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Background Electrocautery is a common surgical technique and is often used during shoulder arthroplasty to elevate or transect the subscapularis tendon. The relative amount of tissue damage caused by cautery as opposed to sharp transection is not currently known. The purpose of this study was to examine local tissue damage resulting from electrocautery vs. sharp transection with a scalpel. We hypothesized that the electrosurgical unit would cause higher collateral tissue damage and cell death compared with sharp transection. Methods Twelve cadaveric ovine shoulders were randomized to either the electrosurgical or sharp transection group. The infraspinatus tendon was isolated, and a partial-thickness transection was made using either a monopolar electrosurgical device (Bovie) or No. 10 scalpel blade. Tendon explants were then visualized with confocal microscopy to evaluate tissue architecture. A live/dead assay was performed using microscopy imaging analysis software. Comparisons between Bovie and scalpel transection were made using the Mann-Whitney U test, and the cell death percentage at standardized distances from the transection site was compared between groups using a mixed-model analysis. Significance was defined at P Results The cellular and tendon fibril architecture was well maintained beyond the scalpel transection site, whereas Bovie transection disrupted the architecture beyond its transection path. The percentage of dead cells in the Bovie group (74.9% ± 31.2%) was significantly higher than that in the scalpel group (27.6% ± 29.9%, P = .0004). Compared with the transection site, the cell death percentage after Bovie transection significantly declined at 2.5 mm whereas that after scalpel transection significantly declined at 1 mm from the transection site. Conclusion There was a significantly higher dead cell percentage in the Bovie transection group, indicating extensive damage beyond the local incision site, compared with sharp transection. Electrosurgical transection of the ovine infraspinatus tendon ex vivo caused higher cell death and greater tissue architecture disruption compared with sharp scalpel transection.

Phagocytic clearance of dead cells and debris is critical for inflammation resolution and maintenance of tissue homeostasis. Consequently, defective clearance of dead cells and debris is associated with initiation and exacerbation of several autoimmune disorders and chronic inflammatory diseases such as atherosclerosis. The progressive loss of dead cell clearance capacity within the atherosclerotic plaque leads to accumulation of necrotic cells, chronic non-resolving inflammation, and expansion of the necrotic core, which triggers atherosclerotic plaque rupture and clinical manifestation of acute thrombotic cardiovascular adverse events. In this review, we describe the fundamental molecular and cellular mechanisms of dead cell clearance and how it goes awry in atherosclerosis. Finally, we highlight novel therapeutic strategies that enhance dead cell and debris clearance within the atherosclerotic plaque to promote inflammation resolution and atherosclerotic plaque stabilization.

Coronary artery disease is the "first killer" in the world, while the classical treatment for this disease is to implant stent. An ideal vascular stent should be nontoxic with self-expanding characteristics, quick expanding speed, and appropriate mechanical supporting property. However, no existing vascular stent covers all properties. Herein, a two-way shape-memory cellulose vascular stent, which can realize shape adjustments by mild solutions such as water and alcohol, is constructed. The shape-memory characteristics, mechanical properties, cell toxicity, and biocompatibility, are systemically investigated by ex vivo experiment as well as molecule simulation and theoretical modeling, revealing that the achieved bilayer two-way shape-memory films (BSMFs) can be used as an artificial vascular stent. In particular, this vascular stent made from BSMFs shows superb biocompatibility according to live/dead cell viability assays. Ex vivo experiments reveal that the novel vascular stent can support arteria coronaria sinistra, or the left main coronary artery, at the opening state while the cross-section of the vessel becomes two times larger than that of the initial state after implantation. Thus, it is believed that effective and scalable BSMFs can make meritorious fundamental contributions to biomaterials science and practical applications such as vascular stents.

Dielectrophoresis (DEP) is a non-destructive, accurate, and label-free cell manipulating technique and DEP applications have been found in various fields. Assessment of cell viability is one of the important applications and many investigations have been reported. In this paper, cell polarization and its modeling, some key parameters employed for living/dead cell separation, as well as electrode configurations are reviewed. Focus is given to the latest development of DEP devices employed for the assessment of cell viability. Experimentally determined factors for separating living/dead cells, such as the conductivity of suspending medium and the frequency of applied electric field, are summarized. The future directions and potential challenges in this field are also outlined.

To overcome the mechanical drawback of bioink, we proposed a supporter model to enhance the mechanical strength of bioprinted 3D constructs, in which a unit-assembly idea was involved. Based on Computed Tomography images of critical-sized rabbit bone defect, the 3D re-construction was accomplished by a sequenced process using Mimics 17.0, BioCAM and BioCAD software. 3D constructs were bioprinted using polycaprolactone (PCL) ink for the outer supporter under extrusion mode, and cell-laden tricalcium phosphate (TCP)/alginate bioink for the inner filler under air pressure dispensing mode. The relationship of viscosity of bioinks, 3D bioprinting pressure, TCP/alginate ratio and cell survival were investigated by the shear viscosities analysis, live/dead cell test and cell-counting kit 8 measurement. The viscosity of bioinks at 1.0 s−1-shear rate could be adjusted within the range of 1.75 ± 0.29 Pa·s to 155.65 ± 10.86 Pa·s by changing alginate concentration, corresponding to 10 kPa–130 kPa of printing pressure. This design with PCL supporter could significantly enhance the compressive strength and compressive modulus of standardized 3D mechanical testing specimens up to 2.15 ± 0.14 MPa to 2.58 ± 0.09 MPa, and 42.83 ± 4.75 MPa to 53.12 ± 1.19 MPa, respectively. Cells could maintain the high viability (over 80%) under the given printing pressure but cell viability declined with the increase of TCP content. Cell survival after experiencing 7 days of cell culture could be achieved when the ratio of TCP/alginate was 1 : 4. All data supported the feasibility of the supporter and unit-assembly model to enhance mechanical properties of bioprinted 3D constructs.

The outer skin layer in any plant is essential in offering a protective barrier against water loss and pathogen attack. Within fleshy fruit, the skin supports internal cell layers and can provide the initial cues in attracting seed-dispersing animals. The skin of a fruit, termed the exocarp, is a key element of consumer preference and a target for many breeding programs. Across fruiting species there is a huge diversity of exocarp types and these range from a simple single living cell layer (epidermis) often covered with a waxy layer, to complex multicellular suberised and dead cell layers (periderm), with various intermediate russet forms in between. Each exocarp can be interspersed with other structures such as hairs or spines. The epidermis has been well characterised and remains pluripotent with the help of the cells immediately under the epidermis. The periderm, in contrast, is the result of secondary meristematic activity, which replaces the epidermal layers, and is not well characterised in fruits. In this review we explore the structure, composition and mechanisms that control the development of a periderm type fruit exocarp. We draw upon literature from non-fleshy fruit species that form periderm tissue, from which a considerable amount of research has been undertaken.

The damage-associated molecular pattern molecules (DAMPs) released by necrotic cells can trigger inflammatory response, which will facilitate the clearance of these dead cells. Neutrophil mobilization is a very important step for the dead cell clearance, however the detailed mechanisms for DAMPs induce neutrophil mobilization remains largely elusive. In this study, by using a necrotic cell-induced neutrophil mobilization mice model, we found that both neutrophil number and percentage rapidly (as early as 30 min) increased with necrotic cells but not live cell treatment. CXCL1 was rapidly increased in the serum and was responsible for the neutrophil mobilization when treated with necrotic cells. We further demonstrated that the hepatocytes in the liver were the main source of CXCL1 production in response to necrotic cells challenge. However, the hepatocytes did not express CXCL1 when incubating with necrotic cells alone. When Kupffer cells were ablated, the increased CXCL1 levels as well as neutrophil mobilization were abolished with necrotic cells challenge. Moreover, we clarified Kupffer cells-derived TNF-α activates the NF-κB pathway in hepatocytes and promote hepatocytes to express CXCL1. In summary, we showed that the liver is the main source for necrotic cell-induced CXCL1 production and neutrophil mobilization. Kupffer cells in the liver sense DAMPs and release TNF-α to activate the NF-κB pathway in hepatocytes. The interaction between Kupffer cells and hepatocytes is critical for CXCL1 production.

Recognition and removal of apoptotic cells by professional phagocytes, including dendritic cells and macrophages, preserves immune self-tolerance and prevents chronic inflammation and autoimmune pathologies. The diverse array of phagocytes that reside within different tissues, combined with the necessarily prompt nature of apoptotic cell clearance, makes it difficult to study this process in situ. The full spectrum of functions executed by tissue-resident phagocytes in response to homeostatic apoptosis, therefore, remains unclear. Here we show that mouse apoptotic intestinal epithelial cells (IECs), which undergo continuous renewal to maintain optimal barrier and absorptive functions, are not merely extruded to maintain homeostatic cell numbers, but are also sampled by a single subset of dendritic cells and two macrophage subsets within a well-characterized network of phagocytes in the small intestinal lamina propria. Characterization of the transcriptome within each subset before and after in situ sampling of apoptotic IECs revealed gene expression signatures unique to each phagocyte, including macrophage-specific lipid metabolism and amino acid catabolism, and a dendritic-cell-specific program of regulatory CD4

Molecules that fluoresce upon binding DNA are widely used in assaying and visualizing DNA in cells and tissues. However, using light to visualize DNA in animals is limited by the attenuation of light transmission by biological tissues. Moreover, it is now clear that DNA is an important mediator of dead cell clearance, coagulation reactions, and an immunogen in autoimmune lupus. Attaching metals (e.g., superparamagnetic nanoparticles, gadolinium ions, radioactive metal ions) to DNA-binding fluorochromes provides a way of imaging DNA in whole animals, and potentially humans, without light. Imaging metal-bearing, DNA-binding fluorochromes and their target DNA by magnetic resonance imaging may shed light on the many key roles of DNA in health and disease beyond the storage of genetic information.

To evaluate in vitro IGF-I treatment during warming of storage-cooled boar semen and its effect on seminal quality parameters and metabolism in spermatic cells.Semen samples (n=7) warmed after stored at 15°C for 24 or 72h were divided into four equal parts. Different IGF-I concentrations (0, 50, 100 and 150ng/mL) were added to the semen samples. The samples were incubated at 37°C, and assessments were made after 0 and 120min of incubation.For semen samples that were stored for 24h, the addition of IGF-I had no effect (p0.05) on the total motility and intensity of movements by spermatic cells, osmotic resistance, live:dead cell ratio or total spermatic abnormalities. However, incubation with 150ng/mL IGF-I did decrease glutathione peroxidase activity (p0.05) and reduce lipid peroxidation after 120min of incubation. For semen samples stored for 72h and incubated with IGF-I for 120min, there was a linear relationship between the IGF-I concentration and the live:dead ratio (p0.05). There was a quadratic relationship between the IGF-I concentration and both the osmotic resistance (peak results at IGF-I=62.4ng/mL) and glutathione peroxidase activity (peak results at IGF-I=77.8ng/mL). There was no effect on lipid peroxidation (p0.05) after 120min of incubation. Addition of IGF-I also decreased fructose utilization by spermatic cells regardless of semen storage time (p0.05).This study suggests that IGF-I may be beneficial to semen stored for longer periods of time. Adding 150ng/mL IGF-I improved the quality of semen stored for 24h, and adding 78ng/mL IGF-I improved the quality of semen stored for 72h.

The existing models of hepatitis B virus (HBV) infection dynamics are based on the assumption that the populations of viruses and cells are uniformly mixed. However, the real virus infection system is actually not homogeneous and some spatial factors might play a nontrivial role in governing the development of HBV infection and its outcome. For instance, the localized populations of dead cells might adversely affect the spread of infection. To consider this kind of inhomogeneous feature, a simple 2D (dimensional) probability Cellular Automaton model was introduced to study the dynamic process of HBV infection. The model took into account the existence of different types of HBV infectious and non-infectious particles. The simulation results thus obtained showed that the Cellular Automaton model could successfully account for some important features of the disease, such as its wide variety in manifestation and its age dependency. Meanwhile, the effects of the model's parameters on the dynamical process of the infection were also investigated. It is anticipated that the Cellular Automaton model may be extended to serve as a useful vehicle for studying, among many other complicated dynamic biological systems, various persistent infections with replicating parasites.

The lifespan of an acute brain slice is approximately 6–12 hours, limiting potential experimentation time. We have designed a new recovery incubation system capable of extending their lifespan to more than 36 hours. This system controls the temperature of the incubated artificial cerebral spinal fluid (aCSF) while continuously passing the fluid through a UVC filtration system and simultaneously monitoring temperature and pH. The combination of controlled temperature and UVC filtering maintains bacteria levels in the lag phase and leads to the dramatic extension of the brain slice lifespan. Brain slice viability was validated through electrophysiological recordings as well as live/dead cell assays. This system benefits researchers by monitoring incubation conditions and standardizing this artificial environment. It further provides viable tissue for two experimental days, reducing the time spent preparing brain slices and the number of animals required for research.

A flow cytometer can quickly perform numerous quantitative, sensitive measurements on each individual cell within a large, heterogeneous population. The modern commercially available analytical instruments, which can be found in most hospitals, pathology laboratories, and cell biology research laboratories in the industrially developed countries, can now routinely measure fluorescence simultaneously at four different wavelengths, in addition to light scatter in two directions, at rates of thousands of cells per second. Thus, flow cytometry provides a powerful and versatile approach to the measurement of cell death and cell killing. However, it is important to stress that the biological nature of the phenomenon under investigation will determine which assay is appropriate, and at what stage in the process of cell killing it should be applied. This consideration is particularly important in the study of drug- or cell-mediated cytotoxicity.

A structured mathematical model describing the dynamics of hybridoma growth and monoclonal antibody (mAb) production in suspension cultures is presented. The model fits well to experimental data obtained from batch, fed-batch, and continuous cultures with hybridoma cells. Applications of the model for process control are demonstrated. 1. An extended Kalman filter (EKF) was designed to estimate the state of the process. The oxygen consumption rate of the cell culture is monitored on-line and is used as the only measurement information for the EKF. This EKF is able to provide good estimates for living and dead cell densities and the medium composition. 2. The mathematical model can be applied for the optimization of fed-batch cultures.

The live/dead fluorescent assay provides a quick method for assessing the proportion of live and dead cells in cell culture systems or tissues and is widely used. Dead cells are detected by the fluorescence produced when propidium iodide (PI) binds to DNA; PI and similar molecules are excluded from live cells but can penetrate dead cells because of their loss of membrane integrity. Here we investigated the effect of serum in the culture medium on the reliability of the method. We assessed viability of chondrocytes with/without serum using both a live/dead assay kit and also trypan blue staining. We found that after 2 days of culture, the DNA-binding dye PI could no longer detect dead cells if serum was present but they were readily detected in serum-free medium or if an inhibitor to DNase I was added to the serum-containing medium. Dead cells could be detected by trypan blue staining in all cultures. Hence dead cells are no longer detected as the DNase I present in serum degrades their DNA. DNA-binding dyes may thus not give a reliable estimate of the number of dead cells in systems that have been cultured in the presence of serum for several days.

In vitro studies on Swiss mice, inoculated with dead or live sarcoma 180-A ascites cells, were carried out to monitor the changes, if any, in the longitudinal (T1) and transverse (T2) relaxation times of tissue water protons following hyperthermic treatment and subsequent thermotolerance. Both relaxation processes exhibited biexponential relaxation curves. With increasing size of the tumours, the longitudinal relaxation behaviour changed from bi- to mono-exponential. This was not observed for the transverse relaxation phenomenon. Inoculation with either dead or live cells caused an immediate increase in the both relaxation times. In the case of dead cell inoculation, the increase lasted for only 24 h after which the relaxation times became the same as for the uninoculated controls. The transverse relaxation time increased as a result of exposure to hyperthermia. During development of thermotolerance, both the relaxation times decreased.

In the present study, melatonin was tested in subsequent doses in glutamate induced neurotoxicity in cerebellar granular cell culture of rat pups. Glutamate at 10(-7) M was found to induce neuronal cell death. The dead cell score was 2.75+/-0.7 in the control, while it was found to be 35.12+/-1.8 in the glutamate-administered group (P0.0001). Melatonin very potently blocked glutamate neurotoxicity at all doses tested, with 10(-3) M, the highest dose tested, being the most effective. Glutamate may exert a neuroprotective effect by blocking one or more steps of the oxidation cascade in neurons and this effect may be blocked by melatonin.

Several different assays are commonly used to evaluate survival of cells inside tissues or three-dimensional carriers, but their accuracy and reliability have not been evaluated. Here, we compare three methods for cell viability (CV) determination: (i) lactate dehydrogenase (LDH) staining on cryosections, (ii) calcein AM/ethidium homodimer-1 (CaAM/EthH) staining, and (iii) carrier digestion and trypan blue (TB) assay. Living and dead cell populations were generated from bovine chondrocytes and combined to produce approximately 0%, 25%, 50%, 75%, and 100% CV mixtures. CV ratios were measured with TB assay (MIX) before seeding cells into fibrin carriers. CV was then determined using the three methods (n=5/method). Custom-written macros were used to process LDH- and CaAM/EthH-stained images, and hand counting with hemocytometer was used for the TB method. Absolute error and intraclass correlation (ICC) were used for accuracy and reliability evaluation. All methods estimated CV values close to MIX values. TB method was the most accurate (ICC=0.99) followed by CaAM/EthH (ICC=0.98) and LDH (ICC=0.97). As for absolute quantification of living and dead cells, TB and LDH methods performed well (ICC=0.75-0.96), whereas CaAM/EthH largely overestimated cell numbers (living, ICC=0.30; dead, ICC=0.30). Although TB was the most accurate, LDH and CaAM/EthH provide valuable information on cell shape and spatial distribution of cells in tissue or a scaffold. © Copyright 2008, Mary Ann Liebert, Inc.

Assessment of cell viability is a key issue in monitoring in vitro engineered tissue constructs. In this study we describe a fully automated, quantitative, and nondestructive approach, which is particularly suitable for tissue engineering. The approach offers several advantages above existing methods. Living and dead cell numbers can be separately determined for both isolated cells and cells that form networks during tissue formation. Moreover, viability can be locally monitored in time throughout the three-dimensional tissue. The viability assay is based on a dual fluorescent staining technique using CellTracker Green (CTG) for detection of living cells and propidium iodide (PI) for dead cells. CTG and PI images are created with a confocal laser scanning microscope. To determine the number of living cells, CTG fluorescence intensity is determined from the CTG image. Thereby, novel image-processing techniques have been developed, normalizing for various undesired influences that alter measurements of absolute CTG fluorescence intensities. Dead cell numbers are determined from the PI image, using an improved computerized counting method. The approach was first evaluated on C2C12 monolayers, of which images were taken directly after probe addition and 24 h later. Results show that at both times, computed living and dead cell numbers highly correlate with manually counted cell numbers (r > 0.996). Next, the approach was applied for monitoring viability in three-dimensional engineered skeletal muscle tissue constructs, which were subjected to unfavorable environmental conditions. This example illustrated that local viability can be quantitatively, nondestructively, and locally monitored in three-dimensional tissue constructs, making it a promising tool in the field of tissue engineering.

Cell viability may be judged by morphological changes or by changes in membrane permeability and/or physiological state inferred from the exclusion of certain dyes or the uptake and retention of others. This unit presents methods based on dye exclusion, esterase activity, and mitochondrial membrane potential, as well as protocols for determining the pre-fixation viability of fixed cells either before or after fixation with amine-reactive dyes suitable for a range of excitation wavelengths. Membrane-impermeable dead cell and live cell dyes as well as dye-exclusion procedures for microscopy are also included. Curr. Protoc. Cytom. 64:9.2.1-9.2.26. © 2013 by John Wiley & Sons, Inc. Keywords: cytometry; flow cytometry; imaging; probes; toxicology; assessment of cell toxicity

This unit addresses the difficult and important area of differentiation between viable and nonviable cells, a question of particular interest for DNA studies. The basic protocol describes the determination of DNA content using pyronin Y combined with dead cell discrimination using 7-AAD. Alternate protocols present methods for DNA content and viability assessment and simultaneous surface or intracellular antigen expression.

Equine embryos recovered on Day 6 after ovulation were cooled to +4 degrees C, or frozen with AFP alone or together with glycerol. Twenty embryos (140-200 microm in diameter) were randomly assigned to 6 treatment groups. In the first 3 groups, the embryos were cooled from room temperature to +4 degrees C at a rate of 3 degrees C/min and warmed again at a rate of 32 degrees C/min in a programmable freezer. In the second 3 groups, the embryos were frozen using a standard protocol, stored in liquid nitrogen for 5-7 days and then thawed in a 37 degrees C waterbath. After cooling/warming or freezing/thawing all the embryos were stained with DAPI. The percentage of dead cell area was significantly lower in the cooling groups than in the freezing groups and no significant differences were apparent between the cryoprotectants used in the study.

Radiation-induced changes in the surface morphology of rat thymocytes in suspension were evaluated by scanning electron microscopy. Viable (erythrosin B unstainable) cells were gene. rally rounded with their surface exhibiting undulations of short microvilli. Exposing the cell suspension to 1 kR X-rays and 4 hour incubation at 37°C caused disappearance of microvilli. Association of dead (erythrosin B stainable) thymocytes with smooth, occasionally porous, surface without any digitations was confirmed by the examination of enriched dead cell populations.

The contact potential difference at the cell membranein vitro-air interface was measured using the dynamic oondensator method. The potential differences were found to be different for various types of living and dead cells.

A novel organoid culture was developed in which hepatocytes maintain high liver functions for more than several weeks in vitro. The main disadvantage of tissue-engineered organoids is the lack of a blood vessel structure between the aggregated cells. Because of depletion of oxygen, the thickness from the surface of an organoid at which hepatocytes can survive is limited. This study showed that a rat hepatocyte organoid that forms by using centrifugal force in a hollow fiber (HF) had a survival limit thickness of about 80 - 100 μm from the surface of the organoid. Based on the value, we designed an elliptic HF having less than 150 μm minor diameter by using a simple annealing method. All hepatocytes were supplied with oxygen and formed an organoid without a dead cell layer in this HF. A hepatocyte organoid in an elliptic HF maintained ammonia removal activity twice as high as in the original HF for at least one month during culture. Albumin secretion activity of an organoid in an elliptic HF was also maintained for at least one month and was the same level as that of liver in a living body. In conclusion, organoid culture by using an elliptic HF seems to be a promising technique to develop a hybrid artificial liver.

An appropriately modified cell spectrophotometer was used successfully for performing automatic counts of live and dead cells in the cytotoxic test, with trypan blue staining as an indicator of dead cells and light scattering to identify viable cells.