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Biomedical subjects

T D Allen

Publications and source records attributed to T D Allen.

At least 127 records · Page 7Linked to original sources

Novel substratum-dependent dendritic morphology and motility in epithelial cells.

Qualitative and quantitative light microscopy including time-lapse observations, and scanning electron microscopy are applied to study the response of cultured primary amnion epithelial cells to surfaces coated with a fibronectin-containing aggregate extracted from placenta (PSF). Cell shapes are compared with those observed in medium containing serum and amniotic fluid. Cells respond rapidly to PSF in serum-free medium to give unusual dendritic structures. Cell bodies remain rounded with numerous long and complex processes appearing and disappearing over a period of 6 to 6 1/2 h. This behaviour is associated with an 18-fold increased motility relative to control cells. Few attachments to substratum occur beneath cell bodies or along dendritic processes, points of adhesion being largely confined to the termini of extensions. Later in the response to PSF, a reduction in cell extensions is accompanied by flattening of the cell body and more conventional shapes reminiscent of control cells are restored. In serum, cells spread more slowly to give well-flattened discoid or polygonal morphologies. In amniotic fluid, blebbed, bipolar and asymmetrical cells are observed with some dendritic extensions, reminiscent of those seen on PSF. The profound influence of substratum-associated ligands on cellular behavior is noted.

Amnion↗

An ultrastructural review of collagen gels, a model system for cell-matrix, cell-basement membrane and cell-cell interactions.

Collagen gels, prepared from rat tail tendon, have been shown to be a useful system for the provision of a more physiologically relevant culture milieu than the rigid inert substratum of tissue culture plastic. The gel is penetrable by cells, but this depends on their origin, either mesenchymal or epithelial, normal or tumour derived. Furthermore, endothelial cells form a basement membrane on the gel surface from which they can be removed with detergent, providing a cell secreted substratum for testing the invasive ability of tumour derived cell lines. Various interactions between the cell and gel occur depending on cell type. Normal fibroblasts plated within the gel will stress and contract the matrix if the gel is freed and allowed to float in the culture medium. The fibroblasts also migrate to the surface and encapsulate the entire gel. Normal lymphocytes rapidly migrate into the gel and continue to move through the gel in a random manner, demonstrating gross changes in surface morphology. Studies are in progress to monitor the behaviour of leukaemic lymphocytes within the gels. Collagen gels will also support in vitro haemopoiesis, providing a useful environment for the positive selection of stromal cells. As an in vitro model for invasion and metastasis the plating of melanoma cells onto a monolayer of endothelium on the gels allows investigation of the interactions between the normal cells which form a barrier to circulating tumour cells and the tumour cells themselves.

Animals↗

The regulation of growth and development of normal and leukaemic cells.

Numerous significant advances have been made in recent years in understanding those factors and conditions which influence the growth and differentiation of haemopoietic cells. This communication is primarily concerned with defining lineage-independent and lineage-restricted growth factors and some of the aberrations which may be involved in leukaemia.

Animals↗

Effect of hydrocortisone on osteoclasts generated in cat bone marrow cultures.

The generation of osteoclasts in cultures of cat bone marrow was completely inhibited for 4 weeks with 10(-6)M hydrocortisone (HC) and partially inhibited with 10(-7) to 10(-9)M in a dose-dependent fashion. This effect was completely reversible when cultures were exposed for only 2 weeks to 10(-9) or 10(-8)M HC. However, cultures in which higher concentrations (10(-7) to 10(-5)M) were maintained for the same period did not show complete recovery in terms of numbers of osteoclasts and number of nuclei per cell after withdrawal of HC, suggesting that precursor cells of osteoclasts were also damaged by HC. To study the effects of HC on osteoclasts already present in the cultures, 10(-6)M was added to 4-week-old untreated cultures. The number of osteoclasts decreased rapidly and a gross morphological response was also apparent (rounding of the cells leading to detachment from the substratum and inhibition of cell fusion), indicating that the generation as well as the survival of osteoclasts in vitro are sensitive to HC. The morphological changes observed under optical and electron microscopy correspond to those of the reported inactive form of osteoclasts, and suggest that their function may also be altered by HC.

Animals↗

The contraction of collagen matrices by dermal fibroblasts.

Floating collagen gel cultures containing human foreskin fibroblasts have been observed to undergo a rapid contraction process. The initial rate of contraction (i.e., within the first 2 hr) was observed to be a linear function of cell number within the concentration range of 10(5)-10(6) cells/gel. Observation of thick, deresined sections of such contracting gels in the SEM, as well as observation of thin sections in the TEM, suggest that the fibroblasts exert a tension upon the surrounding collagen fibers. These observations further indicate that the fibroblasts migrate from the interior regions of the gel matrix and eventually form a monolayer of cells encapsulating the contracted collagen disc. These observations are discussed in terms of the possible mechanisms involved in gel contraction.

Cells, Cultured↗

Lymphocyte migration into three-dimensional collagen matrices: a quantitative study.

Lymphocytes have been plated onto the surface of three-dimensional gels of native collagen fibers, and their distribution throughout the three-dimensional collagen matrix has been determined in a quantitative fashion at various times thereafter. Information regarding the total number of applied cells may be obtained by this means. Lymphocyte penetration into the collagen gel does not appear to involve the expression of collagenolytic activity, nor does it require the presence of serum. Analysis of the kinetics of lymphocyte penetration into the gel matrix indicates that lymphocytes are migrating in a "random-walk" fashion. Our objective has been to establish a model system for studying the cell-matrix and cell-cell interactions which influence the pattern of lymphocyte recirculation in vivo and the results presented here are discussed in this context.

Cell Adhesion↗

The fragile X: a scanning electron microscope study.

Scanning electron microscopy (SEM) has been used to study the fragile X chromosome. The fragile site appears as an isochromatid gap in the majority of cases, confirming light microscope (LM) observations. SEM has allowed a more precise location of the fragile site to the Xq27 . 3 region.

Chromosome Mapping↗

Stromal colonies from mouse marrow: characterization of cell types, optimization of plating efficiency and its effect on radiosensitivity.

Modifications to the colony assay in vitro for stromal progenitor cells in mouse femoral marrow have been studied so as to optimize the efficiency of colony formation (CFE). The highest reproducible concentration achieved was about 30 colonies (containing fibroblasts, macrophages and endothelioid cells) per 10(6) nucleated marrow cells (range 20-50) in mice 3-4 months old, and higher by 50% in mice 14-15 months old. Each of many slight technical modifications could reduce these values by more than 30%. The importance of optimization was demonstrated by a reduced radiosensitivity when the CFE was reduced by a factor of 3 using alpha-medium stored at 4 degrees C for 15 days. The D0 value was 3.9 +/- 0.8 Gy compared to 1.6 +/- 0.1 Gy using freshly prepared medium, and this could be due to the selection of a radioresistant subpopulation. The modifications studied may partly explain the marked variations in CFE and in radiosensitivity reported in the literature.

Animals↗

Effects of culture conditions on the proliferation, morphology and migration of bovine aortic endothelial cells.

Various culture conditions, such as the presence of ascorbic acid, initial plating density and the nature of the substratum (plastic, gelatin or native collagen gels), influenced the growth, morphology and migration of three cloned populations of adult bovine aorta endothelial cells. Aorta endothelial cells showed two distinctive and reversible morphological phenotypes. Cells presenting a free apical surface were polygonal and formed sheets of overlapping or non-overlapping cells, depending on the culture conditions. When the cells were able to establish adhesive interactions over their entire cell surface they adopted an elongated shape and formed meshworks of interconnected 'sprouting' cells. The cells were capable of migrating into a collagen gel from both their basal and apical surfaces. Once in the gel, they formed characteristic, compact, three-dimensional meshworks.

Animals↗

Lest we forget.

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Female↗

The application of scanning electron microscopy to cells in culture: selected methodologies.

Cell culture is one of the most widely used techniques in modern biology. As a result, morphology of culture systems at the ultrastructural level is also becoming more widespread. Whilst offering an optimum in terms of fixation accessibility, cells in tissue culture can present problems in terms of handling for scanning electron microscopy, particularly when they are grown on, or in semi-solid media, or on gels e.g. collagen. The examination of cultured cell suspensions or mixed cell populations may also require careful handling to produce meaningful results in terms of surface morphology and cellular interaction. Cells specifically cultured on carbon films can also be used for information of internal cell structure as well as surface topography, often simultaneously, if a suitable imaging system is available. This communication reviews a wide spectrum of experimental manipulations of cultured material to provide an introduction to the gathering of morphological information from material in tissue culture.

Animals↗

Long term bone marrow cultures: an ultrastructural review.

Long-term liquid cultures of bone marrow support the growth and proliferation of self renewing haemopoietic stem cells and the majority of myeloid lineages. The maturation of granulocyte elements occurs in normal cultures, but this may be shifted to erythropoiesis by the addition of a stimulus such as serum from anaemic mice. The continued production of stem cells and differentiating populations of either granulocytes or erythroid cells is dependent upon the establishment and maintenance of an adherent layer in the cultures which arise from the stromal cells in the original marrow inoculum. To date, the presence of adipocytes, fibroblasts, reticulum cells, and endothelial cells has been established amongst the stromal cells; and neutrophil and basophil granulocytes, megakaryocytes, full erythroid differentiation, and monocytes and macrophages have been identified in the haemopoietic lineages. Interactions at the cellular level appear to occur between the lipid synthesising adipocytes and developing granulocytes, and a central macrophage and erythroblasts, forming an 'in vitro' erythroblastic islet. These associations may form aspects of an 'in vitro' haemopoietically inductive microenvironment.

Animals↗

Clonal preadipocyte cell lines with different phenotypes derived from murine marrow stroma: factors influencing growth and adipogenesis in vitro.

We have isolated continuously growing cell lines derived from mouse bone marrow stroma. These cell lines were independently obtained, and though they showed morphologies ranging from the epithelioid to the fibroblastoid patterns, they all differentiated into adipocytes. Subclones obtained from two cell lines had a very high frequency (90-100%) of differentiation into adipocytes after two or three weeks of arrested growth. Though extensive accumulation of lipid often mechanically impaired mitosis, the cells committed to adipocytes did not suffer an irreversible loss of proliferative capacity. Adipogenesis was obtained in conditions similar to those required for fat cell formation in long-term bone marrow culture. The cell lines were found to be insensitive to insulin as a signal of adipocyte differentiation. The ultrastructural characteristics of the preadipocytes and fat cells are also similar to those of the fat cells developing in long-term bone marrow culture. As such, these cell lines should prove useful for analysing cell/cell interactions in haemopoiesis.

Adipose Tissue↗

Ultrastructural aspects of erythropoietic differentiation in long-term bone marrow culture.

Long-term liquid cultures of mouse bone marrow produce stem cell (CFU-S) and differentiated granulocytes for many months. Addition of AMS (anaemic mouse serum) to the cultures almost entirely eliminates the granulopoietic activity and stimulates erythropoiesis, with full erythroid maturation and the production of adult haemoglobin. Ultrastructural analysis of in situ fixed material reveals the cell shape and surface morphology of the erythroid maturation series, and the generation of erythroblastic islands in vitro. Each erythroblastic island consists of one or more synchronously maturing cohorts of erythroid cells undergoing four or five divisions between proerythroblast and normoblast. Each island is centered on a macrophage, which interacts with the developing erythroid population in several ways. Expelled nuclei are phagocytosed by te macrophage, which also has large areas of closely apposed membrane with the erythroid cells, gap junctions, and possible reciprocal vesicular activity. Changes in the adherent layer (stromal cells) also occur with the transition from granulopoiesis to erythropoiesis. There is a reduction in the endothelial cell cover, and mobilisation of lipid from the granulopoietic associated apidocytes.

Animals↗

High-resolution scanning electron microscopy of human metaphase chromosomes.

Human metaphase chromosomes, prepared for light microscopy were examined by scanning electron microscopy. Use of an osmium impregnation technique eliminated the need for sputter-coating and allowed high-resolution visualization of uncoated specimens. Chromosomes were of three-dimensional cylindrical profile, with well-defined chromatids and centromeres. Prior to Giemsa-banding a smooth surface morphology was observed. Relaxation of chromosome integrity by Giemsa-banding pretreatment allowed resolution of several orders of chromosome structure not previously demonstrated by scanning electron microscopy. The observed organization of the chromatin fibres allowed parallels to be drawn with the radial loop model of chromosome construction as described by Marsden and Laemmli.

Chromosome Banding↗