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Annals of Translational Medicine Mar 2020Although the cuff technique in rat lung transplantation (LTx) has a long history, it remains technically challenging. We have developed key tricks and modifications in...
BACKGROUND
Although the cuff technique in rat lung transplantation (LTx) has a long history, it remains technically challenging. We have developed key tricks and modifications in the devices and the cuff technique that optimize the rat LTx model to achieve successful operations during a short learning period.
METHODS
Altogether, 180 consecutive rats underwent orthotopic left LTx performed by a single surgeon using our modified devices and procedures. Allogeneic and syngeneic transplantation were performed using Lewis rats as recipients and Brown Norway and Lewis rats as donors. Allogeneic recipients were treated with cyclosporine during the first week. Recipients were sacrificed at various time points after ≥2 weeks.
RESULTS
A special cuff-preparation plate was created using a petri dish and two foam blocks. This modified plate stabilizes the preparation and prevents donor lung compression. A "┴"-shaped incision was carved into the front wall of the pulmonary artery (PA) using micro-scissors. "V"-shaped incisions were made from the inferior-to-superior branches of the pulmonary vein (PV) and bronchus. A "pendulum model" was applied at implantation to make the hilar anastomosis tension-free and technically easier to perform. There were no intraoperative complications. Ten rats (5.6%) experienced partial or full pulmonary atelectasis. Five deaths (2.8%) due to pleural effusion occurred during the follow-up period. The operative times for heart-lung block retrieval, cuff preparation, cold ischemia, warm ischemia, and total procedure time were 8.4±0.8, 11.6±1.5, 25.1±2.2, 8.1±1.2, and 46.7±2.8 min, respectively.
CONCLUSIONS
The key tricks and improvements we made in the cuff technique for rat LTx provided the advantages of expeditiousness, a low complication rate, and a high success rate.
PubMed: 32355851
DOI: 10.21037/atm.2020.02.46 -
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 -
Micromachines Sep 2020In this paper, we report on a novel biocompatible micromechanical bioreactor (actuator and sensor) designed for the in situ manipulation and characterization of live...
In this paper, we report on a novel biocompatible micromechanical bioreactor (actuator and sensor) designed for the in situ manipulation and characterization of live microtissues. The purpose of this study was to develop and validate an application-targeted sterile bioreactor that is accessible, inexpensive, adjustable, and easily fabricated. Our method relies on a simple polydimethylsiloxane (PDMS) molding technique for fabrication and is compatible with commonly-used laboratory equipment and materials. Our unique design includes a flexible thin membrane that allows for the transfer of an external actuation into the PDMS beam-based actuator and sensor placed inside a conventional 35 mm cell culture Petri dish. Through computational analysis followed by experimental testing, we demonstrated its functionality, accuracy, sensitivity, and tunable operating range. Through time-course testing, the actuator delivered strains of over 20% to biodegradable electrospun poly (D, L-lactide-co-glycolide) (PLGA) 85:15 non-aligned nanofibers (~91 µm thick). At the same time, the sensor was able to characterize time-course changes in Young's modulus (down to 10-150 kPa), induced by an application of isopropyl alcohol (IPA). Furthermore, the actuator delivered strains of up to 4% to PDMS monolayers (~30 µm thick), simultaneously characterizing their elastic modulus up to ~2.2 MPa. The platform repeatedly applied dynamic (0.23 Hz) tensile stimuli to live Human Dermal Fibroblast (HDF) cells for 12 hours (h) and recorded the cellular reorientation towards two angle regimes, with averages of -58.85° and +56.02°. The device biocompatibility with live cells was demonstrated for one week, with no signs of cytotoxicity. We can conclude that our PDMS bioreactor is advantageous for low-cost tissue/cell culture micromanipulation studies involving mechanical actuation and characterization. Our device eliminates the need for an expensive experimental setup for cell micromanipulation, increasing the ease of live-cell manipulation studies by providing an affordable way of conducting high-throughput experiments without the need to open the Petri dish, reducing manual handling, cross-contamination, supplies, and costs. The device design, material, and methods allow the user to define the operational range based on their targeted samples/application.
PubMed: 32993158
DOI: 10.3390/mi11100892 -
Membranes Jun 2022Crosslinked carboxymethyl rice starch (CLCMRS), prepared via dual modifications of native rice starch (NRS) with chloroacetic acid and sodium trimetaphosphate, was...
Crosslinked carboxymethyl rice starch (CLCMRS), prepared via dual modifications of native rice starch (NRS) with chloroacetic acid and sodium trimetaphosphate, was employed to facilitate the disintegration of hydroxypropylmethylcellulose (HPMC) orodispersible films (ODFs), with or without the addition of glycerol. Fabricated by using the solvent casting method, the composite films, with the HPMC--LCMRS ratios of 9:1, 7:1, 5:1 and 4:1, were then subjected to physicochemical and mechanical evaluations, including weight, thickness, moisture content and moisture absorption, swelling index, transparency, folding endurance, scanning electron microscopy, Fourier transform infrared spectroscopy, tensile strength, elongation at break, and Young’s modulus, as well as the determination of disintegration time by using the Petri dish method (PDM) and slide frame and bead method (SFM). The results showed that HPMC-CLCMRS composite films exhibited good film integrity, uniformity, and transparency with up to 20% CLCMRS incorporation (4:1 ratio). Non-plasticized composite films showed no significant changes in the average weight, thickness, density, folding endurance (96−122), tensile strength (2.01−2.13 MPa) and Young’s modulus (10.28−11.59 MPa) compared to HPMC film (135, 2.24 MPa, 10.67 MPa, respectively). On the other hand, the moisture content and moisture absorption were slightly higher, whereas the elongation at break (EAB; 4.31−5.09%) and the transparency (4.73−6.18) were slightly lowered from that of the HPMC film (6.03% and 7.03%, respectively). With the addition of glycerol as a plasticizer, the average weight and film thickness increased, and the density decreased. The folding endurance was improved (to >300), while the transparency remained in the acceptable range. Although the tensile strength of most composite films decreased (0.66−1.75 MPa), they all exhibited improved flexibility (EAB 7.27−11.07%) while retaining structural integrity. The disintegration times of most composite films (PDM 109−331, SFM 70−214 s) were lower than those of HPMC film (PDM 345, SFM 229 s). In conclusion, the incorporation of CLCMRS significantly improved the disintegration time of the composite films whereas it did not affect or only slightly affected the physicochemical and mechanical characteristics of the films. The 5:1 and 4:1 HPMC:CLCMRS composite films, in particular, showed promising potential application as a film base for the manufacturing of orodispersible film dosage forms.
PubMed: 35736301
DOI: 10.3390/membranes12060594 -
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 -
SLAS Technology Apr 2023The spot assay of the budding yeast Saccharomyces cerevisiae is an experimental method that is used to evaluate the effect of genotypes, medium conditions, and...
The spot assay of the budding yeast Saccharomyces cerevisiae is an experimental method that is used to evaluate the effect of genotypes, medium conditions, and environmental stresses on cell growth and survival. Automation of the spot assay experiments from preparing a dilution series to spotting to observing spots continuously has been implemented based on large laboratory automation devices and robots, especially for high-throughput functional screening assays. However, there has yet to be an affordable solution for the automated spot assays suited to researchers in average laboratories and with high customizability for end-users. To make reproducible spot assay experiments widely available, we have automated the plate-based yeast spot assay of budding yeast using Opentrons OT-2 (OT-2), an affordable liquid-handling robot, and a flatbed scanner. We prepared a 3D-printed mount for the Petri dish to allow for precise placement of the Petri dish inside the OT-2. To account for the uneven height of the agar plates, which were made by human hands, we devised a method to adjust the z-position of the pipette tips based on the weight of each agar plate. During the incubation of the agar plates, a flatbed scanner was used to automatically take images of the agar plates over time, allowing researchers to quantify and compare the cell density within the spots at optimal time points a posteriori. Furthermore, the accuracy of the newly developed automated spot assay was verified by performing spot assays with human experimenters and the OT-2 and quantifying the yeast-grown area of the spots. This study will contribute to the introduction of automated spot assays and the automated acquisition of growth processes in conventional laboratories that are not adapted for high-throughput laboratory automation.
Topics: Humans; Saccharomyces cerevisiae; Agar; Robotics; Automation; Genotype
PubMed: 36503082
DOI: 10.1016/j.slast.2022.12.001