Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 9 de 9
Filtrar
1.
Proc Natl Acad Sci U S A ; 119(4)2022 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-35046027

RESUMO

Production of high-energy lipids by microalgae may provide a sustainable energy source that can help tackle climate change. However, microalgae engineered to produce more lipids usually grow slowly, leading to reduced overall yields. Unfortunately, culture vessels used to select cells based on growth while maintaining high biomass production, such as well plates, water-in-oil droplet emulsions, and nanowell arrays, do not provide production-relevant environments that cells experience in scaled-up cultures (e.g., bioreactors or outdoor cultivation farms). As a result, strains that are developed in the laboratory may not exhibit the same beneficial phenotypic behavior when transferred to industrial production. Here, we introduce PicoShells, picoliter-scale porous hydrogel compartments, that enable >100,000 individual cells to be compartmentalized, cultured in production-relevant environments, and selected based on growth and bioproduct accumulation traits using standard flow cytometers. PicoShells consist of a hollow inner cavity where cells are encapsulated and a porous outer shell that allows for continuous solution exchange with the external environment. PicoShells allow for cell growth directly in culture environments, such as shaking flasks and bioreactors. We experimentally demonstrate that Chlorella sp., Saccharomyces cerevisiae, and Chinese hamster ovary cells, used for bioproduction, grow to significantly larger colony sizes in PicoShells than in water-in-oil droplet emulsions (P < 0.05). We also demonstrate that PicoShells containing faster dividing and growing Chlorella clonal colonies can be selected using a fluorescence-activated cell sorter and regrown. Using the PicoShell process, we select a Chlorella population that accumulates chlorophyll 8% faster than does an unselected population after a single selection cycle.


Assuntos
Técnicas de Cultura de Células , Ensaios de Triagem em Larga Escala/métodos , Nanopartículas , Nanotecnologia , Animais , Biocombustíveis , Células CHO , Cricetulus , Citometria de Fluxo , Microalgas/metabolismo , Técnicas Analíticas Microfluídicas
3.
Am J Physiol Lung Cell Mol Physiol ; 315(5): L673-L681, 2018 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-30160518

RESUMO

The asthma-obesity syndrome represents a major public health concern that disproportionately contributes to asthma severity and induces insensitivity to therapy. To date, no study has shown an intrinsic difference between human airway smooth muscle (HASM) cells derived from nonobese subjects and those derived from obese subjects. The objective of this study was to address whether there is a greater response to agonist-induced calcium mobilization, phosphorylation of myosin light chain (MLC), and greater shortening in HASM cells derived from obese subjects. HASM cells derived from nonobese and obese subjects were age and sex matched. Phosphorylation of MLC was measured after having been stimulated by carbachol. Carbachol- or histamine-induced mobilization of calcium and cell shortening were assessed in HASM cells derived from nonobese and obese donors. Agonist-induced MLC phosphorylation, mobilization of calcium, and cell shortening were greater in obese compared with non-obese-derived HASM cells. The MLC response was comparable in HASM cells derived from obese nonasthma and nonobese fatal asthma subjects. HASM cells derived from obese female subjects were more responsive to carbachol than HASM cells derived from obese male subjects. Insulin pretreatment had little effect on these responses. Our results show an increase in agonist-induced calcium mobilization associated with an increase in MLC phosphorylation and an increase in ASM cell shortening in favor of agonist-induced hyperresponsiveness in HASM cells derived from obese subjects. Our studies suggest that obesity induces a retained phenotype of hyperresponsiveness in cultured human airway smooth muscle cells.


Assuntos
Asma/fisiopatologia , Carbacol/farmacologia , Histamina/farmacologia , Contração Muscular/efeitos dos fármacos , Músculo Liso/patologia , Obesidade/complicações , Sistema Respiratório/patologia , Adulto , Asma/etiologia , Asma/metabolismo , Cálcio/metabolismo , Cardiotônicos/farmacologia , Estudos de Casos e Controles , Células Cultivadas , Feminino , Agonistas dos Receptores Histamínicos/farmacologia , Humanos , Masculino , Músculo Liso/efeitos dos fármacos , Cadeias Leves de Miosina/metabolismo , Prognóstico , Sistema Respiratório/efeitos dos fármacos
4.
Small ; 14(44): e1803315, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30369052

RESUMO

Microalgae are an attractive feedstock organism for sustainable production of biofuels, chemicals, and biomaterials, but the ability to rationally engineer microalgae to enhance production has been limited. To enable the evolution-based selection of new hyperproducing variants of microalgae, a method is developed that combines phase-transitioning monodisperse gelatin hydrogel droplets with commercial flow cytometric instruments for high-throughput screening and selection of clonal populations of cells with desirable properties, such as high lipid productivity per time traced over multiple cell cycles. It is found that gelatin microgels enable i) the growth and metabolite (e.g., chlorophyll and lipids) production of single microalgal cells within the compartments, ii) infusion of fluorescent reporter molecules into the hydrogel matrices following a sol-gel transition, iii) selection of high-producing clonal populations of cells using flow cytometry, and iv) cell recovery under mild conditions, enabling regrowth after sorting. This user-friendly method is easily integratable into directed cellular evolution pipelines for strain improvement and can be adopted for other applications that require high-throughput processing, e.g., cellular secretion phenotypes and intercellular interactions.


Assuntos
Gelatina/química , Microalgas/metabolismo , Microfluídica/métodos , Biocombustíveis , Biomassa
5.
Methods Mol Biol ; 2689: 53-64, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37430046

RESUMO

Microfluidics enables the creation of monodisperse, micron-scale aqueous droplets, or other compartments. These droplets serve as picolitre-volume reaction chambers which can be utilized for various chemical assays or reactions. Here we describe the use of a microfluidic droplet generator to encapsulate single cells within hollow hydrogel microparticles called PicoShells. The PicoShell fabrication utilizes a mild pH-based crosslinking modality of an aqueous two-phase prepolymer system, avoiding the cell death and unwanted genomic modifications that accompany more typical, ultraviolet light crosslinking techniques. The cells are grown inside of these PicoShells into monoclonal colonies in any number of environments, including scaled production environments using commercially relevant incubation methods. Colonies can be phenotypically analyzed and/or sorted using standard, high-throughput laboratory techniques, namely, fluorescence-activated cell sorting (FACS). Cell viability is maintained throughout particle fabrication and analysis, and cells exhibiting a desired phenotype can be selected and released for re-culturing and downstream analysis. Large-scale cytometry runs are of particular use when measuring the protein expression of heterogeneous cells in response to environmental stimuli, notably to identify targets early in the drug discovery process. The sorted cells can also be encapsulated multiple times to direct the evolution of a cell line to a desired phenotype.


Assuntos
Ensaios de Triagem em Larga Escala , Hidrogéis , Microfluídica , Análise da Expressão Gênica de Célula Única , Hidrogéis/síntese química , Ensaios de Triagem em Larga Escala/instrumentação , Ensaios de Triagem em Larga Escala/métodos , Análise da Expressão Gênica de Célula Única/instrumentação , Análise da Expressão Gênica de Célula Única/métodos , Citometria de Fluxo , Leveduras/genética , Leveduras/crescimento & desenvolvimento , Leveduras/metabolismo , Microfluídica/instrumentação , Microfluídica/métodos , Células Clonais/fisiologia
6.
Life (Basel) ; 12(8)2022 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-36013347

RESUMO

Studying microbes at the single-cell level in space can accelerate human space exploration both via the development of novel biotechnologies and via the understanding of cellular responses to space stressors and countermeasures. High-throughput technologies for screening natural and engineered cell populations can reveal cellular heterogeneity and identify high-performance cells. Here, we present a method to desiccate and preserve microbes in nanoliter-scale compartments, termed PicoShells, which are microparticles with a hollow inner cavity. In PicoShells, single cells are confined in an inner aqueous core by a porous hydrogel shell, allowing the diffusion of nutrients, wastes, and assay reagents for uninhibited cell growth and flexible assay protocols. Desiccated PicoShells offer analysis capabilities for single-cell derived colonies with a simple, low resource workflow, requiring only the addition of water to rehydrate hundreds of thousands of PicoShells and the single microbes encapsulated inside. Our desiccation method results in the recovery of desiccated microparticle morphology and porosity after a multi-week storage period and rehydration, with particle diameter and porosity metrics changing by less than 18% and 7%, respectively, compared to fresh microparticles. We also recorded the high viability of Saccharomyces cerevisiae yeast desiccated and rehydrated inside PicoShells, with only a 14% decrease in viability compared to non-desiccated yeast over 8.5 weeks, although we observed an 85% decrease in initial growth potential over the same duration. We show a proof-of-concept for a growth rate-based analysis of single-cell derived colonies in rehydrated PicoShells, where we identified 11% of the population that grows at an accelerated rate. Desiccated PicoShells thus provide a robust method for cell preservation before and during launch, promising a simple single-cell analysis method for studying heterogeneity in microbial populations in space.

7.
ACS Nano ; 16(5): 7242-7257, 2022 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-35324146

RESUMO

Techniques to analyze and sort single cells based on functional outputs, such as secreted products, have the potential to transform our understanding of cellular biology as well as accelerate the development of next-generation cell and antibody therapies. However, secreted molecules rapidly diffuse away from cells, and analysis of these products requires specialized equipment and expertise to compartmentalize individual cells and capture their secretions. Herein, we describe methods to fabricate hydrogel-based chemically functionalized microcontainers, which we call nanovials, and demonstrate their use for sorting single viable cells based on their secreted products at high-throughput using only commonly accessible laboratory infrastructure. These nanovials act as solid supports that facilitate attachment of a variety of adherent and suspension cell types, partition uniform aqueous compartments, and capture secreted proteins. Solutions can be exchanged around nanovials to perform fluorescence immunoassays on secreted proteins. Using this platform and commercial flow sorters, we demonstrate high-throughput screening of stably and transiently transfected producer cells based on relative IgG production. Chinese hamster ovary cells sorted based on IgG production regrew and maintained a high secretion phenotype over at least a week, yielding >40% increase in bulk IgG production rates. We also sorted hybridomas and B lymphocytes based on antigen-specific antibody production. Hybridoma cells secreting an antihen egg lysozyme antibody were recovered from background cells, enriching a population of ∼4% prevalence to >90% following sorting. Leveraging the high-speed sorting capabilities of standard sorters, we sorted >1 million events in <1 h. IgG secreting mouse B cells were also sorted and enriched based on antigen-specific binding. Successful sorting of antibody-secreting B cells combined with the ability to perform single-cell RT-PCR to recover sequence information suggests the potential to perform antibody discovery workflows. The reported nanovials can be easily stored and distributed among researchers, democratizing access to high-throughput functional cell screening.


Assuntos
Hidrogéis , Análise de Célula Única , Cricetinae , Camundongos , Animais , Células CHO , Hidrogéis/metabolismo , Cricetulus , Hibridomas , Análise de Célula Única/métodos , Antígenos/metabolismo , Imunoglobulina G/metabolismo , Citometria de Fluxo/métodos
8.
ACS Sens ; 5(2): 385-394, 2020 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-31902202

RESUMO

Nucleic acid amplification assays including loop-mediated isothermal amplification (LAMP) are routinely used in diagnosing diseases and monitoring water and food quality. The results of amplification in these assays are commonly measured with an analog fluorescence readout, which requires specialized optical equipment and can lack quantitative precision. Digital analysis of amplification in small fluid compartments based on exceeding a threshold fluorescence level can enhance the quantitative precision of nucleic acid assays (i.e., digital nucleic acid amplification assays), but still requires specialized optical systems for fluorescence readout and the inclusion of a fluorescent dye. Here, we report Fractal LAMP, an automated method to detect amplified DNA in subnanoliter scale droplets following LAMP in a label-free manner. Our computer vision algorithm achieves high accuracy detecting DNA amplification in droplets by identifying LAMP byproducts that form fractal structures observable in brightfield microscopy. The capabilities of Fractal LAMP are further realized by developing a Bayesian model to estimate DNA concentrations for unknown samples and a bootstrapping method to estimate the number of droplets required to achieve target limits of detection. This digital, label-free assay has the potential to lower reagent and reader cost for nucleic acid measurement while maintaining high quantitative accuracy over 3 orders of magnitude of concentration.


Assuntos
Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos
9.
Lab Chip ; 18(17): 2575-2582, 2018 08 21.
Artigo em Inglês | MEDLINE | ID: mdl-30046787

RESUMO

Hydrogel droplets encapsulating cells and molecules provide a unique platform in biochemistry, biology, and medicine, including single-cell and single-molecule analysis, directed molecular evolution, and detection of cellular secretions. The ability to prepare hydrogel droplets with high monodispersity can lead to synchronization of populations, more controlled biomaterials, and more quantitative assays. Here, we present an inertial microfluidic device for passive, continuous, and high-throughput sorting of hydrogel droplets by size. The sorting is achieved due to size-dependent lateral inertial equilibrium positions: hydrogel droplets of different sizes have different equilibrium positions under the combined effects of shear-gradient lift and wall-effect lift forces. We apply this separation technique to isolate smaller hydrogel droplets containing microalgal colonies from larger empty droplets. We found that hydrogel droplets containing microalga Euglena gracilis (E. gracilis) shrink as cells grow and divide, while empty hydrogel droplets retain their size. Cell-laden hydrogel droplets were collected with up to 93.6% purity, and enrichment factor up to 5.51. After sorting, we were able to recover cells from hydrogel droplets without significantly affecting cell viability.


Assuntos
Hidrogéis/química , Hidrogéis/isolamento & purificação , Dispositivos Lab-On-A-Chip , Microalgas/citologia
SELEÇÃO DE REFERÊNCIAS
Detalhe da pesquisa