Growth orientation of heart cells on nylon monofilament. Determination of the volume-to-surface area ratio and intracellular potassium concentration

A new method is described for orienting the growth of embryonic chick heart cells as thin annuli about nylon monofilament. Analytical measurements of cell water, intracellular potassium, cell volume, and cell surface area incorporate several new techniques and provide the quantitative basis for characterizing the respective cell types in the preparation. The measurements support the hypothesis that tissue culture methodology does not alter the morphological and physiological properties of cardiac muscle cells. The preparations are ideally suited for radiotracer studies since tissue mass can be increased while retaining a relatively short diffusional distance.     

Fast quantification of immunohistochemistry tissue microarrays in lung carcinoma

Tissue microarrays (TMAs) are an effective tool for high-throughput molecular analysis of tissues to help identify new diagnostic and prognostic markers and targets in human cancers. We have developed a fully automated method for rapid, continuous and quantitative analysis of TMAs based on immunohistochemistry. The method deals with complex and varying tissue architectures, segments tumour cells from normal cells, conducts cell compartmentalisation, identifies nuclei and cytoplasm and produces three different continuous measurements of marker expression levels within tumour cell nuclei, tumour cell cytoplasm and total tumour cell protein expression. We have demonstrated this method using three independent protein markers (BAK, BAX and a novel biomarker, named KS) over 7 TMAs, involving 2 BAK stained TMAs with 229 tumour tissue cores, 2 BAX stained TMAs with 229 tumour tissue cores and 3 KS stained TMAs with 373 tumour cores of lung carcinomas. We validated the automated method, showing that the automated scoring is significantly correlated with the pathologist-based scoring.     

Improved Methods for the Measurement of Total Cell Surface Area in Leaf Mesophyll Tissue

Two improved methods are described for the measurement of thetotal surface area of mesophyll cells in leaf tissue. The firstinvolves stereological measurements on suspensions of enzymicallyisolated mesophyll cells; the second involves measurements ofthe total perimeter of mesophyll cell profiles on transversesections of the leaf, with allowance for curvature of the cellsurfaces by means of theoretically based correction factors.Application of the two methods to the mesophyll tissue of tobaccoleaves gave results which were in very good agreement with eachother. Both methods avoid the inaccuracies and uncertaintiesof previous methods for measurement of mesophyll cell surfaceareas. Key words: Mesophyll, Surface: Area     

Preparation of cells from paraffin-embedded tissue for cytometry and cytomorphologic evaluation

A method is described for the preparation of monolayer smears from paraffin-embedded tissue. The smears are suitable for automated image analysis and DNA measurements while still allowing interpretation of nuclear morphology. The proposed technique uses enzyme treatment and syringing for cell dispersal. The preparation of cell monolayers is performed by cytocentrifugation. After staining the specimens with gallocyanin, nuclear DNA can be measured. Automated DNA measurements using the Leyden Television Analysis System (LEYTAS) showed coefficients of variation of 4.5% for the diploid cell population of suspended benign tissue. After DNA measurements, the specimens are counterstained using orange G and eosin. Since gallocyanin has spectral properties similar to those of hematoxylin, the obtained end product is comparable to specimens stained according to the routinely used Papanicolaou procedure. Using this technique, image cytometry can be applied to paraffin-embedded tissue in combination with conventional cytomorphologic study of the cells.     

Osteogenic differentiation of preconditioned bone marrow mesenchymal stem cells with lipopolysaccharide on modified poly-l-lactic-acid nanofibers

Tissue engineering is an interdisciplinary expertise that involves the use of nanoscaffolds for repairing, modifying, and removing tissue defects and formation of new tissues. Mesenchymal stem cells (MSCs) can differentiate into a variety of cell types, and they are attractive candidates for tissue engineering. In the current study, the electrospinning process was used for nanofiber preparation, based on a poly-l -lactic-acid (PLLA) polymer. The surface was treated with O ₂ plasma to enhance hydrophilicity, cell attachment, growth, and differentiation potential. The nanoscaffolds were preconditioned with lipopolysaccharide (LPS) to enhance induction of differentiation. The nanoscaffolds were categorized by contact angle measurements and scanning electron microscopy. The MTT assay was used to analyze the rate of growth and proliferation of cells. Osteogenic differentiation of cultured MSCs was evaluated on nanofibers using common osteogenic markers, such as alkaline phosphatase activity, calcium mineral deposition, quantitative real-time polymerase chain reaction, and immunocytochemical analysis. Based on the in vitro results, primed MSCs with LPS on the PLLA nanoscaffold significantly enhanced the proliferation and osteogenesis of MSCs. Also, the combination of LPS and electrospun nanofibers can provide a new and suitable matrix to support stem cells’ differentiation for bone tissue engineering.     

Various seeding methods for tissue development of human umbilical-cord-derived mesenchymal stem cells in 3-dimensional PET matrix

Mesenchymal stem cells derived from human umbilical cords (hUCMSCs) are attractive as a new cell source for tissue engineering. It is essential to investigate and optimize the seeding process of these cells for the success of cell culture and tissue regeneration in vitro. In this study, a static seeding method (SSM), a centrifugal seeding method (CSM), and a novel method-cycling filtration seeding method (CFSM) are evaluated in terms of seeding efficiency, cell damage, and distribution inside the scaffolds, cell proliferation, and osteogenic differentiation. Cells were seeded on three-dimensional (3-D) nonwoven PET discs at a density of 1×10⁴ cells/disc, followed by 21 days of cell culture and 20 days of osteogenic differentiation. Cells grown in 3-D conditions exhibited higher metabolic activity than those grown on a 2-D control surface. The CSM and CFSM groups showed higher seeding efficiency, proliferation capacity, and differentiation potential. H&E staining indicated a more uniform spatial distribution of cells in CFSM groups. LDH level measurements suggested that more cell damage was caused by the CFSM process. Above all, the results showed that the cells maintained their proliferation ability and differentiation potential ex vivo during approximately 7 weeks of culture. The CSM and CFSM are recommended for hUCMSC tissue engineering, although the seeding parameters still require further investigation and optimization.     

Short-lived, transitory cell-cell interactions foster migration-dependent aggregation

During embryonic development, motile cells aggregate into cohesive groups, which give rise to tissues and organs. The role of cell migration in regulating aggregation is unclear. The current paradigm for aggregation is based on an equilibrium model of differential cell adhesivity to neighboring cells versus the underlying substratum. In many biological contexts, however, dynamics is critical. Here, we provide evidence that multicellular aggregation dynamics involves both local adhesive interactions and transport by cell migration. Using time-lapse video microscopy, we quantified the duration of cell-cell contacts among migrating cells that collided and adhered to another cell. This lifetime of cell-cell interactions exhibited a monotonic decreasing dependence on substratum adhesivity. Parallel quantitative measurements of cell migration speed revealed that across the tested range of adhesive substrata, the mean time needed for cells to migrate and encounter another cell was greater than the mean adhesion lifetime, suggesting that aggregation dynamics may depend on cell motility instead of the local differential adhesivity of cells. Consistent with this hypothesis, aggregate size exhibited a biphasic dependence on substratum adhesivity, matching the trend we observed for cell migration speed. Our findings suggest a new role for cell motility, alongside differential adhesion, in regulating developmental aggregation events and motivate new design principles for tuning aggregation dynamics in tissue engineering applications.     

Cellular composition of the cyclic corpus luteum of the cow

The cellular composition of CL from 6 cows on approximately Day 12 of the oestrous cycle, after synchronization with cloprostenol, was studied by ultrastructural morphometry. Point-count measurements of volume density (mean +/- s.d.) showed that large luteal cells occupied 40.2 +/- 7.0% of the luteal tissue, and small luteal cells 27.7 +/- 6.3%. Of the total of 393.4 +/- 52.0 x 10(3) cells per mm3 of luteal tissue, large luteal cells made up only 3.5% and small luteal cells 26.7%, a ratio of 1:7.6. Endothelial cells/pericytes, at 52.3%, were the most numerous cell type. The mean volume per large luteal cell was 29.6 +/- 6.3 x 10(3) microns 3, while that of small luteal cells was 2.7 +/- 0.4 x 10(3) microns 3. In spherical form, these volumes would represent mean diameters of 38.4 microns and 17.2 microns respectively, and are consistent with published measurements on dispersed luteal cells. However, the values for cell numbers are much higher than published values based on luteal tissue dispersion, suggesting that dispersion may result in substantial and possibly selective losses of luteal cells.     

种子细胞——组织工程连载之三

种子细胞也是组织工程的核心研究内容,获得足够数量和质量的种子细胞是开展体外组织工程的必要基础。用于组织工程的种子细胞必须具有形成新组织结构的能力,主要来源于自体、同种异体或异种,在具体应用时各有利弊。一些成体干细胞由于不存在伦理争议以及发育分化条件相对简单等优势是重要的种子细胞,包括造血干细胞、骨髓干细胞、神经干细胞、脂肪干细胞、皮肤干细胞。人胚胎干细胞及其组织工程要真正在临床医学中得到应用,还有很长的一段路要走。其他一些细胞也可以作为组织工程种子细胞,包括内皮细胞、上皮细胞、成纤维细胞、骨细胞、成骨细胞、角质细胞、前脂肪细胞、脂肪细胞、肌腱细胞等。这些细胞已分化,分裂能力有限,但仍应用于组织工程。理想的种子细胞具有一定标准。     

Preparation of monolayer smears from paraffin-embedded tissue for image cytometry

A method is described for the preparation of monolayer smears from paraffin-embedded tissue suitable for automated image analysis and DNA measurements. The proposed technique uses enzyme treatment and syringing for cell dispersal. Slide preparation is performed by centrifugal cytology. After Feulgen staining the quality of the monolayer smears is sufficiently high to enable visual morphologic evaluation. Automated DNA measurements using the Leyden television analysis system (LEYTAS) show coefficients of variation (CV) of 4.5% for the diploid cell population of the suspended tissue. This is approximately the same as the CV in fresh material from the same tumor. Formalin fixed trout red blood cells are used as reference cells. By applying image cytometry to paraffin-embedded tissue this method allows retrospective studies of, for instance, the significance of DNA content with regard to the behavior of a tumor.     

Localization of ionic pathways in the teleost opercular membrane by extracellular recording with a vibrating probe

We have adapted the vibrating probe extracellular recording technique to use on an epithelium under voltage clamp in an Ussing chamber. The vibrating probe allows very low drift measurements of current density immediately over the epithelial surface. These measurements allowed sites of electrogenic transport in the epithelium to be localized with a spatial resolution of 5 micrometers. The technique was applied to the opercular membrane of the teleost fish, the tilapia, Sarotherodon mossambicus. The mitochondrion-rich "chloride cells" were shown to be the only sites of electrogenic ion transport in this heterogeneous epithelium. Cell sampling experiments demonstrated variable negative short-circuit currents associated with nearly all of approximately 300 chloride cells examined, which appeared to account for all of the tissue short-circuit current. Current-voltage relations for individual cells were also measured. Conductance associated with chloride cells (i.e. cellular and junctional pathways) accounted for all but 0.5 mS/cm2 of the tissue conductance, with the balance apparently accounted for by leak pathways near the edge of the tissue. Current and conductance associated with other cell types was at least 50-fold smaller than for the chloride cell. Chloride-free solutions reduced chloride cell current and conductance by 98 and 95%, respectively.     

A comparison of the size of photoreceptor cells from the retina of the domestic pig as determined by light and scanning electron microscopy

While gathering data on the visual pigments of numerous species, many light micrographs have been taken of the photoreceptor cells containing the visual pigment after spectral recordings have been made from these cells. These micrographs are used to view the cells and obtain measurements of their size. Usually the morphology of the photoreceptor cells is retained adequately so that such measurements can be taken. Occasionally it is necessary to partially fix the retinal tissue which aids in the maintenance of photoreceptor morphology while allowing visual pigment spectral recordings to be made. We have found that primate retinal tissue, as well as some other mammalian tissue, disintegrates rapidly. Although partial fixation allows spectral recordings to be made before the micrographs are taken, the treatment does not always adequately preserve cell morphology for quality micrographs to be obtained. In these cases, visual pigment recordings are made from pieces of unfixed and partially fixed retinal tissue; an additional piece of the same retinal material is well-fixed, embedded and thick-sectioned for light microscopy.Preparing retinal material for sectioning is lengthy and time consuming so an alternative tissue preparation technique was sought. Material can be processed for the scanning electron microscope (SEM) more rapidly than for sectioning, however severe tissue shrinkage occurs during this process. It was found that although shrinkage does occur in the retinal tissue prepared for SEM, the relative proportions of the photoreceptor cells are maintained extremely well. Using the critical point drying method (CPD) pig retinal tissue was prepared for SEM. Scanning electron micrographs of the pig photoreceptors were taken for cell measurement. Since these micrographs could be made at higher magnification than is available by light microscopy, a more detailed view of the pig photoreceptor cells was obtained. Cell measurements made from the light and the scanning electron micrographs indicate that an approximate shrinkage of 50% occurs in the SEM prepared material.     

Dedifferentiated fat cells: A cell source for regenerative medicine

The identification of an ideal cell source for tissue regeneration remains a challenge in the stem cell field. The ability of progeny cells to differentiate into other cell types is important for the processes of tissue reconstruction and tissue engineering and has clinical, biochemical or molecular implications. The adaptation of stem cells from adipose tissue for use in regenerative medicine has created a new role for adipocytes. Mature adipocytes can easily be isolated from adipose cell suspensions and allowed to dedifferentiate into lipid-free multipotent cells, referred to as dedifferentiated fat (DFAT) cells. Compared to other adult stem cells, the DFAT cells have unique advantages in their abundance, ease of isolation and homogeneity. Under proper condition in vitro and in vivo, the DFAT cells have exhibited adipogenic, osteogenic, chondrogenic, cardiomyogenc, angiogenic, myogenic, and neurogenic potentials. In this review, we first discuss the phenomena of dedifferentiation and transdifferentiation of cells, and then dedifferentiation of adipocytes in particular. Understanding the dedifferentiation process itself may contribute to our knowledge of normal growth processes, as well as mechanisms of disease. Second, we highlight new developments in DFAT cell culture and summarize the current understanding of DFAT cell properties. The unique features of DFAT cells are promising for clinical applications such as tissue regeneration.     

In vivo extraction of Arabidopsis cell turgor pressure using nanoindentation in conjunction with finite element modeling

Turgor pressure in plant cells is involved in many important processes. Stable and normal turgor pressure is required for healthy growth of a plant, and changes in turgor pressure are indicative of changes taking place within the plant tissue. The ability to quantify the turgor pressure of plant cells in vivo would provide opportunities to understand better the process of pressure regulation within plants, especially when plant stress is considered, and to understand the role of turgor pressure in cellular signaling. Current experimental methods do not separate the influence of the turgor pressure from the effects associated with deformation of the cell wall when estimates of turgor pressure are made. In this paper, nanoindentation measurements are combined with finite element simulations to determine the turgor pressure of cells in vivo while explicitly separating the cell‐wall properties from the turgor pressure effects. Quasi‐static cyclic tests with variable depth form the basis of the measurements, while relaxation tests at low depth are used to determine the viscoelastic material properties of the cell wall. Turgor pressure is quantified using measurements on Arabidopsis thaliana under three pressure states (control, turgid and plasmolyzed) and at various stages of plant development. These measurements are performed on cells in vivo without causing damage to the cells, such that pressure changes may be studied for a variety of conditions to provide new insights into the biological response to plant stress conditions.     

Kinetic and Physical Studies of Cell Death Induced By Chemotherapeutic Agents Or Hyperthermia

The kinetics of three physical parameters: cell density, relative cytoplasmic viscosity and DNA stability to denaturation have been measured during the period preceding cell death induced by hyperthermia, methylprednisolone and a series of cancer chemotherapeutic agents. This series of measurements employed cultured human lymphoblastoid cells as an experimental system to establish the changes that can be observed in the early stages of cell death, prior to applying such measurements to tissue biopsies from solid human tumours. Cell death, induced by hyperthermia up to 43°C, methylprednisolone, vincristine, 5-fluorouracil, BCNU and melphalan, showed essentially identical and reproducible changes corresponding to those which characterize programmed cell death (apoptosis). Such changes could also be observed following hyperthermia above 43°C, but reproducibility was poor and increasing damage to the cell membranes was evident. In cells treated with adriamycin or methotrexate, cell sub-populations showing an increase in cell density were not detected. Measurements of DNA stability were readily performed by flow cytofluorometry thus allowing rapid quantitation of the fraction of cells in the early stages of cell death. Modified flow cytometric instrumentation would further allow measurement of cytoplastic viscosity as an additional parameter to indicate entry into programmed cell death. This suggests that these measurements could readily be applied to cell suspensions derived from tumour tissue biopsies for a more accurate assessment of tumour growth rate, and to allow monitoring of response to therapy in sequential tumour biopsies.     

Reliability of computerized image analysis for the evaluation of serial synovial biopsies in randomized controlled trials in rheumatoid arthritis

Analysis of biomarkers in synovial tissue is increasingly used in the evaluation of new targeted therapies for patients with rheumatoid arthritis (RA). This study determined the intrarater and inter-rater reliability of digital image analysis (DIA) of synovial biopsies from RA patients participating in clinical trials. Arthroscopic synovial biopsies were obtained before and after treatment from 19 RA patients participating in a randomized controlled trial with prednisolone. Immunohistochemistry was used to detect CD3⁺ T cells, CD38⁺ plasma cells and CD68⁺ macrophages. The mean change in positive cells per square millimetre for each marker was determined by different operators and at different times using DIA. Nonparametric tests were used to determine differences between observers and assessments, and to determine changes after treatment. The intraclass correlations (ICCs) were calculated to determine the intrarater and inter-rater reliability. Intrarater ICCs showed good reliability for measuring changes in T lymphocytes (R = 0.87), plasma cells (R = 0.62) and macrophages (R = 0.73). Analysis by Bland–Altman plots showed no systemic differences between measurements. The smallest detectable changes were calculated and their discriminatory power revealed good response in the prednisolone group compared with the placebo group. Similarly, inter-rater ICCs also revealed good reliability for measuring T lymphocytes (R = 0.68), plasma cells (R = 0.69) and macrophages (R = 0.72). All measurements identified the same cell types as changing significantly in the treated patients compared with the placebo group. The measurement of change in total positive cell numbers in synovial tissue can be determined reproducibly for various cell types by DIA in RA clinical trials.     

Optimisation des matériaux hybrides à base de cellules souches pour la réparation de grands défauts osseux

The limitations imposed to both autogenous and allogenous bone grafts led to the development of new strategies for the treatment of large bone defects. The approach of bone tissue engineering aims to restore damaged bone tissue by combining osteocompetent cells such as mesenchymal stromal cells (MSC), and material scaffolds like ceramics. However, the therapeutic effectiveness of cell constructs has not yet met that of autologous bone grafts, in part due to the high death rate of cells (loaded onto the material scaffold) upon their implantation into the injured site. In order to improve the therapeutic functionality of these cell constructs, different strategies can be implemented. In this context, the Glassbone project aimed to optimize the conditions for preparation of tissue engineered products by approaching three aspects: identification of optimal ceramic scaffold relevant to bone formation; survival of implanted cells post-implantation, and finally cell preconditioning to promote cell viability in vivo. Such project will pave the way for the development of new “pro-survival” tissue engineered materials for optimal tissue regeneration.     

Cancer cell detection in tissue sections using AFM

Currently, cancer diagnosis relies mostly on morphological examination of exfoliated, aspirated cells or surgically removed tissue. As long as standard diagnosis is concerned, this classical approach seems to be satisfactory. In the recent years, cancer progression has been shown to be accompanied by alterations in mechanical properties of cells. This offers the detection of otherwise unnoticed cancer cell disregarded by histological analysis due to insignificant manifestations. One of techniques, sensitive to changes in mechanical properties, is the atomic force microscopy, which detects cancer cells through their elastic properties. Such measurements were applied to tissue sections collected from patients suffering from various cancers. Despite of heterogeneity and complexity of cancer cell sections, the use of the Young's modulus as an indicator of cell elasticity allow for detection of cancer cells in tissue slices.     

Elastic modulus and hydraulic permeability of MDCK monolayers

The critical role of cell mechanics in tissue health has led to the development of many in vitro methods that measure the elasticity of the cytoskeleton and whole cells, yet the connection between these local cell properties and bulk measurements of tissue mechanics remains unclear. To help bridge this gap, we have developed a monolayer indentation technique for measuring multi-cellular mechanics in vitro. Here, we measure the elasticity of cell monolayers and uncover the role of fluid permeability in these multi-cellular systems, finding that the resistance of fluid transport through cells controls their force–response at long times.     

Mesenchymal progenitor cells in adult human dental pulp and their ability to form bone when transplanted into immunocompromised mice

The technique of tissue engineering is developing for the restoration of lost tissues. This new technique requires cells that fabricate tissue. Mesenchymal stem cells in bone marrow have been used as the cell source for this technique; however, dental pulp cells have recently been shown to possess stem-cell-like properties. We earlier demonstrated that dental pulp cells proliferate and produce an extracellular matrix that subsequently becomes mineralized in vitro. We now report that such dental pulp cells (first to eighth passage) produced bone instead of dentin when those cells were implanted into subcutaneous sites in immunocompromised mice with HA/TCP powder as their carrier. This evidence shows that dental pulp cells are the common progenitors of odontoblasts and osteoblasts, or dental pulp cells are mesenchymal stem cells themselves. It is expected that dental pulp cells can be a useful candidate cell source for tissue engineering, and contain the potential of new therapeutic approaches for the restoration of damaged or diseased tissue.