dMSCC: a microfluidic platform for microbial single-cell cultivation of Corynebacterium glutamicum under dynamic environmental medium conditions

Microbial cells are often exposed to rapidly fluctuating environmental conditions. A novel microfluidic system for the cultivation of single cells and small cell clusters is presented under dynamic environment conditions.

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dMSCC: A microfluidic platform for microbial single-cell cultivation under dynamic environmental medium conditions

10.1101/2020.07.10.188938 ◽

2020 ◽

Author(s):

Sarah Täuber ◽

Corinna Golze ◽

Phuong Ho ◽

Eric von Lieres ◽

Alexander Grünberger

Keyword(s):

Growth Rate ◽

Single Cell ◽

Environmental Conditions ◽

Cellular Response ◽

Large Scale ◽

Single Cells ◽

Model Organism ◽

Colony Growth ◽

Cell Cultivation ◽

Natural Habitats

AbstractIn nature and in technical systems, microbial cells are often exposed to rapidly fluctuating environmental conditions. These conditions can vary in quality, e.g., existence of a starvation zone, and quantity, e.g., average residence time in this zone. For strain development and process design, cellular response to such fluctuations needs to be systematically analysed. However, the existing methods for physically emulating rapidly changing environmental conditions are limited in spatio-temporal resolution. Hence, we present a novel microfluidic system for cultivation of single cells and small cell clusters under dynamic environmental conditions (dynamic microfluidic single-cell cultivation (dMSCC)). This system enables to control nutrient availability and composition between two media with second to minute resolution. We validate our technology using the industrially relevant model organism Corynebacterium glutamicum. The organism was exposed to different oscillation frequencies between nutrient excess (feasts) and scarcity (famine). Resulting changes in cellular physiology, such as the colony growth rate and cell morphology were analysed and revealed significant differences with growth rate and cell length between the different conditions. dMSCC also allows to apply defined but randomly changing nutrient conditions, which is important for reproducing more complex conditions from natural habitats and large-scale bioreactors. The presented system lays the foundation for the cultivation of cells under complex changing environmental conditions.

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The cytomorphologic spectrum of small-cell carcinoma and large-cell neuroendocrine carcinoma in body cavity effusions: A study of 68 cases

CytoJournal ◽

10.4103/1742-6413.86816 ◽

2011 ◽

Vol 8 ◽

pp. 18 ◽

Cited By ~ 17

Author(s):

Walid E. Khalbuss ◽

Huaitao Yang ◽

Qian Lian ◽

Abdelmonem Elhosseiny ◽

Liron Pantanowitz ◽

...

Keyword(s):

Cell Carcinoma ◽

Single Cell ◽

Small Cell Carcinoma ◽

Neuroendocrine Carcinoma ◽

Large Cell ◽

Body Cavity ◽

Small Cell ◽

Cell Clusters ◽

Cell Pattern ◽

Ancillary Studies

Background: Small-cell carcinoma (SCC) and large-cell neuroendocrine carcinoma (LCNEC) are uncommon in serous body cavity effusions. The purpose of this study is to examine the cytomorphological spectrum of SCC and LCNEC in body cavity serous fluids. Materials and Methods: We have 68 cases from 53 patients who had metastatic SCC or LCNEC diagnoses. All cytology slides and the available clinical data, histological follow-up, and ancillary studies were reviewed. Results: A total of 68 cases (60 pleural, 5 peritoneal, and 3 pericardial effusions) from 53 patients with an average age of 73 years (age range 43-92 years) were reported as diagnostic or suspicious of SCC (52 cases) or LCNEC (16 cases). The primary site was lung in 56 cases, pancreas in 6 cases, and 2 cases each from cervix, colon, and the head and neck region. Of the 68 cases, 48 cases had no history of malignancy of the same type. Ancillary studies were used in 46 cases (68%) including flow cytometric studies in 5 cases. There were three predominant cytomorphological patterns observed including small-cell clusters with prominent nuclear molding (33 cases, 49%), large-cell clusters mimicking non-small-cell carcinoma (18 cases, 26%), and single-cell pattern mimicking lymphoma (17 cases, 25%). Significant apoptosis was seen in 22 cases (33%) and marked tumor cell cannibalism was seen in 11 cases (16%). Nucleoli were prominent in 16 cases (24%). The most frequent neuroendocrine markers performed were synaptophysin and chromogranin. Conclusions: The most common cytomorphologic patterns seen in body cavity effusions of SCC and LCNEC were small-cell clusters with nuclear molding. However, in 51% of the cases either a predominant single-cell pattern mimicking lymphoma or large-cell clusters mimicking non-small carcinoma were noted. In our experience, effusions were the first manifestation of disease in the majority of patients diagnosed with neuroendocrine carcinoma. Therefore, familiarity with the cytomorphological spectrum of neuroendocrine carcinomas in fluid cytology may help in rapidly establishing an accurate diagnosis and in directing appropriate management.

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An acoustofluidic trap and transfer approach for organizing a high density single cell array

Lab on a Chip ◽

10.1039/c8lc00196k ◽

2018 ◽

Vol 18 (14) ◽

pp. 2124-2133 ◽

Cited By ~ 6

Author(s):

Korine A. Ohiri ◽

Sean T. Kelly ◽

Jeffrey D. Motschman ◽

Kevin H. Lin ◽

Kris C. Wood ◽

...

Keyword(s):

Single Cell ◽

Single Cells ◽

Microfluidic System ◽

High Density ◽

Cell Array

We demonstrate a hybrid microfluidic system that combines fluidic trapping and acoustic switching to organize an array of single cells at high density.

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Micropillar array embedded system for single cell encapsulation in hydrogel

MRS Proceedings ◽

10.1557/opl.2015.66 ◽

2015 ◽

Vol 1724 ◽

Author(s):

Kyun Joo Park ◽

Kyoung G. Lee ◽

Seunghwan Seok ◽

Bong Gill Choi ◽

Seok Jae Lee ◽

...

Keyword(s):

Embedded System ◽

Single Cell ◽

Single Cells ◽

Cell Encapsulation ◽

Polyethylene Glycol Diacrylate ◽

Glycol Diacrylate ◽

Practical Applications ◽

Cell Clusters ◽

Single Cell Encapsulation ◽

Micropillar Array

ABSTRACTA cylindrical-shaped micropillar array embedded microfluidic device was proposed to enhance the dispersion of cell clusters and the efficiency of single cell encapsulation in hydrogel. Different sizes of micropillar arrays act as a sieve to break Escherichia coli (E. coli) aggregates into single cells in polyethylene glycol diacrylate (PEGDA) solution. We applied the external force for the continuous breakup of cell clusters, resulting in the production of more than 70% of single cells into individual hydrogel particles. This proposed strategy and device will be a useful platform to utilize genetically modified microorganisms in practical applications.

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Rigid and Conductive Dual Nanoprobe for Single Cell Analysis

Jurnal Teknologi ◽

10.11113/jt.v69.3304 ◽

2014 ◽

Vol 69 (8) ◽

Author(s):

Abdul Hafiz Mat Sulaiman ◽

Mohd Ridzuan Ahmad

Keyword(s):

Single Cell ◽

Single Cell Analysis ◽

Single Cells ◽

Electrical Measurement ◽

Microfluidic System ◽

General Information ◽

Aluminium Copper ◽

Element Approach ◽

Cell Surgery ◽

Electrical Characterizations

Electrical property characterization of a single cell can be used to infer about its physiological condition, e.g. cell viability. Due to that, a dual nanoprobe-microfluidic system for electrical properties measurement of single cells has been proposed. This paper is concerned about the mechanical and electrical characterizations of the dual nanoprobe. Electrical and mechanical characterizations were conducted to measure the resistance and the strength of the dual nanoprobe for five different metals i.e. Aluminium, Copper, Silver, Tungsten, and Zinc using finite element approach. From the findings, Tungsten’s nanoprobe has the highest strength while the resistance values for the five materials are not significantly different. Therefore, Tungsten is selected as the most recommended metal for the dual nanoprobe. We also performed single cell electrical measurement to test the functionality of the sensor. This work provides general information of the nanoprobe which can be used as a framework in other applications involving Nano devices i.e. cell surgery and drug delivery.

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Single-cell RNA sequencing analysis of mouse follicular somatic cells

Biology of Reproduction ◽

10.1093/biolre/ioab163 ◽

2021 ◽

Author(s):

Sen Li ◽

Lei-Ning Chen ◽

Hai-Jing Zhu ◽

Xie Feng ◽

Feng-Yun Xie ◽

...

Keyword(s):

Single Cell ◽

Single Cells ◽

Somatic Cells ◽

Metabolic Reprogramming ◽

Follicle Development ◽

Sequencing Analysis ◽

Cell Clusters ◽

New Genes ◽

Variable Genes ◽

Cell Cell

Abstract Within the development of ovarian follicle, in addition to cell proliferation and differentiation, sophisticated cell–cell cross talks are established among follicular somatic cells such as granulosa cells (GCs) and theca cells. To systematically reveal the cell differentiation and signal transductions in follicular somatic cells, we collected the mouse follicular somatic cells from secondary to ovulatory stage, and analyzed the single cell transcriptomes. Having data filtered and screened, we found 6883 high variable genes in 4888 single cells. Then follicular somatic cells were clustered into 26 cell clusters, including 18 GC clusters, 4 theca endocrine cell (TEC) clusters, and 4 other somatic cell clusters, which include immune cells and Acta2 positive theca externa cells. From our data, we found there was metabolic reprogramming happened during GC differentiation. We also found both Cyp19a1 and Cyp11a1 could be expressed in TECs. We analyzed the expression patterns of genes associated with cell–cell interactions such as steroid hormone receptor genes, insulin signaling genes, and cytokine/transformation growth factor beta associated genes in all cell clusters. Lastly, we clustered the highly variable genes into 300 gene clusters, which could be used to search new genes involved in follicle development. These transcriptomes of follicular somatic cells provide us potential clues to reveal how mammals regulating follicle development and could help us find targets to improve oocyte quality for women with low fertility.

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Statistical single-cell analysis of cell cycle-dependent quantum dot cytotoxicity and cellular uptake using a microfluidic system

RSC Advances ◽

10.1039/c4ra01665c ◽

2014 ◽

Vol 4 (47) ◽

pp. 24929-24934 ◽

Cited By ~ 14

Author(s):

Jing Wu ◽

Haifang Li ◽

Qiushui Chen ◽

Xuexia Lin ◽

Wu Liu ◽

...

Keyword(s):

Cell Cycle ◽

Quantum Dot ◽

Single Cell ◽

Cellular Uptake ◽

Single Cell Analysis ◽

Single Cells ◽

Microfluidic System ◽

Cell Analysis ◽

System P ◽

Cycle Dependent

The response of single cells in different cell cycle phases to QD cytotoxicity studied on a microfluidic device.

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Selective Retrieval of Individual Cells from Microfluidic Arrays Combining Dielectrophoretic Force and Directed Hydrodynamic Flow

Micromachines ◽

10.3390/mi11030322 ◽

2020 ◽

Vol 11 (3) ◽

pp. 322

Author(s):

Pierre-Emmanuel Thiriet ◽

Joern Pezoldt ◽

Gabriele Gambardella ◽

Kevin Keim ◽

Bart Deplancke ◽

...

Keyword(s):

Single Cell ◽

Single Cells ◽

Three Dimensional ◽

Microfluidic Channel ◽

Microfluidic System ◽

Transcriptional Analysis ◽

Molecular Profile ◽

Cell Phenotype ◽

Pneumatic Valves ◽

On Chip

Hydrodynamic-based microfluidic platforms enable single-cell arraying and analysis over time. Despite the advantages of established microfluidic systems, long-term analysis and proliferation of cells selected in such devices require off-chip recovery of cells as well as an investigation of on-chip analysis on cell phenotype, requirements still largely unmet. Here, we introduce a device for single-cell isolation, selective retrieval and off-chip recovery. To this end, singularly addressable three-dimensional electrodes are embedded within a microfluidic channel, allowing the selective release of single cells from their trapping site through application of a negative dielectrophoretic (DEP) force. Selective capture and release are carried out in standard culture medium and cells can be subsequently mitigated towards a recovery well using micro-engineered hybrid SU-8/PDMS pneumatic valves. Importantly, transcriptional analysis of recovered cells revealed only marginal alteration of their molecular profile upon DEP application, underscored by minor transcriptional changes induced upon injection into the microfluidic device. Therefore, the established microfluidic system combining targeted DEP manipulation with downstream hydrodynamic coordination of single cells provides a powerful means to handle and manipulate individual cells within one device.

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Laser induced isolation and cultivation of single microbial cells

10.1101/2020.07.27.224287 ◽

2020 ◽

Author(s):

Peng Liang ◽

Huan Wang ◽

Yun Wang ◽

Yinping Zhao ◽

Wei E. Huang ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Single Cell ◽

Cell Sorting ◽

Single Cells ◽

Lactobacillus Rhamnosus ◽

Microbial Cells ◽

E Coli ◽

Eukaryotic Microorganisms ◽

Cultivation Technique ◽

Single Cell Isolation

AbstractSingle cell isolation and cultivation play an important role in studying physiology, gene expression and functions of microorganisms. Laser Induced Forward Transfer Technique (LIFT) has been applied to isolate single cells but the cell viability after sorting is unclear. We demonstrate that a three-layer LIFT system could be applied to isolate single cells of Gram-negative (E. coli), Gram-positive (Lactobacillus rhamnosus GG, LGG), and eukaryotic microorganisms (Saccharomyces cerevisiae) and the sorted single cells were able to be cultured. The experiment results showed that the average cultivation recovery rate of the ejected single cells were 58% for Saccharomyces cerevisiae, 22% for E. coli, and 74% for Lactobacillus rhamnosus GG (LGG). The identities of the cultured cells from single cell sorting were confirmed by using colony PCR with 16S-rRNA for bacteria and large unit rRNA for yeast and subsequent sequencing. This precise sorting and cultivation technique of live single microbial cells can be coupled with other microscopic approaches (e.g. fluorescent and Raman microscopy) to culture single microorganisms with specific functions, revealing their roles in the natural community.ImportanceSingle cell isolation and cultivation are crucial to recover microorganisms for the study of physiology, gene expression and functions. We developed a laser induced cell sorting technology to precisely isolate single microbial cells from a microscopic slide. More importantly, the isolated single microbial cells are still viable for cultivation. We demonstrate to apply the live sorting method to isolate and cultivate single cells of Gram-negative (E. coli), Gram-positive (Lactobacillus rhamnosus GG, LGG), and eukaryotic microorganisms (Saccharomyces cerevisiae). This precise sorting and cultivation technique can be coupled with other microscopic approaches (e.g. fluorescent and Raman microscopy) to culture specifically targeted single microorganisms from microbial community.Abstract Graphic

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