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1.
Nat Commun ; 11(1): 5104, 2020 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-33037214

RESUMO

Many intestinal pathogens, including Clostridioides difficile, use mucus-derived sugars as crucial nutrients in the gut. Commensals that compete with pathogens for such nutrients are therefore ecological gatekeepers in healthy guts, and are attractive candidates for therapeutic interventions. Nevertheless, there is a poor understanding of which commensals use mucin-derived sugars in situ as well as their potential to impede pathogen colonization. Here, we identify mouse gut commensals that utilize mucus-derived monosaccharides within complex communities using single-cell stable isotope probing, Raman-activated cell sorting and mini-metagenomics. Sequencing of cell-sorted fractions reveals members of the underexplored family Muribaculaceae as major mucin monosaccharide foragers, followed by members of Lachnospiraceae, Rikenellaceae, and Bacteroidaceae families. Using this information, we assembled a five-member consortium of sialic acid and N-acetylglucosamine utilizers that impedes C. difficile's access to these mucosal sugars and impairs pathogen colonization in antibiotic-treated mice. Our findings underscore the value of targeted approaches to identify organisms utilizing key nutrients and to rationally design effective probiotic mixtures.


Assuntos
Clostridium difficile/patogenicidade , Microbioma Gastrointestinal/fisiologia , Monossacarídeos/metabolismo , Acetilglucosamina/metabolismo , Animais , Antibacterianos , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/metabolismo , Separação Celular/métodos , Infecções por Clostridium/microbiologia , Clostridium difficile/genética , Clostridium difficile/crescimento & desenvolvimento , Deutério , Feminino , Mucinas Gástricas/química , Mucinas Gástricas/metabolismo , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/microbiologia , Metagenoma , Camundongos Endogâmicos C57BL , Ácido N-Acetilneuramínico/metabolismo , Polissacarídeos/química , Polissacarídeos/metabolismo , Análise Espectral Raman
2.
PLoS One ; 15(9): e0239593, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32970754

RESUMO

The method for increasing the separation efficiency of particles smaller than 2.5 micrometers by combined ultrasonic agglomeration and swirling flow technique is proposed in the article. The swirling flow creates areas with an increased concentration of particles on the outer radius of the vortex. The ultrasonic exposure on these areas leads to more efficient agglomeration and the formation of agglomerates of many times larger than the original particles. The resulting agglomerates are easily separated from the gas flow. The design of the agglomerator was developed. The vortex velocity is determined, at which ultrasonic exposure on the swirling flow increases the average particle size d32 = 2.5 micrometer to 4.5 times. The ultrasonic exposure on a rectilinear flow can increase the particle size no more than 1.6 times for comparison. The proposed method is compared with inertial gas clearing in a cyclone. It was found that the proposed combined method allows increasing the cleaning efficiency from 46% to 85% at ultrasonic exposure on the swirling flow in the agglomerator and cyclone.


Assuntos
Hidrodinâmica , Nanopartículas/química , Ultrassom/métodos , Aerossóis/metabolismo , Separação Celular/métodos , Tamanho da Partícula , Fenômenos Físicos
3.
J Vis Exp ; (162)2020 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-32865529

RESUMO

The stromal-vascular fraction (SVF) of white adipose tissue (WAT) is remarkably heterogeneous and consists of numerous cell types that contribute functionally to the expansion and remodeling of WAT in adulthood. A tremendous barrier to studying the implications of this cellular heterogeneity is the inability to readily isolate functionally distinct cell subpopulations from WAT SVF for in vitro and in vivo analyses. Single-cell sequencing technology has recently identified functionally distinct fibro-inflammatory and adipogenic PDGFRß+ perivascular cell subpopulations in intra-abdominal WAT depots of adult mice. Fibro-inflammatory progenitors (termed, "FIPs") are non-adipogenic collagen producing cells that can exert a pro-inflammatory phenotype. PDGFRß+ adipocyte precursor cells (APCs) are highly adipogenic both in vitro and in vivo upon cell transplantation. Here, we describe multiple methods for the isolation of these stromal cell subpopulations from murine intra-abdominal WAT depots. FIPs and APCs can be isolated by fluorescence-activated cell sorting (FACS) or by taking advantage of biotinylated antibody-based immunomagnetic bead technology. Isolated cells can be used for molecular and functional analysis. Studying the functional properties of stromal cell subpopulation in isolation will expand our current knowledge of adipose tissue remodeling under physiological or pathological conditions on the cellular level.


Assuntos
Gordura Abdominal/citologia , Adipogenia , Separação Celular/métodos , Células Estromais/citologia , Tecido Adiposo Branco/citologia , Animais , Citometria de Fluxo , Inflamação/patologia , Camundongos , Células Estromais/patologia
4.
Adv Exp Med Biol ; 1255: 7-27, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32949387

RESUMO

Within the last decade, single-cell analysis has revolutionized our understanding of cellular processes and heterogeneity across all disciplines of life science. As the transcriptome, genome, or epigenome of individual cells can nowadays be analyzed at low cost and in high-throughput within a few days by modern techniques, tremendous improvements in disease diagnosis on the one hand and the investigation of disease-relevant mechanisms on the other were achieved so far. This relies on the parallel development of reliable cell capturing and single-cell sequencing approaches that have paved the way for comprehensive single-cell studies. Apart from single-cell isolation methods in high-throughput, a variety of methods with distinct specializations were developed, allowing for correlation of transcriptomics with cellular parameters like electrophysiology or morphology.For all single-cell-based approaches, accurate and reliable isolation with proper quality controls is prerequisite, whereby different options exist dependent on sample type and tissue properties. Careful consideration of an appropriate method is required to avoid incorrect or biased data that may lead to misinterpretations.In this chapter, we will provide a broad overview of the current state of the art in matters of single-cell isolation methods mostly applied for sequencing-based downstream analysis, and their respective advantages and drawbacks. Distinct technologies will be discussed in detail addressing key parameters like sample compatibility, viability, purity, throughput, and isolation efficiency.


Assuntos
Separação Celular/métodos , Análise de Célula Única/métodos , Animais , Genoma , Humanos , Transcriptoma
5.
PLoS One ; 15(8): e0237748, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32866195

RESUMO

Soil microbiota are considered a source of undiscovered bioactive compounds, yet cultivation of most bacteria within a sample remains generally unsuccessful. Two main reasons behind the unculturability of bacteria are the presence of cells in a viable but not culturable state (such as dormant cells) and the failure to provide the necessary growth requirements in vitro (leading to the classification of some bacterial taxa as yet-to-be-cultured). The present work focuses on the development of a single procedure that helps distinguish between both phenomena of unculturability based on viability staining coupled with flow cytometry and fluorescence-activated cell sorting. In the selected soil sample, the success rate of cultured bacteria was doubled by selecting viable and metabolically active bacteria. It was determined that most of the uncultured fraction was not dormant or dead but likely required different growth conditions. It was also determined that the staining process introduced changes in the taxonomic composition of the outgrown bacterial biomass, which should be considered for further developments. This research shows the potential of flow cytometry and fluorescence-activated cell sorting applied to soil samples to improve the success rate of bacterial cultivation by estimating the proportion of dormant and yet-to-be-cultured bacteria and by directly excluding dormant cells from being inoculated into growth media.


Assuntos
Bactérias/crescimento & desenvolvimento , Técnicas Microbiológicas/métodos , Microbiota/fisiologia , Microbiologia do Solo , Bactérias/química , Bactérias/genética , Biomassa , Separação Celular/métodos , Meios de Cultura , DNA Bacteriano/isolamento & purificação , Estudos de Viabilidade , Citometria de Fluxo/métodos , Corantes Fluorescentes/química , Filogenia , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Coloração e Rotulagem/métodos
6.
PLoS One ; 15(8): e0237308, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32790691

RESUMO

The isolation and analysis of circulating tumor cells (CTC) has the potential to provide minimally invasive diagnostic, prognostic and predictive information. Widespread clinical implementation of CTC analysis has been hampered by a lack of comparative investigation between different analytic methodologies in clinically relevant settings. The objective of this study was to evaluate four different CTC isolation techniques-those that rely on surface antigen expression (EpCAM or CD45 using DynaBeads® or EasySep™ systems) or the biophysical properties (RosetteSep™ or ScreenCell®) of CTCs. These were evaluated using cultured cells in order to calculate isolation efficiency at various levels including; inter-assay and inter-operator variability, protocol complexity and turn-around time. All four techniques were adequate at levels above 100 cells/mL which is commonly used for the evaluation of new isolation techniques. Only the RosetteSep™ and ScreenCell® techniques were found to provide adequate sensitivity at a level of 10 cells/mL. These techniques were then applied to the isolation and analysis of circulating tumor cells blood drawn from metastatic breast cancer patients where CTCs were detected in 54% (15/28) of MBC patients using the RosetteSep™ and 75% (6/8) with ScreenCell®. Overall, the ScreenCell® method had better sensitivity.


Assuntos
Neoplasias da Mama/secundário , Separação Celular/métodos , Células Neoplásicas Circulantes/patologia , Adulto , Idoso , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Molécula de Adesão da Célula Epitelial/análise , Feminino , Humanos , Antígenos Comuns de Leucócito/análise , Pessoa de Meia-Idade
7.
J Vis Exp ; (161)2020 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-32773760

RESUMO

Epithelial organoid models serve as valuable tools to study the basic biology of an organ system and for disease modeling. When grown as organoids, epithelial progenitor cells can self-renew and generate differentiating progeny that exhibit cellular functions similar to those of their in vivo counterparts. Herein we describe a step-by-step protocol to isolate region-specific progenitors from human lung and generate 3D organoid cultures as an experimental and validation tool. We define proximal and distal regions of the lung with the goal of isolating region-specific progenitor cells. We utilized a combination of enzymatic and mechanical dissociation to isolate total cells from the lung and trachea. Specific progenitor cells were then fractionated from the proximal or distal origin cells using fluorescence associated cell sorting (FACS) based on cell type-specific surface markers, such as NGFR for sorting basal cells and HTII-280 for sorting alveolar type II cells. Isolated basal or alveolar type II progenitors were used to generate 3D organoid cultures. Both distal and proximal progenitors formed organoids with a colony forming efficiency of 9-13% in distal region and 7-10% in proximal region when plated 5000 cell/well on day 30. Distal organoids maintained HTII-280+ alveolar type II cells in culture whereas proximal organoids differentiated into ciliated and secretory cells by day 30. These 3D organoid cultures can be used as an experimental tool for studying the cell biology of lung epithelium and epithelial mesenchymal interactions, as well as for the development and validation of therapeutic strategies targeting epithelial dysfunction in a disease.


Assuntos
Técnicas de Cultura de Células , Separação Celular/métodos , Células Epiteliais/citologia , Pulmão/citologia , Organoides/citologia , Células-Tronco/citologia , Diferenciação Celular , Fracionamento Celular , Humanos , Organoides/metabolismo , Coloração e Rotulagem
8.
J Vis Exp ; (162)2020 08 08.
Artigo em Inglês | MEDLINE | ID: mdl-32831316

RESUMO

Chick ciliary ganglia (CG) are part of the parasympathetic nervous system and are responsible for the innervation of the muscle tissues present in the eye. This ganglion is constituted by a homogenous population of ciliary and choroidal neurons that innervate striated and smooth muscle fibers, respectively. Each of these neuronal types regulate specific eye structures and functions. Over the years, neuronal cultures of the chick ciliary ganglia were shown to be effective cell models in the study of muscle-nervous system interactions, which communicate through cholinergic synapses. Ciliary ganglion neurons are, in its majority, cholinergic. This cell model has been shown to be useful comparatively to previously used heterogeneous cell models that comprise several neuronal types, besides cholinergic. Anatomically, the ciliary ganglion is localized between the optic nerve (ON) and the choroid fissure (CF). Here, we describe a detailed procedure for the dissection, dissociation and in vitro culture of ciliary ganglia neurons from chick embryos. We provide a step-by-step protocol in order to obtain highly pure and stable cellular cultures of CG neurons, highlighting key steps of the process. These cultures can be maintained in vitro for 15 days and, hereby, we show the normal development of CG cultures. The results also show that these neurons can interact with muscle fibers through neuro-muscular cholinergic synapses.


Assuntos
Técnicas de Cultura de Células , Separação Celular/métodos , Gânglios Parassimpáticos/citologia , Neurônios , Animais , Embrião de Galinha , Gânglios Parassimpáticos/metabolismo , Neurônios/fisiologia
9.
J Vis Exp ; (160)2020 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-32628176

RESUMO

We describe the implementation of spinal cord injury in mice to elicit detrusor-sphincter dyssynergia, a functional bladder outlet obstruction, and subsequent bladder wall remodeling. To facilitate assessment of the cellular composition of the bladder wall in non-injured control and spinal cord injured mice, we developed an optimized dissociation protocol that supports high cell viability and enables the detection of discrete subpopulations by flow cytometry. Spinal cord injury is created by complete transection of the thoracic spinal cord. At the time of tissue harvest, the animal is perfused with phosphate-buffered saline under deep anesthesia and bladders are harvested into Tyrode's buffer. Tissues are minced prior to incubation in digestion buffer that has been optimized based on the collagen content of mouse bladder as determined by interrogation of publicly available gene expression databases. Following generation of a single cell suspension, material is analyzed by flow cytometry for assessment of cell viability, cell number and specific subpopulations. We demonstrate that the method yields cell populations with greater than 90% viability, and robust representation of cells of mesenchymal and epithelial origin. This method will enable accurate downstream analysis of discrete cell types in mouse bladder and potentially other organs.


Assuntos
Separação Celular/métodos , Traumatismos da Medula Espinal/patologia , Bexiga Urinária/patologia , Animais , Calibragem , Sobrevivência Celular , Análise de Dados , Matriz Extracelular/metabolismo , Feminino , Citometria de Fluxo , Camundongos , Perfusão , Traumatismos da Medula Espinal/cirurgia , Transcriptoma/genética
10.
Nat Commun ; 11(1): 3452, 2020 07 10.
Artigo em Inglês | MEDLINE | ID: mdl-32651381

RESUMO

The advent of image-activated cell sorting and imaging-based cell picking has advanced our knowledge and exploitation of biological systems in the last decade. Unfortunately, they generally rely on fluorescent labeling for cellular phenotyping, an indirect measure of the molecular landscape in the cell, which has critical limitations. Here we demonstrate Raman image-activated cell sorting by directly probing chemically specific intracellular molecular vibrations via ultrafast multicolor stimulated Raman scattering (SRS) microscopy for cellular phenotyping. Specifically, the technology enables real-time SRS-image-based sorting of single live cells with a throughput of up to ~100 events per second without the need for fluorescent labeling. To show the broad utility of the technology, we show its applicability to diverse cell types and sizes. The technology is highly versatile and holds promise for numerous applications that are previously difficult or undesirable with fluorescence-based technologies.


Assuntos
Separação Celular/métodos , Análise Espectral Raman/métodos , Animais , Humanos
11.
PLoS One ; 15(7): e0229967, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32645012

RESUMO

Phthalic acid esters (phthalates) are male reproductive toxicants, which exert their most potent toxicity during fetal development. In the fetal rat, exposure to phthalates reduces testosterone biosynthesis, alters the development of seminiferous cords and other male reproductive tissues, and induces the formation of abnormal multinucleated germ cells (MNGs). Identification of MNGs is a time-intensive process, and it requires specialized training to identify MNGs in histological sections. As a result, MNGs are not routinely quantified in phthalate toxicity experiments. In order to speed up and standardize this process, we have developed an improved method for automated detection of MNGs. Using hand-labeled histological section images with human-identified MNGs, we trained a convolutional neural network with a U-Net architecture to identify MNGs on unlabeled images. With unseen hand-labeled images not used in model training, we assessed the performance of the model, using five different configurations of the data. On average, the model reached near human accuracy, and in the best model, it exceeded it. The use of automated image analysis will allow data on this histopathological endpoint to be more readily collected for analysis of phthalate toxicity. Our trained model application code is available for download at github.com/brown-ccv/mngcount.


Assuntos
Automação Laboratorial/métodos , Núcleo Celular , Separação Celular/métodos , Células Germinativas/patologia , Processamento de Imagem Assistida por Computador , Animais , Feminino , Masculino , Redes Neurais de Computação , Ácidos Ftálicos/toxicidade , Ratos Sprague-Dawley , Testículo/patologia
12.
Proc Natl Acad Sci U S A ; 117(29): 16839-16847, 2020 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-32641515

RESUMO

Circulating tumor cell (CTC)-based liquid biopsies provide unique opportunities for cancer diagnostics, treatment selection, and response monitoring, but even with advanced microfluidic technologies for rare cell detection the very low number of CTCs in standard 10-mL peripheral blood samples limits their clinical utility. Clinical leukapheresis can concentrate mononuclear cells from almost the entire blood volume, but such large numbers and concentrations of cells are incompatible with current rare cell enrichment technologies. Here, we describe an ultrahigh-throughput microfluidic chip, LPCTC-iChip, that rapidly sorts through an entire leukapheresis product of over 6 billion nucleated cells, increasing CTC isolation capacity by two orders of magnitude (86% recovery with 105 enrichment). Using soft iron-filled channels to act as magnetic microlenses, we intensify the field gradient within sorting channels. Increasing magnetic fields applied to inertially focused streams of cells effectively deplete massive numbers of magnetically labeled leukocytes within microfluidic channels. The negative depletion of antibody-tagged leukocytes enables isolation of potentially viable CTCs without bias for expression of specific tumor epitopes, making this platform applicable to all solid tumors. Thus, the initial enrichment by routine leukapheresis of mononuclear cells from very large blood volumes, followed by rapid flow, high-gradient magnetic sorting of untagged CTCs, provides a technology for noninvasive isolation of cancer cells in sufficient numbers for multiple clinical and experimental applications.


Assuntos
Separação Celular/métodos , Ensaios de Triagem em Larga Escala/métodos , Microfluídica/métodos , Células Neoplásicas Circulantes/classificação , Linhagem Celular Tumoral , Separação Celular/instrumentação , Ensaios de Triagem em Larga Escala/instrumentação , Humanos , Leucaférese/métodos , Campos Magnéticos , Microfluídica/instrumentação
13.
PLoS One ; 15(7): e0234986, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32634135

RESUMO

Glioblastoma is a common, malignant brain tumor whose disease incidence increases with age. Glioblastoma stem-like cells (GSCs) are thought to contribute to cancer therapy resistance and to be responsible for tumor initiation, maintenance, and recurrence. This study utilizes both SNP array and gene expression profiling to better understand GSCs and their relation to malignant disease. Peripheral blood and primary glioblastoma tumor tissue were obtained from patients, the latter of which was used to generate GSCs as well as a CD133pos./CD15pos. subpopulation. The stem cell features of GSCs were confirmed via the immunofluorescent expression of Nestin, SOX2, and CD133. Both tumor tissue and the isolated primary cells shared unique abnormal genomic characteristics, including a gain of chromosome 7 as well as either a partial or complete loss of chromosome 10. Individual genomic differences were also observed, including the loss of chromosome 4 and segmental uniparental disomy of 9p24.3→p21.3 in GSCs. Gene expression profiling revealed 418 genes upregulated in tumor tissue vs. CD133pos./CD15pos. cells and 44 genes upregulated in CD133pos./CD15pos. cells vs. tumor tissue. Pathway analyses demonstrated that upregulated genes in CD133pos./CD15pos. cells are relevant to cell cycle processes and cancerogenesis. In summary, we detected previously undescribed genomic and gene expression differences when comparing tumor tissue and isolated stem-like subpopulations.


Assuntos
Glioblastoma/patologia , Células-Tronco Neoplásicas/patologia , Antígeno AC133/análise , Separação Celular/métodos , Células Cultivadas , Perfilação da Expressão Gênica , Humanos , Antígenos CD15/análise , Polimorfismo de Nucleotídeo Único/genética , Manejo de Espécimes , Regulação para Cima
14.
J Vis Exp ; (161)2020 07 08.
Artigo em Inglês | MEDLINE | ID: mdl-32716369

RESUMO

The isolation and culture of primary melanocytes from skin tissues is very important for biological research and has been widely used for clinical applications. Isolating primary melanocytes from skin tissues by the conventional method usually takes about 3 to 4 weeks to passage sufficiently. More importantly, the tissues used are usually newborn foreskins and it is still a challenge to efficiently isolate primary melanocytes from adult tissues. We recently developed a new isolation method for melanocytes that adds Y-27632, a Rho kinase inhibitor, to the initial culture medium for 48 h. Compared with the conventional protocol, this new method dramatically increases the yield of melanocytes and shortens the time required to isolate melanocytes from foreskin tissues. We now describe this new method in more detail using adult epidermis to efficiently culture primary melanocytes. Importantly, we show that melanocytes obtained from adult tissues prepared by this new method can function normally. This new protocol will significantly benefit studies of pigmentation defects and melanomas using primary melanocytes prepared from easily accessed adult skin tissues.


Assuntos
Amidas/farmacologia , Separação Celular/métodos , Melanócitos , Piridinas/farmacologia , Pele/citologia , Pele/efeitos dos fármacos , Adulto , Células Cultivadas , Meios de Cultura/química , Células Epidérmicas , Humanos , Melanócitos/citologia
15.
J Vis Exp ; (160)2020 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-32597837

RESUMO

Studying isolated cells from mucosa-associated lymphoid tissues (MALT) allows understanding of immune cells response in pathologies involving mucosal immunity, because they can model host-pathogen interactions in the tissue. While isolated cells derived from tissues were the first cell culture model, their use has been neglected because tissue can be hard to obtain. In the present protocol, we explain how to easily process and culture tonsillar mononuclear cells (TMCs) from healthy human tonsils to study innate immune responses upon activation, mimicking viral infection in mucosal tissues. Isolation of TMCs from the tonsils is quick, because the tonsils barely have any epithelium and yield up to billions of all major immune cell types. This method allows detection of cytokine production using several techniques, including immunoassays, qPCR, microscopy, flow cytometry, etc., similar to the use of peripheral mononuclear cells (PBMCs) from blood. Furthermore, TMCs show a higher sensitivity to drug testing than PBMCs, which needs to be considered for future toxicity assays. Thus, ex vivo TMCs cultures are an easy and accessible mucosal model.


Assuntos
Separação Celular/métodos , Imunidade Inata/imunologia , Imunidade nas Mucosas/imunologia , Leucócitos Mononucleares/citologia , Tecido Linfoide/imunologia , Tonsila Palatina/citologia , Citometria de Fluxo , Humanos , Leucócitos Mononucleares/imunologia , Tecido Linfoide/citologia , Tonsila Palatina/imunologia
16.
J Vis Exp ; (160)2020 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-32597844

RESUMO

The isolation and culturing of cardiac myocytes from mice has been essential for furthering the understanding of cardiac physiology and pathophysiology. While isolating myocytes from neonatal mouse hearts is relatively straightforward, myocytes from the adult murine heart are preferred. This is because compared to neonatal cells, adult myocytes more accurately recapitulate cell function as it occurs in the adult heart in vivo. However, it is technically difficult to isolate adult mouse cardiac myocytes in the necessary quantities and viability, which contributes to an experimental impasse. Furthermore, published procedures are specific for the isolation of either atrial or ventricular myocytes at the expense of atrial and ventricular non-myocyte cells. Described here is a detailed method for isolating both atrial and ventricular cardiac myocytes, along with atrial and ventricular non-myocytes, simultaneously from a single mouse heart. Also provided are the details for optimal cell-specific culturing methods, which enhance cell viability and function. This protocol aims not only to expedite the process of adult murine cardiac cell isolation, but also to increase the yield and viability of cells for investigations of atrial and ventricular cardiac cells.


Assuntos
Técnicas de Cultura de Células/métodos , Separação Celular/métodos , Átrios do Coração/citologia , Ventrículos do Coração/citologia , Miócitos Cardíacos/citologia , Envelhecimento , Animais , Técnicas de Cultura de Células/instrumentação , Sobrevivência Celular , Células Cultivadas , Camundongos
17.
J Vis Exp ; (159)2020 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-32510487

RESUMO

The principal cells in the peripheral nervous system are the Schwann cells (SCs) and the fibroblasts. Both these cells distinctly express the sensory and motor phenotypes involved in different patterns of neurotrophic factor gene expression and other biological processes, affecting nerve regeneration. The present study has established a protocol to obtain highly purified rat sensory and motor SCs and fibroblasts more rapidly. The ventral root (motor nerve) and the dorsal root (sensory nerve) of neonatal rats (7-days-old) were dissociated and the cells were cultured for 4-5 days, followed by isolation of sensory and motor fibroblasts and SCs by combining differential digestion and differential adherence methods sequentially. The results of immunocytochemistry and flow cytometry analyses showed that the purity of the sensory and motor SCs and fibroblasts were >90%. This protocol can be used to obtain a large number of sensory and motor fibroblasts/SCs more rapidly, contributing to the exploration of sensory and motor nerve regeneration.


Assuntos
Separação Celular/métodos , Fibroblastos/citologia , Neurônios Motores/citologia , Células de Schwann/citologia , Células Receptoras Sensoriais/citologia , Animais , Regeneração Nervosa , Fenótipo , Ratos
18.
J Vis Exp ; (160)2020 06 12.
Artigo em Inglês | MEDLINE | ID: mdl-32597868

RESUMO

Human implantation, the apposition and adhesion to the uterine surface epithelia and subsequent invasion of the blastocyst into the maternal decidua, is a critical yet enigmatic biological event that has been historically difficult to study due to technical and ethical limitations. Implantation is initiated by the development of the trophectoderm to early trophoblast and subsequent differentiation into distinct trophoblast sublineages. Aberrant early trophoblast differentiation may lead to implantation failure, placental pathologies, fetal abnormalities, and miscarriage. Recently, methods have been developed to allow human embryos to grow until day 13 post-fertilization in vitro in the absence of maternal tissues, a time-period that encompasses the implantation period in humans. This has given researchers the opportunity to investigate human implantation and recapitulate the dynamics of trophoblast differentiation during this critical period without confounding maternal influences and avoiding inherent obstacles to study early embryo differentiation events in vivo. To characterize different trophoblast sublineages during implantation, we have adopted existing two-dimensional (2D) extended culture methods and developed a procedure to enzymatically digest and isolate different types of trophoblast cells for downstream assays. Embryos cultured in 2D conditions have a relatively flattened morphology and may be suboptimal in modeling in vivo three-dimensional (3D) embryonic architectures. However, trophoblast differentiation seems to be less affected as demonstrated by anticipated morphology and gene expression changes over the course of extended culture. Different trophoblast sublineages, including cytotrophoblast, syncytiotrophoblast and migratory trophoblast can be separated by size, location, and temporal emergence, and used for further characterization or experimentation. Investigation of these early trophoblast cells may be instrumental in understanding human implantation, treating common placental pathologies, and mitigating the incidence of pregnancy loss.


Assuntos
Separação Celular/métodos , Implantação do Embrião , Embrião de Mamíferos/citologia , Trofoblastos/citologia , Animais , Biomarcadores/metabolismo , Blastocisto/citologia , Forma Celular , Células Cultivadas , Gonadotropina Coriônica/farmacologia , Feminino , Humanos , Gravidez , Imagem com Lapso de Tempo , Fixação de Tecidos , Tripsina/metabolismo , Vitrificação
19.
Nat Protoc ; 15(7): 2163-2185, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32572244

RESUMO

Circulating tumor cells (CTCs) enable noninvasive liquid biopsy and identification of cancer. Various approaches exist for the capture and release of CTCs, including microfluidic methods and those involving magnetic beads or nanostructured solid interfaces. However, the concomitant cell damage and fragmentation that often occur during capture make it difficult to extensively characterize and analyze living CTCs. Here, we describe an aptamer-trigger-clamped hybridization chain reaction (atcHCR) method for the capture of CTCs by porous 3D DNA hydrogels. The 3D environment of the DNA networks minimizes cell damage, and the CTCs can subsequently be released for live-cell analysis. In this protocol, initiator DNAs with aptamer-toehold biblocks specifically bind to the epithelial cell adhesion molecule (EpCAM) on the surface of CTCs, which triggers the atcHCR and the formation of a DNA hydrogel. The DNA hydrogel cloaks the CTCs, facilitating quantification with minimal cell damage. This method can be used to quantitively identify as few as 10 MCF-7 cells in a 2-µL blood sample. Decloaking of tumor cells via gentle chemical stimulus (ATP) is used to release living tumor cells for subsequent cell culture and live-cell analysis. We also describe how to use the protocol to encapsulate and release cells of cancer cell lines, which can be used in preliminary experiments to model CTCs. The whole protocol takes ~2.5 d to complete, including downstream cell culture and analysis.


Assuntos
Separação Celular/métodos , DNA/química , Hidrogéis/química , Células Neoplásicas Circulantes/patologia , Cápsulas , Sobrevivência Celular , Humanos , Células MCF-7 , Hibridização de Ácido Nucleico
20.
J Vis Exp ; (159)2020 05 23.
Artigo em Inglês | MEDLINE | ID: mdl-32510488

RESUMO

The neurosphere assay is an extremely useful in vitro technique for studying the inherent properties of neural stem/progenitor cells (NSPCs) including proliferation, self-renewal and multipotency. In the postnatal and adult brain, NSPCs are mainly present in two neurogenic niches: the subventricular zone (SVZ) lining the lateral ventricles and the subgranular zone of the hippocampal dentate gyrus (DG). The isolation of the neurogenic niches from postnatal brain allows obtaining a higher amount of NSPCs in culture with a consequent advantage of higher yields. The close contact between cells within each neurosphere creates a microenvironment that may resemble neurogenic niches. Here, we describe, in detail, how to generate SVZ- and DG-derived neurosphere cultures from 1-3-day-old (P1-3) mice, as well as passaging, for neurosphere expansion. This is an advantageous approach since the neurosphere assay allows a fast generation of NSPC clones (6-12 days) and contributes to a significant reduction in the number of animal usage. By plating neurospheres in differentiative conditions, we can obtain a pseudomonolayer of cells composed of NSPCs and differentiated cells of different neural lineages (neurons, astrocytes and oligodendrocytes) allowing the study of the actions of intrinsic or extrinsic factors on NSPC proliferation, differentiation, cell survival and neuritogenesis.


Assuntos
Técnicas de Cultura de Células/métodos , Separação Celular/métodos , Células-Tronco Neurais/citologia , Neurogênese , Animais , Astrócitos/citologia , Diferenciação Celular , Proliferação de Células , Sobrevivência Celular , Hipocampo/citologia , Ventrículos Laterais/citologia , Camundongos , Neurônios/citologia , Oligodendroglia/citologia
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