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Biomedical Microdevices May 2022Three-dimensional cell agglomerates are broadly useful in tissue engineering and drug testing. We report a well-free method to form large (1.4-mm) multicellular clusters...
Three-dimensional cell agglomerates are broadly useful in tissue engineering and drug testing. We report a well-free method to form large (1.4-mm) multicellular clusters using 100-MHz surface acoustic waves (SAW) without direct contact with the media or cells. A fluid couplant is used to transform the SAW into acoustic streaming in the cell-laden media held in a petri dish. The couplant transmits longitudinal sound waves, forming a Lamb wave in the petri dish that, in turn, produces longitudinal sound in the media. Due to recirculation, human embryonic kidney (HEK293) cells in the dish are carried to the center of the coupling location, forming a cluster in less than 10 min. A few minutes later, these clusters may then be translated and merged to form large agglomerations, and even repeatedly folded to produce a roughly spherical shape of over 1.4 mm in diameter for incubation-without damaging the existing intercellular bonds. Calcium ion signaling through these clusters and confocal images of multiprotein junctional complexes suggest a continuous tissue construct: intercellular communication. They may be formed at will, and the method is feasibly useful for formation of numerous agglomerates in a single petri dish.
Topics: Acoustics; Animals; Cell Communication; Culture Media; HEK293 Cells; Humans; Sheep; Sound
PubMed: 35596837
DOI: 10.1007/s10544-022-00617-z -
Journal of Cellular Biochemistry Nov 2010Stem cells always balance between self-renewal and differentiation. Hence, stem cell culture parameters are critical and need to be continuously refined according to...
Stem cells always balance between self-renewal and differentiation. Hence, stem cell culture parameters are critical and need to be continuously refined according to progress in our stem cell biology understanding and the latest technological developments. In the past few years, major efforts have been made to define more precisely the medium composition in which stem cells grow or differentiate. This led to the progressive replacement of ill-defined additives such as serum or feeder cell layers by recombinant cytokines or growth factors. Another example is the control of the oxygen pressure. For many years cell cultures have been done under atmospheric oxygen pressure which is much higher than the one experienced by stem cells in vivo. A consequence of cell metabolism is that cell culture conditions are constantly changing. Therefore, the development of high sensitive monitoring processes and control algorithms is required for ensuring cell culture medium homeostasis. Stem cells also sense the physical constraints of their microenvironment. Rigidity, stiffness, and geometry of the culture substrate influence stem cell fate. Hence, nanotopography is probably as important as medium formulation in the optimization of stem cell culture conditions. Recent advances include the development of synthetic bioinformative substrates designed at the micro- and nanoscale level. On going research in many different fields including stem cell biology, nanotechnology, and bioengineering suggest that our current way to culture cells in Petri dish or flasks will soon be outdated as flying across the Atlantic Ocean in the Lindbergh's plane.
Topics: Animals; Bioreactors; Cell Culture Techniques; Humans; Nanotechnology; Stem Cell Niche; Stem Cells
PubMed: 20803548
DOI: 10.1002/jcb.22847 -
Gels (Basel, Switzerland) May 2023Macroscopic spatial patterns were formed in calcium alginate gels when a drop of a calcium nitrate solution was placed on the center of a sodium alginate solution on a...
Macroscopic spatial patterns were formed in calcium alginate gels when a drop of a calcium nitrate solution was placed on the center of a sodium alginate solution on a petri dish. These patterns have been classified into two groups. One is multi-concentric rings consisting of alternating cloudy and transparent areas observed around the center of petri dishes. The other is streaks extending to the edge of the petri dish, which are formed to surround the concentric bands between the concentric bands and the petri dish edge. We have attempted to understand the origins of the pattern formations using the properties of phase separation and gelation. The distance between two adjacent concentric rings was roughly proportional to the distance from where the calcium nitrate solution was dropped. The proportional factor increased exponentially for the inverse of the absolute temperature of the preparation. The also depended on the concentration of alginate. The pattern characteristics in the concentric pattern agreed with those in the Liesegang pattern. The paths of radial streaks were disturbed at high temperatures. The length of these streaks shortened with increasing alginate concentration. The characteristics of the streaks were similar to those of crack patterns resulting from inhomogeneous shrinkage during drying.
PubMed: 37367115
DOI: 10.3390/gels9060444 -
Biomicrofluidics Jan 2021Many wound-healing assays are used in cell biology and biomedicine; they are often labor intensive and/or require specialized and costly equipment. We describe a...
Many wound-healing assays are used in cell biology and biomedicine; they are often labor intensive and/or require specialized and costly equipment. We describe a contactless method to create wounds with any imaginable 2D pattern in cell monolayers using the micro-jets of either media or an immiscible and biocompatible fluorocarbon (i.e., FC40). We also combine this with another method that allows automation and multiplexing using standard Petri dishes. A dish is filled with a thin film of media overlaid with FC40, and the two liquids are reshaped into an array of microchambers within minutes. Each chamber in such a grid is isolated from others by the fluid walls of FC40. Cells are now added, allowed to grow into a monolayer, and wounds are created using the microjets; then, healing is monitored by microscopy. As arrays of chambers can be made using media and Petri dishes familiar to biologists, and as dishes fit seamlessly into their incubators, microscopes, and workflows, we anticipate that this assay will find wide application in wound healing.
PubMed: 33598064
DOI: 10.1063/5.0043312 -
PloS One 2022This study presents novel biocompatible Polydimethylsiloxane (PDMS)-based micromechanical tweezers (μTweezers) capable of the stiffness characterization and...
This study presents novel biocompatible Polydimethylsiloxane (PDMS)-based micromechanical tweezers (μTweezers) capable of the stiffness characterization and manipulation of hydrogel-based organoids. The system showed great potential for complementing established mechanical characterization methods such as Atomic Force Microscopy (AFM), parallel plate compression (PPC), and nanoindentation, while significantly reducing the volume of valuable hydrogels used for testing. We achieved a volume reduction of ~0.22 μl/sample using the μTweezers vs. ~157 μl/sample using the PPC, while targeting high-throughput measurement of widely adopted micro-mesoscale (a few hundred μm-1500 μm) 3D cell cultures. The μTweezers applied and measured nano-millinewton forces through cantilever' deflection with high linearity and tunability for different applications; the assembly is compatible with typical inverted optical microscopes and fit on standard tissue culture Petri dishes, allowing mechanical compression characterization of arrayed 3D hydrogel-based organoids in a high throughput manner. The average achievable output per group was 40 tests per hour, where 20 organoids and 20 reference images in one 35 mm petri dish were tested, illustrating efficient productivity to match the increasing demand on 3D organoids' applications. The changes in stiffness of collagen I hydrogel organoids in four conditions were measured, with ovarian cancer cells (SKOV3) or without (control). The Young's modulus of the control group (Control-day 0, E = 407± 146, n = 4) measured by PPC was used as a reference modulus, where the relative elastic compressive modulus of the other groups based on the stiffness measurements was also calculated (control-day 0, E = 407 Pa), (SKOV3-day 0, E = 318 Pa), (control-day 5, E = 528 Pa), and (SKOV3-day 5, E = 376 Pa). The SKOV3-embedded hydrogel-based organoids had more shrinkage and lowered moduli on day 0 and day 5 than controls, consistently, while SKOV3 embedded organoids increased in stiffness in a similar trend to the collagen I control from day 0 to day 5. The proposed method can contribute to the biomedical, biochemical, and regenerative engineering fields, where bulk mechanical characterization is of interest. The μTweezers will also provide attractive design and application concepts to soft membrane-micro 3D robotics, sensors, and actuators.
Topics: Cell Culture Techniques; Cell Line, Tumor; Female; Humans; Hydrogels; Middle Aged; Organoids; Stress, Mechanical
PubMed: 35073389
DOI: 10.1371/journal.pone.0262950 -
Biomolecules Nov 2021Microbial biofilms formed by pathogenic and antibiotic-resistant microorganisms represent a serious threat for public health in medicine and many industrial branches....
Microbial biofilms formed by pathogenic and antibiotic-resistant microorganisms represent a serious threat for public health in medicine and many industrial branches. Biofilms are involved in many persistent and chronic infections, the biofouling of water and food contamination. Therefore, current research is involved in the development of new treatment strategies. Biofilm is a complex system, and thus all aspects of the measurement and monitoring of its growth and eradication in various conditions, including static and dynamic flow, are issues of great importance. The antibiofilm character of rhamnolipid mixtures produced by four strains was studied under different conditions. For this purpose, the biofilm of opportunistic pathogen was used and treated under static conditions (microscope glass coverslip in a Petri dish) and under dynamic conditions (a single-channel flow cell). The results show that the biological activity of rhamnolipids depends both on their properties and on the conditions of the biofilm formation. Therefore, this aspect must be taken into account when planning the experimental or application design.
Topics: Basidiomycota; Biofilms; Glycolipids
PubMed: 34827725
DOI: 10.3390/biom11111727 -
Veterinary Research 2005Mannheimia haemolytica induced pneumonias are only observed in goats, sheep and cattle. The bacterium produces several virulence factors,whose principal ones are... (Review)
Review
Mannheimia haemolytica induced pneumonias are only observed in goats, sheep and cattle. The bacterium produces several virulence factors,whose principal ones are lipopolysaccharide and leukotoxin. The latter is cytotoxic only for ruminant leukocytes, a phenomenon that is correlated with its ability to bind and interact with the ruminant beta2-integrin Lymphocyte Function-associated Antigen 1. This paper globally reviews all the information available on host-pathogen interactions underlying respiratory mannheimiosis (formerly pasteurellosis), from the stable and the Petri dish to the biochemical cascade of events triggered by the leukotoxin inside ruminant leukocytes. One conclusion can be made: the most widespread cattle respiratory disease with the most important impact on beef production worldwide, is probably due to a tiny ruminant-specific focal variation in the CD18- and/or CD11a-expressing genes.
Topics: Animals; Bacterial Toxins; Cattle; Exotoxins; Lipopolysaccharides; Mannheimia haemolytica; Pasteurellosis, Pneumonic; Virulence Factors
PubMed: 15720968
DOI: 10.1051/vetres:2004065 -
Materials (Basel, Switzerland) Jul 2016The biodegradable cellular capsule, being prepared from simple vaporization of liquid marbles, is an ideal vehicle for the potential application of drug encapsulation...
The biodegradable cellular capsule, being prepared from simple vaporization of liquid marbles, is an ideal vehicle for the potential application of drug encapsulation and release. This paper reports the fabrication of cellular capsules via facile vaporization of Pickering emulsion marbles in an ambient atmosphere. Stable Pickering emulsion (water in oil) was prepared while utilizing dichloromethane (containing poly(l-lactic acid)) and partially hydrophobic silica particles as oil phase and stabilizing agents respectively. Then, the Pickering emulsion marbles were formed by dropping emulsion into a petri dish containing silica particles with a syringe followed by rolling. The cellular capsules were finally obtained after the complete vaporization of both oil and water phases. The technique of scanning electron microscope (SEM) was employed to research the microstructure and surface morphology of the prepared capsules and the results showed the cellular structure as expected. An in vitro drug release test was implemented which showed a sustained release property of the prepared cellular capsules. In addition, the use of biodegradable poly(l-lactic acid) and the biocompatible silica particles also made the fabricated cellular capsules of great potential in the application of sustained drug release.
PubMed: 28773693
DOI: 10.3390/ma9070572 -
Scientific Reports Dec 2022In nature, bacteria prevailingly reside in the form of biofilms. These elaborately organized surface-bound assemblages of bacterial cells show numerous features of...
In nature, bacteria prevailingly reside in the form of biofilms. These elaborately organized surface-bound assemblages of bacterial cells show numerous features of multicellular organization. We recently showed that biofilm growth is a true developmental process, which resembles developmental processes in multicellular eukaryotes. To study the biofilm growth in a fashion of eukaryotic ontogeny, it is essential to define dynamics and critical transitional phases of this process. The first step in this endeavor is to record the gross morphological changes of biofilm ontogeny under standardized conditions. This visual information is instrumental in guiding the sampling strategy for the later omics analyses of biofilm ontogeny. However, none of the currently available visualizations methods is specifically tailored for recording gross morphology across the whole biofilm development. To address this void, here we present an affordable Arduino-based approach for time-lapse visualization of complete biofilm ontogeny using bright field stereomicroscopy with episcopic illumination. The major challenge in recording biofilm development on the air-solid interphase is water condensation, which compromises filming directly through the lid of a Petri dish. To overcome these trade-offs, we developed an Arduino microcontroller setup which synchronizes a robotic arm, responsible for opening and closing the Petri dish lid, with the activity of a stereomicroscope-mounted camera and lighting conditions. We placed this setup into a microbiological incubator that maintains temperature and humidity during the biofilm growth. As a proof-of-principle, we recorded biofilm development of five Bacillus subtilis strains that show different morphological and developmental dynamics.
Topics: Time-Lapse Imaging; Microscopy; Bacteria
PubMed: 36476631
DOI: 10.1038/s41598-022-24431-y -
Materials (Basel, Switzerland) Aug 2019The wound-healing assay is commonly and widely used for investigating collective cell migration under various physical and chemical stimuli. Substrate-coating materials...
The wound-healing assay is commonly and widely used for investigating collective cell migration under various physical and chemical stimuli. Substrate-coating materials are shown to affect the wound-healing process in a cell-type dependent manner. However, experiment-to-experiment variations make it difficult to compare results from different assays. In this paper, a modified barrier wound-healing assay was reported for studying the wound-healing process on different substrates in one single petri dish. In short, half of a dish was covered with the tape, and coating materials, poly-l-lysine and gelatin, were applied to the surface. After peeling off the tape, half of the surface was coated with the desired material. Then a customized barrier was placed inside the dish to create the wound. The results indicated that surface coating did not affect cell proliferation/viability, and the wound-healing rate increased in coated surfaces compared to uncoated ones. The present study provides a platform for further understanding the mechanisms of substrate coating-dependent wound-healing processes.
PubMed: 31470524
DOI: 10.3390/ma12172775