Reconfigurable microfluidic circuits for isolating and retrieving cells of interest

Microfluidic devices are widely used in many fields of biology, but a key limitation is that cells are typically surrounded by solid walls, making it hard to access those that exhibit a specific phenotype for further study. Here, we provide a general and flexible solution to this problem that exploits the remarkable properties of microfluidic circuits with fluid walls - transparent interfaces between culture media and an immiscible fluorocarbon that are easily pierced with pipets. We provide two proofs-of-concept in which specific cell sub-populations are isolated and recovered: i) murine macrophages chemotaxing towards complement component 5a, and ii) bacteria (Pseudomonas aeruginosa) in developing biofilms that migrate towards antibiotics. We build circuits in minutes on standard Petri dishes, add cells, pump in laminar streams so molecular diffusion creates attractant gradients, acquire time-lapse images, and isolate desired sub-populations in real-time by building fluid walls around migrating cells with an accuracy of tens of micrometres using 3D-printed adaptors that convert conventional microscopes into wall-building machines. Our method allows live cells of interest to be easily extracted from microfluidic devices for downstream analyses.

Download Full-text

Identification Pseudomonas aeruginosa by OprD Gene for Differentiation from Other Pseudomonas Species that Isolated from Clinical Samples

International Journal of Drug Delivery Technology ◽

10.25258/ijddt.9.1.8 ◽

2019 ◽

Vol 9 (01) ◽

pp. 46-50

Author(s):

Ashwak B Al-Hashimy ◽

Huda S Alagely ◽

Akeel K Albuaji ◽

Khalid R Majeed

Keyword(s):

Pseudomonas Aeruginosa ◽

General Hospital ◽

Culture Media ◽

Final Diagnosis ◽

Clinical Samples ◽

Bacterial Isolates ◽

Molecular Method ◽

Biochemical Tests ◽

Pseudomonas Species ◽

Specific Sequences

The present study included the collection of 100 samples from various clinical sources for investigating the presence of P. aeruginosa in those sources, the samples have been collected from some hospitals in Baghdad and Hillah city (Al-qassim General Hospital, ,Al-hillah teaching hospital,and Al-hashimya General hospital ) which included wounds, burns, ear and sputum infections. The study was carried out through October 2017 till the end of March 2018. The samples were identified based on the morphological and microscopically characteristics of the colonies when they were culturing or number of culture media as well as biochemical tests, molecular identification were also used as a final diagnostic test for isolates that were positive as they belong to P.aeruginosa bacteria during previous tests based on the OprD gene which has specific sequences for P.aeruginosa bacteria as a detection gene and also consider as virulence factor so it have a synonyms mechanism to antibiotic resistance . The results of the final diagnosis showed that 38 isolates belong to target bacteria were distributed as 18 of burns, 11 isolates of wounds, 6 isolates of ear infection and 3 isolates of sputum, The examination of the sensitivity of all bacterial isolates was done for elected 38 isolation towards the 9 antibiotic by a Bauer - Kirby and the isolates were resistant for a number of antibiotics used such as Ciprofloxacin 65.7%, Norflaxacin 71%, Imipenem 63.1% Meropenem 68.4%, Gentamicin 65.7%, Amikacin 26.3%, Cefepime 68.4%, Ceftazidime 65.7% and Piperacillin 57.8%.Molecular method , All isolates (38) of P. aeruginosa positive for the diagnostic special gene (OprD) genes (100%).

Download Full-text

Nephrotoxicity Testing In Vitro: The Current Situation

Alternatives to Laboratory Animals ◽

10.1177/026119299702500506 ◽

1997 ◽

Vol 25 (5) ◽

pp. 497-503

Author(s):

Jean-Paul Morin ◽

Marc E. De Broe ◽

Walter Pfaller ◽

Gabriele Schmuck

Keyword(s):

Proximal Tubule ◽

Culture Media ◽

Task Force ◽

Primary Cultures ◽

Phenotypic Expression ◽

Transepithelial Transport ◽

Specific Cell ◽

Proximal Tubule Cells ◽

Tubule Cells

An ECVAM task force on nephrotoxicity has been established to advise, in particular, on the follow-up to recommendations made in the ECVAM workshop report on nephrotoxicity testing in vitro. Since this workshop was held, in 1994, there have been several improvements in the techniques used. For example, the duration of renal slice viability, and the maintenance of functional activities in slices, have been improved by using dynamic incubation systems with higher oxygen tensions and more-appropriate cell culture media. Highly differentiated primary cultures of pig, human and rabbit proximal tubule cells have been established by using specific cell isolation procedures and/or selective culture media. To date, the most comparable phenotypic expression and transepithelial transport capacities to proximal tubules in vivo have been obtained with primary cultures of rabbit proximal tubule cells which are grown on bicompartmental supports; in this system, transepithelial substrate gradients are generated and the transepithelial transport of both organic anions and cations is highly active. This in vitro system has been selected by ECVAM for further evaluation and prevalidation. Industrial needs in the area of nephrotoxicity testing have been identified, and recommendations are made at the end of this report concerning possible future initiatives.

Download Full-text

3D printed microfluidic devices: a review focused on four fundamental manufacturing approaches and implications on the field of healthcare

Bio-Design and Manufacturing ◽

10.1007/s42242-020-00112-5 ◽

2021 ◽

Author(s):

Viraj Mehta ◽

Subha N. Rath

Keyword(s):

Microfluidic Devices ◽

3D Printed

Download Full-text

Automated calibration of 3D-printed microfluidic devices based on computer vision

Biomicrofluidics ◽

10.1063/5.0037274 ◽

2021 ◽

Vol 15 (2) ◽

pp. 024102

Author(s):

Junchao Wang ◽

Kaicong Liang ◽

Naiyin Zhang ◽

Hailong Yao ◽

Tsung-Yi Ho ◽

...

Keyword(s):

Computer Vision ◽

Microfluidic Devices ◽

Automated Calibration ◽

3D Printed

Download Full-text

Development of single-cell-level microfluidic technology for long-term growth visualization of living cultures of Mycobacterium smegmatis

Microsystems & Nanoengineering ◽

10.1038/s41378-021-00262-1 ◽

2021 ◽

Vol 7 (1) ◽

Author(s):

Han Wang ◽

Gloria M. Conover ◽

Song-I Han ◽

James C. Sacchettini ◽

Arum Han

Keyword(s):

Drug Treatment ◽

Single Cell ◽

Mycobacterium Smegmatis ◽

Culture Media ◽

Low Cost ◽

Bacterial Pathogen ◽

Growth Dynamics ◽

Time Lapse ◽

Cell Phenotype

AbstractAnalysis of growth and death kinetics at single-cell resolution is a key step in understanding the complexity of the nonreplicating growth phenotype of the bacterial pathogen Mycobacterium tuberculosis. Here, we developed a single-cell-resolution microfluidic mycobacterial culture device that allows time-lapse microscopy-based long-term phenotypic visualization of the live replication dynamics of mycobacteria. This technology was successfully applied to monitor the real-time growth dynamics of the fast-growing model strain Mycobacterium smegmatis (M. smegmatis) while subjected to drug treatment regimens during continuous culture for 48 h inside the microfluidic device. A clear morphological change leading to significant swelling at the poles of the bacterial membrane was observed during drug treatment. In addition, a small subpopulation of cells surviving treatment by frontline antibiotics was observed to recover and achieve robust replicative growth once regular culture media was provided, suggesting the possibility of identifying and isolating nonreplicative mycobacteria. This device is a simple, easy-to-use, and low-cost solution for studying the single-cell phenotype and growth dynamics of mycobacteria, especially during drug treatment.

Download Full-text

Imaging and Analysis of Pseudomonas aeruginosa Swarming and Rhamnolipid Production

Applied and Environmental Microbiology ◽

10.1128/aem.06644-11 ◽

2011 ◽

Vol 77 (23) ◽

pp. 8310-8317 ◽

Cited By ~ 27

Author(s):

Joshua D. Morris ◽

Jessica L. Hewitt ◽

Lawrence G. Wolfe ◽

Nachiket G. Kamatkar ◽

Sarah M. Chapman ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Fluorescent Protein ◽

Nile Red ◽

Temporal Distribution ◽

Time Lapse ◽

Content Type ◽

Rhamnolipid Production ◽

Serovar Typhimurium ◽

Surface Motility ◽

Green Fluorescent

ABSTRACTMany bacteria spread over surfaces by “swarming” in groups. A problem for scientists who study swarming is the acquisition of statistically significant data that distinguish two observations or detail the temporal patterns and two-dimensional heterogeneities that occur. It is currently difficult to quantify differences between observed swarm phenotypes. Here, we present a method for acquisition of temporal surface motility data using time-lapse fluorescence and bioluminescence imaging. We specifically demonstrate three applications of our technique with the bacteriumPseudomonas aeruginosa. First, we quantify the temporal distribution ofP. aeruginosacells tagged with green fluorescent protein (GFP) and the surfactant rhamnolipid stained with the lipid dye Nile red. Second, we distinguish swarming ofP. aeruginosaandSalmonella entericaserovar Typhimurium in a coswarming experiment. Lastly, we quantify differences in swarming and rhamnolipid production of severalP. aeruginosastrains. While the best swarming strains produced the most rhamnolipid on surfaces, planktonic culture rhamnolipid production did not correlate with surface growth rhamnolipid production.

Download Full-text

Rapid Production of PDMS Microdevices for Electrodriven Separations and Microfluidics by 3D-Printed Scaffold Removal

Separations ◽

10.3390/separations8050067 ◽

2021 ◽

Vol 8 (5) ◽

pp. 67

Author(s):

Alena Šustková ◽

Klára Konderlová ◽

Ester Drastíková ◽

Stefan Sützl ◽

Lenka Hárendarčíková ◽

...

Keyword(s):

Organic Dyes ◽

Microfluidic Devices ◽

Proof Of Concept ◽

Magnetic Microparticles ◽

Mechanical Removal ◽

Glass Slides ◽

Rapid Production ◽

3D Printed

In our work, we produced PDMS-based microfluidic devices by mechanical removal of 3D-printed scaffolds inserted in PDMS. Two setups leading to the fabrication of monolithic PDMS-based microdevices and bonded (or stamped) PDMS-based microdevices were designed. In the monolithic devices, the 3D-printed scaffolds were fully inserted in the PDMS and then carefully removed. The bonded devices were produced by forming imprints of the 3D-printed scaffolds in PDMS, followed by bonding the PDMS parts to glass slides. All these microfluidic devices were then successfully employed in three proof-of-concept applications: capture of magnetic microparticles, formation of droplets, and isotachophoresis separation of model organic dyes.

Download Full-text

Conditioning of a culture substratum by the ectodermal layer promotes attachment and oriented locomotion by amphibian gastrula mesodermal cells

Journal of Cell Science ◽

10.1242/jcs.59.1.43 ◽

1983 ◽

Vol 59 (1) ◽

pp. 43-60 ◽

Cited By ~ 1

Author(s):

N. Nakatsuji ◽

K.E. Johnson

Keyword(s):

Cell Migration ◽

Time Lapse ◽

Ambystoma Maculatum ◽

Specific Cell ◽

Animal Pole ◽

Micron Diameter ◽

The Relationship ◽

Extracellular Fibrils ◽

Scanning Electron

We have found that ectodermal fragments of Ambystoma maculatum gastrulae deposit immense numbers of 0.1 micron diameter extracellular fibrils on plastic coverslips. When migrating mesodermal cells from A. maculatum gastrulae are seeded on such conditioned plastic substrata, they attach and begin migrating after 15–30 min in vitro. We did a detailed analysis of the relationship between fibril orientation and cell migration using time-lapse cinemicrography, scanning electron microscopy, and a microcomputer with a graphics tablet and morphometric program. We found that cells move in directions closely related to the orientation of fibrils. Usually fibrils are oriented in dense arrays with a predominance of fibrils running parallel to the blastopore-animal pole axis of the explant, and cells move preferentially along lines parallel to the blastopore-animal pole axis. When fibrils are unaligned, cells move at random. We have also shown that cells move with a slightly stronger tendency towards the animal pole direction. These results are discussed concerning the mechanism of specific cell migration during amphibian gastrulation.

Download Full-text

Comparative study of membrane filtration and enrichment media for the isolation and enumeration of Pseudomonas aeruginosa from sewage, surface water, and swimming pools

Canadian Journal of Microbiology ◽

10.1139/m85-130 ◽

1985 ◽

Vol 31 (8) ◽

pp. 686-692 ◽

Cited By ~ 10

Author(s):

A. H. Havelaar ◽

M. During ◽

E. H. M. Delfgou-Van Asch

Keyword(s):

Pseudomonas Aeruginosa ◽

Membrane Filtration ◽

Culture Media ◽

Polluted Water ◽

Most Probable Number ◽

Cellulose Ester ◽

Swimming Pools ◽

Probable Number ◽

Membrane Filters ◽

Swimming Water

The recovery of Pseudomonas aeruginosa on several selective culture media was tested using raw sewage and secondary sewage effluent samples as well as spiked chlorinated imitation swimming water and samples from whirlpools. mPA-medium B gave good recovery of both vital and chlorine-injured P. aeruginosa and selectivity was greater than 90% when analysing whirlpool samples. It is therefore the medium recommended for examination of chlorinated swimming pools. When analysing sewage polluted water with the mPA-B medium, reduced selectivity was noted from low verification rates and from overgrowth by competitive flora. A modified medium (mPA-D; addition of cetrimide, omission of sulphapyridine and actidione) was more selective and sufficiently recovered noninjured cells. Chlorine-injured cells were completely inhibited, however. C-390 (9-chloro-9-(4-diethylaminophenyl)-10-phenylacridan) was confirmed to be highly selective for P. aeruginosa when used in spread plates at a concentration of 30 μg/mL; P. aeruginosa was slightly inhibited. However, the medium could not be used with conventional membrane filtration techniques, because cellulose ester filters interfered with the selective action of C-390. Selectivity could be improved by using Gelman Tuffryn (polysulphone) filters and increasing the C-390 concentration to 120 μg/mL. At this concentration, however, the medium was strongly inhibitory to P. aeruginosa; resuscitation only partially improved recovery. Two other membrane filtration media were tested. Both cetrimide – nalidixic acid agar and Drake's medium No. 19 were inhibitory to chlorine-injured cells. Several types of membrane filters were tested and there was little difference between them. In the most-probable-number technique, recovery of P. aeruginosa was shown to be excellent when using asparagine broth. Malachite green broth was strongly inhibitory to chlorine-injured P. aeruginosa.

Download Full-text