte activity uncovers novel single-cell and population-level physiology

2018 ◽  
Author(s):  
Yizhi Wang ◽  
Nicole V. DelRosso ◽  
Trisha Vaidyanathan ◽  
Michael Reitman ◽  
Michelle K. Cahill ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Single Cells ◽  
Region Of Interest ◽  
Population Level ◽  
Analytical Framework ◽  
Model Organisms ◽  
Imaging Data ◽  
Fluorescent Indicators ◽  
Event Based

AbstractRecent work examining astrocytic physiology centers on fluorescence imaging approaches, due to development of sensitive fluorescent indicators and observation of spatiotemporally complex calcium and glutamate activity. However, the field remains hindered in fully characterizing these dynamics, both within single cells and at the population-level, because of the insufficiency of current region-of-interest-based approaches to describe activity that is often spatially unfixed, size-varying, and propagative. Here, we present a paradigm-shifting analytical framework that releases astrocyte biologists from ROI-based tools. Astrocyte Quantitative Analysis (AQuA) software enables users to take an event-based approach to accurately capture and quantify the irregular activity observed in astrocyte imaging datasets. We apply AQuA to a range of ex vivo and in vivo imaging data, and uncover previously undescribed physiological phenomena in each. Since AQuA is data-driven and based on machine learning principles, it can be applied across model organisms, fluorescent indicators, experimental modes, and imaging resolutions and speeds, enabling researchers to elucidate fundamental astrocyte physiology.

scholarly journals Expression profiling of single cells and patient cohorts identifies multiple immunosuppressive pathways and an altered NK cell phenotype in glioblastoma

2019 ◽  
Author(s):  
Helen J. Close ◽  
Lucy F. Stead ◽  
Jérémie Nsengimana ◽  
Katrina A. Reilly ◽  
Alastair Droop ◽  
...  
Keyword(s):  
Nk Cells ◽  
Expression Profiling ◽  
Neural Progenitor Cells ◽  
Nk Cell ◽  
Ex Vivo ◽  
Single Cells ◽  
Tumour Microenvironment ◽  
Cell Phenotype ◽  
Tumour Immunity ◽  
Gene And Protein Expression

AbstractGlioblastoma (GBM) is an aggressive cancer with a very poor prognosis. Generally viewed as weakly immunogenic, GBM responds poorly to current immunotherapies. To better understand this problem we used a combination of NK cell functional assays together with gene and protein expression profiling to define the NK cell response to GBM and explore immunosuppression in the GBM microenvironment. In addition, we used transcriptome data from patient cohorts to classify GBM according to immunological profiles. We show that glioma stem-like cells, a source of post-treatment tumour recurrence, express multiple immunomodulatory cell surface molecules and are targeted in preference to normal neural progenitor cells by natural killer (NK) cells ex vivo. In contrast, GBM-infiltrating NK cells express reduced levels of activation receptors within the tumour microenvironment, with hallmarks of TGF-β mediated inhibition. This NK cell inhibition is accompanied by expression of mutiple immune checkpoint molecules on T cells. Single cell transcriptomics demonstrated that both tumour and haematopoietic-derived cells in GBM express multiple, diverse mediators of immune evasion. Despite this, immunome analysis across a patient cohort identifies a spectrum of immunological activity in GBM, with active immunity marked by co-expression of immune effector molecules and feedback inhibitory mechanisms. Our data show that GBM is recognised by the immune system but that anti-tumour immunity is restrained by multiple immunosuppressive pathways, some of which operate in the healthy brain. The presence of immune activity in a subset of patients suggests that these patients will more likely benefit from combination immunotherapies directed against multiple immunosuppressive pathways.

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