Cell Area Ratio Research Articles

Development of cylindrical microfibrous scaffold using melt-spinning method for vascular tissue engineering

In this report, we demonstrated fabrication of cylindrical scaffold made of poly (lactide-co-caprolactone) (PLCL) fibers for vascular tissue engineering by the melt-spinning method with use of a commercially available cotton candy machine. This method was able to generate microfibrous cylindrical scaffold with the fiber diameter ranged of 1–17 µm. It was found that the microfibrous scaffold showed greater mechanical properties including elastic modulus and ring tensile strength than those of cylindrical microporous PLCL scaffold prepared by the phase separation method. Biological test using hMSCs also demonstrated that the microfibrous scaffold exhibited better cells growth behavior including larger cell area and aspect ratio than the microporous scaffold after 3 h culture. Cell proliferation on the microfibrous scaffold was significantly higher than that on the microporous scaffold during 7 days culture. In conclusion, microfibrous scaffold with improved mechanical and biological properties was successfully fabricated using the melt-spinning method.

Improvement of islet function and survival by integration of perfluorodecalin into microcapsules in vivo and in vitro.

Hypoxic injury of islets is a major obstacle for encapsulated islet transplantation into the peritoneal cavity. To improve oxygen delivery to encapsulated islets, we integrated 20% of the oxygen carrier material, perfluorodecalin (PFD), in alginate capsules mixed with islets (PFD-alginate). Integration of PFD clearly improved islet viability and decreased reactive oxygen species production compared to islets encapsulated with alginate only (alginate) and naked islets exposed to hypoxia in vitro. In PFD-alginate capsules, HIF-1α expression was minimal, and insulin expression was well maintained. Furthermore, the best islet function represented by glucose-stimulated insulin secretion was observed for the PFD-alginate capsules in hypoxic condition. For the in vivo study, the same number of naked islets and encapsulated islets (alginate and PFD-alginate) was transplanted into streptozotocin-induced diabetic mice. Nonfasting blood glucose levels and the area under the curve for glucose based on intraperitoneal glucose tolerance tests in the PFD-alginate group were lower than in the alginate group. The harvested islets stained positive for insulin in all groups, but the ratio of dead cell area was 4 times higher in the alginate group than in the PFD-alginate group. In conclusion, integration of PFD in alginate microcapsules improved islet function and survival by minimizing the hypoxic damage of islets after intraperitoneal transplantation.

188P - A clinical validation study of RASKET-B: A multiple detection kit for RAS and BRAF gene mutations in colorectal cancer

Background: The detection of RAS and BRAF gene mutations has been essential to determine the treatment strategy for metastatic colorectal cancer (CRC). A multiplex kit simultaneously detecting both gene mutations is required, although few genetic assays with high-quality assurance are established. We evaluated RAS/BRAF KIT (RASKET-B), a multiplex assay using PCR-reverse sequence specific oligonucleotide (PCR-rSSO) and xMAP technology to concurrently detect 48 types of RAS mutations as well as the BRAF V600E mutation in a short turnaround time (4.5 h/96 specimens). Methods: Formalin-fixed paraffin-embedded tissues were obtained from 309 consenting patients with histologically-confirmed CRC. The primary endpoint was the concordance rate (CR) between RAS mutations identified with RASKET-B and a PCR-rSSO method kit (RASKET) and between BRAF mutations identified with direct sequencing (DS), respectively. As the secondary endpoints, we evaluated the CR between RASKET-B and DS for RAS mutations and between RASKET-B and the pyrosequencing (PYRO) method for BRAF V600E mutation. Results: We registered 302 samples, all of which were tested using the RASKET-B and reference methods. In RAS genes, 142 samples (47.0%) were detected by RASKET-B: 114 KRAS exon 2 and 28 other RAS mutations. The CR between RASKET-B and RASKET or RASKET-B and DS were 100% (95%CI: 99-100%) and 97.4% (95%CI: 95-99%), respectively. In BRAF genes, 18 samples (6.0%) were detected using RASKET-B. The CR between RASKET-B and DS or RASKET-B and PYRO were 100% (95%CI: 99-100%) and 99.7% (95%CI: 98-100%), respectively. The result of RASKET-B was consistent with that of DS. We also analyzed RAS and BRAF mutation ratios for each tumor cell ratio and tumor cell area ratio (0-25%, 25-50%, 50-75%, 75-100% respectively) and report it. Conclusions: RASKET-B provides rapid, precise and simultaneous detections of RAS and BRAF mutations in CRC. Clinical trial indentification: Study ID: UMIN000022742. Legal entity responsible for the study: Medical & Biological Laboratories Co., Ltd. Funding: Medical & Biological Laboratories Co., Ltd. Disclosure: W. Okamoto: MSD, K. Muro: Takeda, Chugai Pharma, Yakult Honsha, Merck Serono, Taiho Pharmaceutical, Lilly., H. Taniguchi: Takeda, Chugai Pharma, Merck Serono, Taiho Pharmaceutical, Bayer, Lilly Japan, Yakult Honsha., H. Hara: Chugai Pharma, Taiho Pharmaceutical, Merck Serono, Yakult Honsha, Lilly, Ono Pharmaceutical, Chugai Pharma., T. Satoh: Bayer Yakuhin, Lilly, Ono Pharmaceutical, Takara Bio, Merck Serono, Chugai Pharma, Bristol-Myers Squibb, Takeda, Yakult Honsha. All other authors have declared no conflicts of interest.

Femtosecond laser multi-patterning of zirconia for screening of cell-surface interactions

Yttria-stabilised tetragonal zirconia polycrystals (3Y-TZP) bioinert ceramics combine excellent strength and toughness, good aesthetics, high resistance to corrosion and absence of allergic reaction. However, improved osseointegration is needed as higher marginal bone loss was sometimes reported. In the present work, 3Y-TZP multi-patterned samples for rapid screening of cell-surface interactions were fabricated by femtosecond laser micromachining. Pits with well-defined edges and micrometric precision in pit diameter, depth and spacing were produced, as determined by white light interferometry. Pits showed a nanometric granular texture on the sidewalls and ripples at pit bottom, as attested by scanning electron microscopy. Focused ion beam analyses indicated limited laser-induced damage. Micropatterns impacted human mesenchymal stem cell (hMSC) size and morphology. Cell area and aspect ratio were mainly influenced by pit diameter, while solidity and circularity were affected by both pit diameter and depth. The pattern 30μm diameter/10μm depth induced the strongest osteoblastic hMSC commitment.

Establishment and characterization of clonal lines with cancer stem- and progenitor-cell properties from monolayer Zajdela hepatoma

The aim of this study was to investigate the biology of cancer stem cells (CSC) from a metastatic tumor. Previously, we explanted the cells of rat ascites Zajdela hepatoma in vitro. We established a permanent monolayer cell line via selection of adhesive cells from multicellular floating islets. In the present work, we cloned these cells by a limiting-dilution method and established five novel clonal sublines of the hepatoma: three holoclonal sublines containing CSC and two meroclonal sublines. After a long-term cultivation (approximately 30 passages, freezing, and thawing), the cell of clonal sublines retained the features of CSC. They have a tumor-initiating potential and produce mainly holoclones upon recloning in the complete growth medium and large nonadhesive hepatospheres in the serum-free medium. Morphometric analysis showed that the cells of holoclonal and meroclonal sublines differed in the cell shape, area, nucleus size, and nuclear-cytoplasmic ratio. We have found for the first time that holoclonal cells of Zajdela hepatoma have a fibroblast-like morphology and form contacts with each other due to membrane protrusions. We suggest that the fibroblast-like morphology of CSC is an attribute of a metastatic tumor and demonstrates the capability of these cells for individual migration.

Immunocytochemical identification and ontogeny of adenohypophyseal cells in a cave fish, Phreatichthys andruzzii (Cypriniformes: Cyprinidae).

The morphogenesis of the pituitary gland and the chronological appearance of adenohypophyseal cells were investigated for the first time in the Somalian cave fish Phreatichthys andruzzii by immunocytochemistry. The adult adenohypophysis contained: a rostral pars distalis, with prolactin (PRL) cells arranged in follicles and adrenocorticotropic (ACTH) cells, a proximal pars distalis with somatotropic (GH), β-thyrotropic (TSH), β-gonadotropic type I (FSH) and type II (LH) cells and a pars intermedia with α-somatolactin (SL), α-melanotropic (MSH) and β-endorphin (END) cells. All regions were deeply penetrated by neurohypophyseal branches. At hatching (24 h post-fertilization) the pituitary was an oval cell mass, close to the ventral margin of diencephalon. The first immunoreactive cells appeared as follows: PRL at 0·5 days after hatching (dah), GH and SL at 1·5 dah, END at 2 dah, TSH, ACTH and MSH at 2·5 dah, FSH at 28 dah and LH at 90 dah. The neurohypophysis appeared at 5 dah and branched extensively inside the adenohypophysis at 130 dah, but there was no boundary between rostral pars distalis and proximal pars distalis at this stage. The potential indices of prolactin and growth hormone production increased until 28 and 60 dah, respectively. The potential index of growth hormone production correlated positively with total length. Activity of PRL and GH cells, measured as ratio of cell area to nucleus area, was significantly higher in juveniles than in larvae.

Image-based correlation between the meso-scale structure and deformation of closed-cell foam

In the correlation between structural parameters and compressive behaviour of cellular materials, previous studies have mostly focused on averaged structural parameters and bulk material properties for different samples. This study focuses on the meso-scale correlation between structure and deformation in a 2D foam sample generated from a computed tomography slice of Alporas™ foam, for which quasi-static compression was simulated using 2D image-based finite element modelling. First, a comprehensive meso-scale structural characterisation of the 2D foam was carried out to determine the size, aspect ratio, orientation and anisotropy of individual cells, as well as the length, straightness, inclination and thickness of individual cell walls. Measurements were then conducted to obtain the axial distributions of local structural parameters averaged laterally to compression axis. Second, the meso-scale deformation was characterised by cell-wall strain, cell area ratio, digital image correlation strain and local compressive engineering strain. According to the results, the through-width sub-regions over an axial length between the average (lower bound) and the maximum (upper bound) of cell size should be used to characterise the meso-scale heterogeneity of the cell structure and deformation. It was found that the first crush band forms in a sub-region where the ratio of cell-wall thickness to cell-wall length is a minimum, in which the collapse deformation is dominated by the plastic bending and buckling of cell walls. Other morphological parameters have secondary effect on the initiation of crush band in the 2D foam. The finding of this study suggests that the measurement of local structural properties is crucial for the identification of the “weakest” region which determines the initiation of collapse and hence the corresponding collapse load of a heterogeneous cellular material.

Computer-assisted morphometric image analysis of cells of normal oral epithelium and oral squamous cell carcinoma.

Context:Oral squamous cell carcinoma (OSCC) is a malignant neoplasm of epithelial tissue origin. OSCC is traditionally graded into well, moderate and poorly differentiated. The assessment of cellular and nuclear features is very important in the histological grading of OSCC.Aims:To establish morphometric study as an important prognostic indicator of OSCC.Settings and Design:The present study is undertaken for computer-assisted morphometric evaluation of the following parameters: changes in cell perimeter, nuclear perimeter (NP), cell area (CA), nuclear area (NA) and nuclear-cytoplasmic ratio (N:C) in twenty cases of OSCC and ten cases of normal oral mucosa.Subjects and Methods:The hematoxylin- and eosin-stained sections were observed under microscope for dysplastic features. For morphometric analysis, images were captured with a 2MP camera attached to a NLCD 307 microscope (Lawrence and Mayo India Pvt., Ltd.,) with a ×40 objective used for morphometric analysis. The images were classified, transferred and stored in the computer for image analysis.Statistical Analysis Used:One-way analysis of variance is used for comparing the parameter for multiple groups followed by Mann-Whitney U-test for pair-wise comparisons.Results:Highly significant difference was seen between cases and controls with respect to CA, NP and NA. Highly significant difference is seen in N:C ratio between the means of cases and control groups.Conclusions:Techniques of image analysis offer an opportunity to quantify the nuclear and cell changes associated with malignancy and provide an objective basis for grading dysplasia and tumors.

Surface modified cellulose scaffolds for tissue engineering

We report the ability of cellulose to support cells without the use of matrix ligands on the surface of the material, thus creating a two-component system for tissue engineering of cells and materials. Sheets of bacterial cellulose, grown from a culture medium containing Acetobacter organism were chemically modified with glycidyltrimethylammonium chloride or by oxidation with sodium hypochlorite in the presence of sodium bromide and 2,2,6,6-tetramethylpipiridine 1-oxyl radical to introduce a positive, or negative, charge, respectively. This modification process did not degrade the mechanical properties of the bulk material, but grafting of a positively charged moiety to the cellulose surface (cationic cellulose) increased cell attachment by 70% compared to unmodified cellulose, while negatively charged, oxidised cellulose films (anionic cellulose), showed low levels of cell attachment comparable to those seen for unmodified cellulose. Only a minimal level of cationic surface derivitisation (ca 3% degree of substitution) was required for increased cell attachment and no mediating proteins were required. Cell adhesion studies exhibited the same trends as the attachment studies, while the mean cell area and aspect ratio was highest on the cationic surfaces. Overall, we demonstrated the utility of positively charged bacterial cellulose in tissue engineering in the absence of proteins for cell attachment.

520P - B cells and natural killer cells in lymph node (LN) are independent predictors of LN size, the number of retrieved LNs and survival in stage II colon cancer

In colon cancer, patients with many retrieved lymph nodes (LNs) have good outcomes. We previously reported that the longest diameter of retrieved LNs correlates with the number of retrieved LNs and is thus a predictor of outcomes (Int J Colorectal Dis 2015). LNs can be divided into three main regions: the cortex, the paracortex, and the medulla, and consist mainly of T cells, B cells, natural killer (NK) cells, and histiocytes. We studied the relations of the T-cell region, B-cell region, and the number of NK cells to the number of retrieved LNs, LN size, and outcomes. The study group comprised 320 patients with stage II colon cancer who underwent curative resection from 1991 through 2003. All operations were performed by the same team and LN dissection was performed to the origin of the feeding artery. LN with maximum long axis diameter (maximum LN) was selected in each patient and immunostained with CD3 as a T-cell marker, CD20 as a B-cell marker, and CD56 as an NK-cell marker. CD3-positive area ratio and CD20-positive area ratio were calculated using the image analyzer. The number of CD56-positive cells were counted at 6 sites and the mean number per 0.093 mm2 was calculated. The number of retrieved LNs was 14.8 ± 10.1 (mean ± SD). The mean CD3-positive area ratio was 0.39 ± 0.08. The mean CD20-positive area ratio was 0.42 ± 0.10. The mean number of CD56-positive cells was 10.7 ± 9.6. Multivariate analysis showed that the diameter of the maximum LN significantly correlated with the following variables (in the order of the strongest to weakest): number of CD56-positive cells, age, tumor location, CD20-positive area ratio and sex. The number of retrieved LNs positively correlated with tumor location, age, number of CD56-positive cells, and CD20-positive area ratio. The median follow-up was 118 months. Multivariate analysis showed that age, tumor location, T stage, CD20-positive area ratio, and the number of CD56-positive cells were independent prognostic factors of survival. B cell area ratio and the number of NK cells in maximum LNs were independent prognostic factors related to LN size, the number of retrieved LNs and survival in Stage II colon cancer.

Influence of Clinically Relevant Mechanical Forces on Vascular Smooth Muscle Cells Under Chronic High Glucose: An In Vitro Dynamic Disease Model

In this study, we subjected vascular smooth muscle cells (VSMC) to acute and chronic high glucose conditions under physiologically relevant levels of cyclic strain and low wall shear forces to compare phenotypic modulation and thus conceptualize a dynamic-disease test model which captures cellular response more accurately in comparison with static cultures. P2-P6 rat aortic smooth muscle cells were seeded on type I collagen-coated silicone membranes and subjected to 0-7% cyclic strain at 1Hz and 0.3dynes/cm(2) shear stress from flow for 24hr under acute (25mM d-glucose, 84hr) and chronic high glucose conditions (25mM d-glucose, 3-4weeks). Samples were analyzed for cell proliferation, percent apoptosis, cellular hypertrophy, and expression levels of smooth muscle contractile state-associated markers with 0.05 level of significance. Concomitant application of cyclic strain and flow shear resulted in an overall increase in proliferation of VSMCs under both acute and chronic high glucose conditions as compared with normal glucose control (P<0.0001). Application of both cyclic strain and cyclic strain shear resulted in a significant increase in percent apoptosis with chronic high glucose treatment in comparison with both normal glucose controls (P<0.0001) and acute high glucose (P<0.0001). Cellular hypertrophy as estimated by measuring cell area and aspect ratio revealed a significantly altered morphology due to concomitant loading under chronic high glucose conditions with significantly higher cell area (P<0.0001) and lower aspect ratio (P<0.0001) indicative of a relatively rounded morphology as compared with normal glucose controls. Western blot analysis demonstrated reduced expression of SM α-actin (P<0.0001), calponin (P<0.0001), and SM22α (P=0.0008) for concomitant loading under chronic high glucose treatment as compared with normal glucose controls. Concomitant application of cyclic strain and low wall shear stress resulted in greater phenotypic modulation of VSMCs due to chronic high glucose treatment as compared with normal glucose controls, thus implicating cellular-response differences which may impact progression of in-stent restenosis in diabetic patients with poorly controlled hyperglycemia. Similarity of VSMC response from our study to existing preclinical models of diabetes and reports of altered phenotype of VSMCs isolated from diabetic patients substantiate the relevance of our dynamic disease test model.

Computer aided morphometric analysis of oral leukoplakia and oral squamous cell carcinoma

We compared the changes in the cells in the basal layer of normal mucosa, oral leukoplakia with dysplasia and different grades of oral squamous cell carcinoma (OSCC) using computer aided image analysis of tissue sections. We investigated three morphometric parameters: nuclear area (NA), cell area (CA) and their ratio (NA:CA). NA and NA:CA ratio showed a statistically significant increase from dysplasia to increasing grades of OSCC. Nuclear size was useful for differentiating normal tissue, potentially malignant leukoplakia and OSCC.

3D printed scaffold architecture controls stem cell differentiation

Event Abstract Back to Event 3D printed scaffold architecture controls stem cell differentiation Murat Guvendiren1*, Carmelo De Maria2, Francesca Montemurro2, Giovanni Vozzi2* and Joachim Kohn1 1 Rutgers University, New Jersey Center for Biomaterials, United States 2 University of Pisa, Department of Ingegneria dell'Informazione (DII), Italy Introduction: Chemistry, mechanics, and topography of a biomaterial strongly regulate the behavior of cells on it, such as attachment, proliferation and differentiation. On 2D substrates cell area and aspect ratio have been shown to control stem cell differentiation[1],[2]. The surface geometry of the substrate has been shown to determine spatial patterning of cell fate[3],[4]. However, the majority of studies have focused on 2D substrates and the use of 3D scaffolds is rather limited. In this study, we used 3D printed scaffolds with a range of designs composed of square, hexagonal or octagonal grids to investigate stem cell response to 3D architecture. Methods: 3D scaffolds were printed using a pressure-activated microsyringe system. Scaffolds were fabricated from poly(tyrosol carbonate) (PTyC) or poly(L-Lactic acid) (PLLA) (10wt% in THF). Each scaffold was composed of three layers with identical design (square, hexagonal and octagonal). Each layer was shifted by one half unit with respect to the previous layer (Fig. 1 insets) to create a 3D porous structure composed of three layers. Human mesenchymal stem cells (hMSCs, from Texas A&M) at passage 3 were seeded (6.5x103 cells/cm2). Alamar Blue assay was performed at day 1, 7 and 14 to evaluate cellular activity. One sample from each group was fixed to evaluate cell morphology. For differentiation studies, hMSCs were cultured 1 day in growth media followed by 14 days in mixed adipogenic/osteogenic induction media. Cells were fixed and stained for F-actin (rhodamine phalloidin), nuclei (Hoechst), and alkaline phosphatase (Fast Blue RR/napthol solution). All measurements were done using NIH ImageJ. Results and Discussion: These adipogenic cells represent a cell population with AR < 2 and cell area < 4000 mm2. The cellular morphology for osteogenic population was significantly different for the square pattern scaffolds (AR < 2 and mean cell area = 8000 mm2) when compared to hexagonal AR =3.7 and cell area = 5500 mm2) and octagonal (AR = 3.5, cell area = 5300 mm2) pattern scaffolds. This indicates an increase in cellular elongation on curved design (in the case of hexagonal and octagonal) when compared to linear design. The percentage of osteogenic cells was significantly higher on scaffolds with hexagonal and octagonal patterns (~80%) as compared to those with square patterns (~65%). These adipogenic cells represent cell population with AR < 2 and cell area < 4000 mm2. The cellular morphology for osteogenic population was significantly different for square scaffolds (AR < 2 and mean cell area = 8000 mm2) when compared to hexagonal AR =3.7 and cell area = 5500 mm2) and octagonal (AR = 3.5, cell area = 5300 mm2) scaffolds. This indicates an increase in cellular elongation on curved design (in the case of hexagonal and octagonal) when compared to linear design. The percentage of osteogenic cells was significantly higher on hexagonal and octagonal scaffolds (~80%) as compared to linear (~65%). Conclusions: Our results show that hMSC cell spreading (area and aspect ratio) can be controlled by 3D scaffold architecture. Cells were highly elongated on hexagonal and octagonal scaffolds leading to a significantly higher osteogenic phenotype as compared to square scaffolds. This study shows the importance of scaffold architecture on stem cell differentiation, and can aid as a first step to develop scaffolds that promote osteogenic differentiation by controlling design. This work was supported by Award Number P41EB001046 from the National Institute of Biomedical Imaging and Bioengineering.

Structural Solution to Enhance the Sensitivity of a Self-Powered Pressure Sensor for an Artificial Tactile System.

Structural design factors of sensor units have been studied in order to enhance the sensitivity of pressure sensors based on utilizing a piezoelectric material for an artificial tactile sensor. In this study, we have primarily demonstrated the effect of a square pattern array design in a pressure sensor using ZnO nanowires. Nanowires grown on the edge of cells can be bent easily because of growth direction, density control, and buckling effect. Since smaller square pattern arrays induce a higher circumference to cell area ratio, if one sensor unit consists of many micro-level square pattern arrays, the design enhances the piezoelectric efficiency and the sensitivity. As a result, 20 μ m×20 μ m cell arrays showed three times higher pressure sensitivity than 250 μ m×250 μ m cell array structures at a pressure range from 4 kPa to 14 kPa. The induced piezoelectric voltage with the same pressure level also increased drastically. Therefore, the square pattern array design is more appropriate for a high-sensitive pressure sensor than a simple one-body cell design for tactile systems, and it has the advantage of better power efficiency, which is also important for artificial tactile systems. This suggested cell array design can be applied to various systems using piezoelectric nanowires.

Fibroblast Cell Cultivation on Wooden Pulp Cellulose Hydrogels for Cytocompatibility Scaffold Method

Fibroblast cell cultivation method was evaluated by using natural polymers sourced from pulp cellulose in their hydrogel forms. The pulp sourced was offered as an alternative for the preparation of hydrogel films when the cellulose was dissolved in dimethylacetamide/lithium choride (DMAc/LiCl) solution and converted hydrogels having flexible and transparent properties. The cultivation of the fibroblast cells was investigated on the hydrogels obtained in different LiCl concentration in the range of 4 to 12 wt%. Regarding the cytocompatibility, when NIH 3T3 fibroblast cells were used for cell adhesion assays, the growing cells showed higher density and aspect ratio on the hydrogel films than the observed on the commercial polystyrene dish (PS dish) used for cell cultivation. The mechanical and surface tests showed that the hydrogel films had elongation around 20 and 40 %, tensile strength from 48 to 67 N/mm2, and high water content value from 200 to 320 %. The results showed that cell addition and spreading on the hydrogel was higher compared with that in the PS dish used as control. Moreover, according with cell morphology tests, the values of cell area, long axis, and aspect ratio were higher than the registered on PS dish. These exhibited that the cellulose hydrogel films prepared with wooden pulp provided good cytocompatibility for its application in tissue engineering.

High content imaging for the morphometric diagnosis and immunophenotypic prognosis of canine lymphomas.

The common, symptomatic form of canine lymphoma (multicentric, medium-to-large cell, advanced) is consistently diagnosed manually and qualitatively by veterinary cytologists mainly based on increased lymphocyte size. The most effective prognostic feature is immunophenotype based on dual-antibody labeling for T versus B cells. High content imaging (HCI) is a novel, semi-automated, fluorescence microscopy and image-analysis technology used in research and predictive toxicology. We tested the hypothesis that HCI could semi-automatize the quantitative diagnosis of canine lymphoma and simultaneously determine immunophenotypic prognosis. Lymphocytes were obtained from lymph nodes of six lymphoma-free and five lymphomatous dogs, stained with antibodies against CD21 and CD3 (B- vs. T-cells), Hoechst-33342 and seeded into 96-well plates. Morphological parameters were examined: (a) cell area, (b) nuclear area, (c) nuclear displacement, (d) cytoplasmic area, (e) nucleus to cell area ratio (N/Cell), (f) nucleus to cytoplasm ratio (N/Cyt), and (g) cell roundness. HCI unequivocally discriminated malignant from benign lymphocytes, and provided immunophenotype. Cell and cytoplasmic area, nuclear displacement and roundness showed biggest differences and the means for each of the samples were not overlapping between the two groups. Mean/median/SD for control versus lymphoma samples were: (a) cell area (µm(2) ): 55.5/53.6/7.5 versus 80.3/75.5/8.7, (b) nuclear area (µm(2) ): 33.4/34.8/3.5 versus 40.2/38.5/5, (c) cytoplasm area (µm(2) ): 22.1/21/6.2 versus 40/38.4/4.9, (d) N/cell: 0.62/0.62/0.06 versus 0.52/0.52/0.03, (e) nuclear displacement (arbitrary units): 0.139/0.149/0.055 versus 0.33/0.30/0.056, (f) N/Cyt: 1.84/1.76/0.44 versus 1.19/1.24/0.17, and (g) roundness index: 1.22/1.21/0.03 versus 1.42/ 1.42/0.11 (P < 0.05 for all). HCI identified several, novel, morphometric parameters that effectively diagnose the common, symptomatic form of canine lymphoma, and also simultaneously determine prognostic immunophenotype.

Quantitative characterization of cell behaviors through cell cycle progression via automated cell tracking.

Cell behaviors are reflections of intracellular tension dynamics and play important roles in many cellular processes. In this study, temporal variations in cell geometry and cell motion through cell cycle progression were quantitatively characterized via automated cell tracking for MCF-10A non-transformed breast cells, MCF-7 non-invasive breast cancer cells, and MDA-MB-231 highly metastatic breast cancer cells. A new cell segmentation method, which combines the threshold method and our modified edge based active contour method, was applied to optimize cell boundary detection for all cells in the field-of-view. An automated cell-tracking program was implemented to conduct live cell tracking over 40 hours for the three cell lines. The cell boundary and location information was measured and aligned with cell cycle progression with constructed cell lineage trees. Cell behaviors were studied in terms of cell geometry and cell motion. For cell geometry, cell area and cell axis ratio were investigated. For cell motion, instantaneous migration speed, cell motion type, as well as cell motion range were analyzed. We applied a cell-based approach that allows us to examine and compare temporal variations of cell behavior along with cell cycle progression at a single cell level. Cell body geometry along with distribution of peripheral protrusion structures appears to be associated with cell motion features. Migration speed together with motion type and motion ranges are required to distinguish the three cell-lines examined. We found that cells dividing or overlapping vertically are unique features of cell malignancy for both MCF-7 and MDA-MB-231 cells, whereas abrupt changes in cell body geometry and cell motion during mitosis are unique to highly metastatic MDA-MB-231 cells. Taken together, our live cell tracking system serves as an invaluable tool to identify cell behaviors that are unique to malignant and/or highly metastatic breast cancer cells.

"High Content Imaging for the Morphometric Diagnosis and Immunophenotypic Prognosis of Canine Lymphomas"

Background: The common, symptomatic form of canine lymphoma (multicentric, medium-to-large cell, advanced) is consistently diagnosed manually and qualitatively by veterinary cytologists based on increased lymphocyte size. The most effective prognostic feature is immunophenotype based on dual-antibody labeling for T versus B cells. High Content Imaging (HCI) is a novel, semi-automated, fluorescence microscopy and image-analysis technology used in research and predictive toxicology. Objective: We tested the hypothesis that HCI could semi-automatise the quantitative diagnosis of canine lymphoma and simultaneously determine immunophenotypic prognosis. Methods: Lymphocytes were obtained from lymph nodes of 6 lymphoma-free and 5 lymphomatous dogs, stained with antibodies against CD21 and CD3 (B- vs. T- cells), Hoechst-33342 and seeded into 96-well plates. Morphological parameters were examined: a) cell area, b) nuclear area, c) nuclear displacement, d) cytoplasmic area, e) nucleus to cell area ratio (N/Cell), f) nucleus to cytoplasm ratio (N/Cyt), g) cell roundness. Results: HCI unequivocally discriminated malignant from benign lymphocytes, and provided immunophenotype. Cell and cytoplasmic area, nuclear displacement and roundness showed biggest differences without value overlap between groups. Mean/ median/SD for control versus lymphoma samples were: a) cell area (µm2 ): 55.5/53.6/7.5 versus 80.3/75.5/8.7, b) nuclear area (µm2 ): 33.4/34.8/3.5 versus 40.2/38.5/5, c) cytoplasm area (µm2 ): 22.1/216.2 versus 40/38.4/4.9, d) N/Cell: 0.62/0.62/0.06 versus 0.52/0.52/0.03, e) nuclear displacement (arbitrary units): 0.139/0.149/0.055 versus 0.33/0.30//0.056, f) N/Cyt: 1.84/1.76/0.44 versus 1.19/1.24/0.17, g) roundness index: 1.22/1.21/0.03 versus 1.42/ 1.42/0.11 (p<0.05). Conclusion: HCI identified several, novel, morphometric parameters that effectively diagnose the common, symptomatic form of canine lymphoma, and also simultaneously determine prognostic immunophenotype. © 2014 Clinical Cytometry Society.

67 : Assessment of β2-adrenergic receptors on leukocyte subpopulations using imaging cytometry

Introduction: β2-Adrenergic receptors (AR) are the primary subtype of adrenergic receptors found on blood leukocytes, and are recognized as key mediators of interaction between the sympathetic nervous system and immune system. This biological pathway is central in understanding neuroimmune signaling in a number of disease states, spanning mental disorders to traumatic brain injury. Traditionally, β2-ARs have been primarily assessed using radio-ligand binding assays, where results are limited in their ability to provide in depth cell-signaling information on specific leukocyte subtypes. As a result, current knowledge of β2-AR distribution, reactivity and potential implications in disease pathology or drug therapy are limited. This study evaluated β2-AR expression on circulating leukocyte subsets with various commercially available antibodies (Abs) using imaging cytometry. Methods: Whole blood was labeled with CD14 and CD16 surface markers for leukocyte subtype phenotyping. Four different β2-AR antibodies, sc-81578 – FITC (Santa Cruz), sc-81577 – PE (Santa Cruz), PA5-19649 (Pierce Biotechnology, USA) and MCA2784 (Serotec, USA) were assessed. DAPI stain was added to all samples to determine nuclear morphology. Based on cell area and aspect ratio, 100,000 single-cell events were acquired on an ImageStreamX Mark II® Cytometer (Amnis, Seattle, WA). Monocytes and lymphocytes were identified using side scatter (SSC) and CD14 expression. Neutrophils and eosinophils were identified using SSC and CD16. Results: β2−AR expression levels on leukocyte subpopulations in whole blood were detectable using image cytometery. Leukocyte subsets identified according to surface markers, size, granularity, cell and nuclear morphology displayed varying amounts of β2-AR expression. Furthermore, the PA5-19649 Ab performed best in terms of binding specificity and signal strength. Conclusion: Imaging cytometry is a valuable technique for evaluating β2-ARs on leukocyte subsets. The capability of this technology to provide in-depth, high throughput cell signaling information offers an attractive alternative to current methods used in the assessment of adrenergic receptors.

Seed Structure Characteristics to Form Ultrahigh Oil Content in Rapeseed

BackgroundRapeseed (Brassica napus L.) is an important oil crop in the world, and increasing its oil content is a major breeding goal. The studies on seed structure and characteristics of different oil content rapeseed could help us to understand the biological mechanism of lipid accumulation, and be helpful for rapeseed breeding.Methodology/Principal FindingsHere we report on the seed ultrastructure of an ultrahigh oil content rapeseed line YN171, whose oil content is 64.8%, and compared with other high and low oil content rapeseed lines. The results indicated that the cytoplasms of cotyledon, radicle, and aleuronic cells were completely filled with oil and protein bodies, and YN171 had a high oil body organelle to cell area ratio for all cell types. In the cotyledon cells, oil body organelles comprised 81% of the total cell area in YN171, but only 53 to 58% in three high oil content lines and 33 to 38% in three low oil content lines. The high oil body organelle to cotyledon cell area ratio and the cotyledon ratio in seed were the main reasons for the ultrahigh oil content of YN171. The correlation analysis indicated that oil content is significantly negatively correlated with protein content, but is not correlated with fatty acid composition.Conclusions/SignificanceOur results indicate that the oil content of YN171 could be enhanced by increasing the oil body organelle to cell ratio for some cell types. The oil body organelle to seed ratio significantly highly positively correlates with oil content, and could be used to predict seed oil content. Based on the structural analysis of different oil content rapeseed lines, we estimate the maximum of rapeseed oil content could reach 75%. Our results will help us to screen and identify high oil content lines in rapeseed breeding.