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PubMed2000

Metabolic imaging in multicellular spheroids of oncogene-transfected fibroblasts.

Four rat embryo fibroblast (REF) cell lines with defined oncogenic transformation were used to study the relationship between tumorigenic conversion, metabolism, and development of cell death in a 3D spheroid system. Rat1 (spontaneously immortalized) and M1 (myc-transfected) fibroblasts represent early nontumorigenic transformation stages, whereas Rat1-T1 (T24Ha-ras-transfected Rat1) and MR1 (myc/T24Ha-ras-co-transfected REF) cells express a highly tumorigenic phenotype. Localized ATP, glucose, and lactate concentrations in spheroid median sections were determined by imaging bioluminescence. ATP concentrations were low in the nonproliferating Rat1 aggregates despite sufficient oxygen and glucose availability and lack of lactate accumulation. In MR1 spheroids, a 50% decrease in central ATP preceded the development of central necrosis at a spheroid diameter of around 800 micrometer. In contrast, the histomorphological emergence of cell death at a diameter of around 500 micrometer in Rat1-T1 spheroids coincided with an initial steep drop in ATP. Concomitantly, reduction in central glucose and increase in lactate before cell death were recorded in MR1 but not in Rat1-T1 spheroids. As shown earlier, myc transfection confers a considerable resistance to hypoxia of MR1 cells in the center of spheroids, which is reflected by their capability to maintain cell integrity and ATP content in a hypoxic environment. The data obtained suggest that small alterations in the genotype of tumor cell lines, such as differences in the immortalization process, lead to substantial differences in morphological structure, metabolism, occurrence of cell death, and tolerance to hypoxia in spheroid culture.

Adenosine Triphosphate↗

PubMed2006

Promotion of angiogenesis in tissue engineering: developing multicellular matrices with multiple capacities.

One of the aims of tissue engineering is to be able to develop multi-tissue organs in the future. This requires the optimization of conditions for the differentiation of multiple cell types and maintenance of the differentiated phenotype within complex engineered tissues. The goal of this study was to develop prototype tissue engineered matrices to support the simultaneous growth of different cell types with a particular focus on the angiogenic process. We examined two different matrix compositions for the promotion of blood vessel and tube formation. A fibrin-based matrix with the addition of a combination of growth factors supported vascular growth and the invasion of inflammatory cells. Using this fibrin matrix, in combination with a collagen-based hydrogel, a simple in vitro model of the cornea with adjacent sclera was developed that was complete with innervation and vascular structures. In addition, we showed that collagen-based matrices were effective in delivering mononuclear endothelial progenitor cells to ischemic tissue in vivo, and allowing these cells to incorporate into vascular structures. It is anticipated that with further development, these matrices have potential for use as delivery matrices for cell transplantation and for in vitro study purposes of multiple cell types.

Acrylamides↗

PubMed2002

Dendritic cells for NK/LAK activation: rationale for multicellular immunotherapy in neuroblastoma patients.

Natural killer (NK)/lymphokine-activated killer (LAK) cell-based immunotherapy could be beneficial against major histocompatibility complex class I-negative tumor residual disease such as neuroblastoma (NB), provided that interleukin 2 (IL-2) or surrogate nontoxic NK cell stimulatory factors could sustain NK cell activation and survival in vivo. Here we show that human monocyte-derived dendritic cells (MD-DCs) promote potent NK/LAK effector functions and long-term survival, circumventing the need for IL-2. This study demonstrates (1) the feasibility of differentiating granulocyte colony-stimulating factor-mobilized hematopoietic peripheral blood stem cells (PBSCs) into high numbers of functional MD-DCs and NK/LAK cells in a series of 12 children with stage 4 neuroblastoma (NB); (2) potent DC-mediated NK cell activation in autologous settings; (3) the reciprocal capacity of NK/LAK cells to turn immature DCs into maturing cells electively capable of triggering NK cell functions; and (4) the unique capacity of maturing DCs to sustain NK cell survival, superior to that achieved in IL-2. These data show a reciprocal interaction between DCs and NK/LAK cells, leading to the amplification of NK cell effector functions, and support the implementation of DC/NK cell-based immunotherapy for purging the graft and/or controlling minimal residual disease after autologous stem cell transplantation.

Cell Survival↗

PubMed1993

Patterns in Dictyostelium discoideum: the role of myosin II in the transition from the unicellular to the multicellular phase.

Dictyostelium discoideum amoebae which lack the myosin II gene are motile and aggregate to form rudimentary mounds, but do not undergo further morphological development (Manstein et al., 1989). Here we use scanning electron microscopy, light microscopy, immunofluorescence and computer analysis of time-lapse video films to study how D. discoideum myosin null cells of strains HS2205 and HS2206 aggregate. Myosin null cells are sufficiently coordinated in their movements to form two-dimensional aggregation streams, although mutant cells within streams lack the elongated shape and parallel orientation of wild-type strains. In the wild-type, cell movements are coordinated, cells usually joining streams that spiral inwards and upwards as the mound extends into the standing papilla. In the aggregates of mutant strains, cell movements are chaotic, only occasionally forming short-term spirals that rotate at less than half the speed of wild-type spirals and frequently change direction. Unlike the situation in the wild-type where spirals continue with mound elongation, cells within the mutant mound eventually cease translocation altogether as the terminal shape of the mound is reached and only intracellular particle movement is observed. Scanning electron micrographs show that the surface of the wild-type mound consists of flattened cells which fit neatly together. The myosin null cell mound has an uneven surface, the orientation of the cells is chaotic and no tip is formed. This is consistent with the results of synergy experiments in which myosin null cells were absent from the tips of chimeric HS2205/AX2 slugs and pre-culminates. Immunofluorescence microscopy using prespore and spore cell markers reveals that a prestalk/prespore pattern forms within the mutant mound but that terminal spore differentiation is incomplete. These results are discussed in relation to the role of myosin II in aggregation and morphogenesis.

Animals↗

PubMed1977

Fine-structural studies of the gametes and embryo of Fucus vesiculosus L. (Phaeophyta). III. Cytokinesis and the multicellular embryo.

Condensation of the chromosomes during the first cell division following fertilization of the brown alga Fucus vesiculosus L. is accompanied by the almost complete disappearance of the nuclear envelope. Golgi vesicles and other small vesicles appear within the spindle, which has paired centrioles at each end. A large amount of rough endoplasmic reticulum is in the surrounding cytoplasm during mitosis, and many vesicles at the spindle margin are encircled by stacks of endoplasmic reticulum. Annulate lamellae are observed during mitosis. The envelope which initially reforms around the chromatin in telophase has unevenly spaced nuclear pores. Cytokinesis results primarily by vesicle addition to a centripetal furrow. Mitochondria and chloroplasts concentrate around the partition site, possibly in association with microfilaments. Fibrillar material is added rapidly to the space between the daughter cells from vesicle discharge of both cells and seems to spread into the older cell wall surrounding the embryo. The rhizoid daughter cell contains numerous mitochondria and hypertrophied Golgi bodies whose vesicles increasingly pack the cell. The thallus daughter cell is packed with a variety of vesicles, and the nucleus is surrounded by many dilated cisternae of rough endoplasmic reticulum. By the four-cell stage, chloroplasts of the rhizoid cells have weakly staining lamellae, while chloroplasts of the thallus cells are actively dividing with deeply staining lamellae.

Cell Division↗

PubMed1970