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 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 Fragile X syndrome patient-derived neurons developing in the mouse brain show FMR1 -dependent phenotypes

2021 ◽  
Author(s):  
Marine A Krzisch ◽  
Hao A Wu ◽  
Bingbing Yuan ◽  
Troy W. Whitfield ◽  
X. Shawn Liu ◽  
...  
Keyword(s):  
Fragile X Syndrome ◽  
Neuronal Development ◽  
Fragile X ◽  
In Vitro Models ◽  
Synaptic Activity ◽  
Human Neurons ◽  
Induced Pluripotent ◽  
And Function ◽  
The Brain

Abnormal neuronal development in Fragile X syndrome (FXS) is poorly understood. Data on FXS patients remain scarce and FXS animal models have failed to yield successful therapies. In vitro models do not fully recapitulate the morphology and function of human neurons. Here, we co-injected neural precursor cells (NPCs) from FXS patient-derived and corrected isogenic control induced pluripotent stem cells into the brain of neonatal immune-deprived mice. The transplanted cells populated the brain and a proportion differentiated into neurons and glial cells. Single-cell RNA sequencing of transplanted cells revealed upregulated excitatory synaptic transmission and neuronal differentiation pathways in FXS neurons. Immunofluorescence analyses showed accelerated maturation of FXS neurons after an initial delay. Additionally, increased percentages of Arc- and Egr1-positive FXS neurons and wider dendritic protrusions of mature FXS striatal medium spiny neurons pointed to an increase in synaptic activity and synaptic strength as compared to control. This transplantation approach provides new insights into the alterations of neuronal development in FXS by facilitating physiological development of cells in a 3D context, and could be used to test new therapeutic compounds correcting neuronal development defects in FXS.

scholarly journals Advances in modelling the human microbiome–gut–brain axis in vitro

2021 ◽  
Author(s):  
Chrysanthi-Maria Moysidou ◽  
Róisín M. Owens
Keyword(s):  
Animal Models ◽  
Cell Biology ◽  
Materials Science ◽  
Human Microbiome ◽  
Paradigm Shifts ◽  
Bidirectional Communication ◽  
Wide Range ◽  
And Function

The human gut microbiome has emerged as a key player in the bidirectional communication of the gut–brain axis, affecting various aspects of homeostasis and pathophysiology. Until recently, the majority of studies that seek to explore the mechanisms underlying the microbiome–gut–brain axis cross-talk, relied almost exclusively on animal models, and particularly gnotobiotic mice. Despite the great progress made with these models, various limitations, including ethical considerations and interspecies differences that limit the translatability of data to human systems, pushed researchers to seek for alternatives. Over the past decades, the field of in vitro modelling of tissues has experienced tremendous growth, thanks to advances in 3D cell biology, materials, science and bioengineering, pushing further the borders of our ability to more faithfully emulate the in vivo situation. The discovery of stem cells has offered a new source of cells, while their use in generating gastrointestinal and brain organoids, among other tissues, has enabled the development of novel 3D tissues that better mimic the native tissue structure and function, compared with traditional assays. In parallel, organs-on-chips technology and bioengineered tissues have emerged as highly promising alternatives to animal models for a wide range of applications. Here, we discuss how recent advances and trends in this area can be applied in host–microbe and host–pathogen interaction studies. In addition, we highlight paradigm shifts in engineering more robust human microbiome-gut-brain axis models and their potential to expand our understanding of this complex system and hence explore novel, microbiome-based therapeutic approaches.

Human-mouse chimeric Fragile X syndrome model reveals FMR1-dependent neuronal phenotypes

2020 ◽  
Author(s):  
Marine Krzisch ◽  
Hao Wu ◽  
Bingbing Yuan ◽  
Troy Whitfield ◽  
Shawn Liu ◽  
...  
Keyword(s):  
Fragile X Syndrome ◽  
Neuronal Development ◽  
Fragile X ◽  
In Vitro Models ◽  
Synaptic Activity ◽  
Human Neurons ◽  
Induced Pluripotent ◽  
And Function ◽  
The Brain

Abstract Abnormal neuronal development in Fragile X syndrome (FXS) is poorly understood. Data on FXS patients remain scarce and FXS animal models have failed to yield successful therapies. In vitro models do not fully recapitulate the morphology and function of human neurons. Here, we co-injected neural precursor cells (NPCs) from FXS patient-derived and corrected isogenic control induced pluripotent stem cells into the brain of neonatal immune-deprived mice. The cells populated the brain and differentiated into neurons and astrocytes. Single-cell RNA sequencing of transplanted cells revealed upregulated excitatory synaptic transmission and neuronal differentiation pathways in FXS neurons. Immunofluorescence analyses showed accelerated maturation of FXS neurons, an increased proportion of Arc-positive FXS neurons and increased dendritic protrusion width of FXS striatal medium spiny neurons. Our data show faster maturation and suggest increased synaptic activity and synaptic strength of FXS transplanted neurons. This model provides new insights into the alterations in FXS neuronal development.

scholarly journals Membrane-associated phase separation: organization and function emerge from a two-dimensional milieu

2021 ◽  
Author(s):  
Jonathon A Ditlev
Keyword(s):  
Phase Separation ◽  
Cell Biology ◽  
Cellular Environment ◽  
Condensate Formation ◽  
Associated Proteins ◽  
Available Technology ◽  
And Function ◽  
Surrounding Membrane

Abstract Liquid‒liquid phase separation (LLPS) of biomolecules has emerged as an important mechanism that contributes to cellular organization. Phase separated biomolecular condensates, or membrane-less organelles, are compartments composed of specific biomolecules without a surrounding membrane in the nucleus and cytoplasm. LLPS also occurs at membranes, where both lipids and membrane-associated proteins can de-mix to form phase separated compartments. Investigation of these membrane-associated condensates using in vitro biochemical reconstitution and cell biology has provided key insights into the role of phase separation in membrane domain formation and function. However, these studies have generally been limited by available technology to study LLPS on model membranes and the complex cellular environment that regulates condensate formation, composition, and function. Here, I briefly review our current understanding of membrane-associated condensates, establish why LLPS can be advantageous for certain membrane-associated condensates, and offer a perspective for how these condensates may be studied in the future.

scholarly journals Activation of the receptor tyrosine kinase RET improves long-term hematopoietic stem cell outgrowth and potency

Blood ◽  
2020 ◽  
Vol 136 (22) ◽  
pp. 2535-2547 ◽  
Author(s):  
W. Grey ◽  
R. Chauhan ◽  
M. Piganeau ◽  
H. Huerga Encabo ◽  
M. Garcia-Albornoz ◽  
...  
Keyword(s):  
Intracellular Signaling ◽  
Culture Conditions ◽  
Cellular Growth ◽  
Great Promise ◽  
Bone Marrow Niche ◽  
Hematopoietic Stem ◽  
Intracellular Signaling Pathway ◽  
Wide Range

Abstract Expansion of human hematopoietic stem cells (HSCs) is a rapidly advancing field showing great promise for clinical applications. Recent evidence has implicated the nervous system and glial family ligands (GFLs) as potential drivers of hematopoietic survival and self-renewal in the bone marrow niche; how to apply this process to HSC maintenance and expansion has yet to be explored. We show a role for the GFL receptor, RET, at the cell surface of HSCs in mediating sustained cellular growth, resistance to stress, and improved cell survival throughout in vitro expansion. HSCs treated with the key RET ligand/coreceptor complex, glial-derived neurotrophic factor and its coreceptor, exhibit improved progenitor function at primary transplantation and improved long-term HSC function at secondary transplantation. Finally, we show that RET drives a multifaceted intracellular signaling pathway, including key signaling intermediates protein kinase B, extracellular signal-regulated kinase 1/2, NF-κB, and p53, responsible for a wide range of cellular and genetic responses that improve cell growth and survival under culture conditions.

Biophysical stimulation for in vitro engineering of functional cardiac tissues

2017 ◽  
Vol 131 (13) ◽  
pp. 1393-1404 ◽  
Author(s):  
Anastasia Korolj ◽  
Erika Yan Wang ◽  
Robert A. Civitarese ◽  
Milica Radisic
Keyword(s):  
Cardiac Tissue ◽  
Culture Media ◽  
Culture Conditions ◽  
Lineage Specification ◽  
Cardiac Tissues ◽  
Cardiac Lineage ◽  
And Function ◽  
Tissue Models

Engineering functional cardiac tissues remains an ongoing significant challenge due to the complexity of the native environment. However, our growing understanding of key parameters of the in vivo cardiac microenvironment and our ability to replicate those parameters in vitro are resulting in the development of increasingly sophisticated models of engineered cardiac tissues (ECT). This review examines some of the most relevant parameters that may be applied in culture leading to higher fidelity cardiac tissue models. These include the biochemical composition of culture media and cardiac lineage specification, co-culture conditions, electrical and mechanical stimulation, and the application of hydrogels, various biomaterials, and scaffolds. The review will also summarize some of the recent functional human tissue models that have been developed for in vivo and in vitro applications. Ultimately, the creation of sophisticated ECT that replicate native structure and function will be instrumental in advancing cell-based therapeutics and in providing advanced models for drug discovery and testing.

scholarly journals hPSC-Derived Enteric Ganglioids Model Human ENS Development and Function

2022 ◽  
Author(s):  
Homa Majd ◽  
Ryan M Samuel ◽  
Jonathan T Ramirez ◽  
Ali Kalantari ◽  
Kevin Barber ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Xenograft Model ◽  
Developmental Relationships ◽  
Drug Candidates ◽  
Effective Strategies ◽  
Wide Range ◽  
Functional Features ◽  
And Function

The enteric nervous system (ENS) plays a central role in gut physiology and mediating the crosstalk between the gastrointestinal (GI) tract and other organs. The human ENS has remained elusive, highlighting the need for an in vitro modeling and mapping blueprint. Here we map out the developmental and functional features of the human ENS, by establishing robust and scalable 2D ENS cultures and 3D enteric ganglioids from human pluripotent stem cells (hPSCs). These models recapitulate the remarkable neuronal and glial diversity found in primary tissue and enable comprehensive molecular analyses that uncover functional and developmental relationships within these lineages. As a salient example of the power of this system, we performed in-depth characterization of enteric nitrergic neurons (NO neurons) which are implicated in a wide range of GI motility disorders. We conducted an unbiased screen and identified drug candidates that modulate the activity of NO neurons and demonstrated their potential in promoting motility in mouse colonic tissue ex vivo. We established a high-throughput strategy to define the developmental programs involved in NO neuron specification and discovered that PDGFR inhibition boosts the induction of NO neurons in enteric ganglioids. Transplantation of these ganglioids in the colon of NO neuron-deficient mice results in extensive tissue engraftment, providing a xenograft model for the study of human ENS in vivo and the development of cell-based therapies for neurodegenerative GI disorders. These studies provide a framework for deciphering fundamental features of the human ENS and designing effective strategies to treat enteric neuropathies.  

scholarly journals The cardiac nanoenvironment: form and function at the nanoscale

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.

scholarly journals Quantitative analysis of tumour spheroid structure

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Alexander P Browning ◽  
Jesse A Sharp ◽  
Ryan J Murphy ◽  
Gency Gunasingh ◽  
Brodie Lawson ◽  
...  
Keyword(s):  
Cell Lines ◽  
Tumour Microenvironment ◽  
Culture Conditions ◽  
Experimental Models ◽  
Seeding Density ◽  
Modelling Framework ◽  
Tumour Spheroid ◽  
Tumour Spheroids ◽  
Wide Range

Tumour spheroids are common in vitro experimental models of avascular tumour growth. Compared with traditional two-dimensional culture, tumour spheroids more closely mimic the avascular tumour microenvironment where spatial differences in nutrient availability strongly influence growth. We show that spheroids initiated using significantly different numbers of cells grow to similar limiting sizes, suggesting that avascular tumours have a limiting structure; in agreement with untested predictions of classical mathematical models of tumour spheroids. We develop a novel mathematical and statistical framework to study the structure of tumour spheroids seeded from cells transduced with fluorescent cell cycle indicators, enabling us to discriminate between arrested and cycling cells and identify an arrested region. Our analysis shows that transient spheroid structure is independent of initial spheroid size, and the limiting structure can be independent of seeding density. Standard experimental protocols compare spheroid size as a function of time; however, our analysis suggests that comparing spheroid structure as a function of overall size produces results that are relatively insensitive to variability in spheroid size. Our experimental observations are made using two melanoma cell lines, but our modelling framework applies across a wide range of spheroid culture conditions and cell lines.

Sign in / Sign up

Export Citation Format

Share Document