Morphological changes in a human scirrhous gastric carcinoma cell line (KATO-III) when cultured in collagen-coated dishes

The morphological differences between cells of a human scirrhous gastric carcinoma cell line (KATO-III) cultured in plastic dishes and in collagen-coated dishes were examined by phase-contrast and electron microscopy. When KATO-III cells were inoculated into plastic dishes, a few cells became attached to the surface of the dishes and the rest remained in suspension. However, when they were inoculated into collagen-coated dishes, they all remained in suspension. In both types of dish, most of the cells in suspension were single although a few were in clusters. The cells in suspension in collagen-coated dishes differed in morphology from those in the plastic dishes. They had abundant cytoplasm, well-developed Golgi complexes, and many microvillus-like cell protrusions. Moreover, they had hemidesmosome-like and desmosome-like structures on their surface and an increased amount of intracytoplasmic desmosome-like structures. The cells in clusters in the collagen-coated dishes were closely connected by junctional complexes, such as tight junctions, desmosomes and interdigitations, whereas those in plastic dishes were linked only by desmosomes. These results suggest that collagen affects the morphology of human scirrhous carcinoma cells.     

Morphological changes in a human scirrhous gastric carcinoma cell line (KATO-III) when cultured in collagen-coated dishes

The morphological differences between cells of a human scirrhous gastric carcinoma cell line (KATO-III) cultured in plastic dishes and in collagen-coated dishes were examined by phase-contrast and electron microscopy. When KATO-III cells were inoculated into plastic dishes, a few cells became attached to the surface of the dishes and the rest remained in suspension. However, when they were inoculated into collagen-coated dishes, they all remained in suspension. In both types of dish, most of the cells in suspension were single although a few were in clusters. The cells in suspension in collagen-coated dishes differed in morphology from those in the plastic dishes. They had abundant cytoplasm, well-developed Golgi complexes, and many microvillus-like cell protrusions. Moreover, they had hemidesmosome-like and desmosome-like structures on their surface and an increased amount of intracytoplasmic desmosome-like structures. The cells in clusters in the collagen-coated dishes were closely connected by junctional complexes, such as tight junctions, desmosomes and interdigitations, whereas those in plastic dishes were linked only by desmosomes. These results suggest that collagen affects the morphology of human scirrhous carcinoma cells.     

Co-expression of differentiation markers in hybrids between friend cells and lymphoid cells and the influence of the cell shape

We have studied the regulation of differentiation within the hemopoietic system by fusing mouse Friend cells (which can be induced to undergo red blood cell differentiation) to various mouse lymphomas and myelomas which express characteristic T and B lymphocyte surface antigens. Our results show that both erythroid and lymphoid differentiation markers can be co-expressed within the same cell. To determine whether this result applies to other differentiation states, we fused suspension Friend cells to three adherent fibroblast cell lines, and isolated both adherent and suspension hybrids. In fact, suspension hybrid clones were inducible for hemoglobin, whereas adherent clones were not. No obvious differences in overall chromosome balance were evident between the adherent and suspension hybrids. A similar correlation between suspension morphology and inducibility of hemoglobin was found in hybrids between suspension Friend cells and an adherent lymphoma line. These results show that different developmental programs can be coexpressed within the same hybrid cell; but the strongly adherent type of morphology is inconsistent with expression of the red blood cell phenotype, both in hybrid cells derived entirely from hemopoietic parental cells and in cells from widely different lineages.     

Phenotypic transformation of the host cell enhances polyoma pseudovirion formation.

Phenotypic transformation of the host cell affected the formation of polyoma pseuodovirions. Polyoma virus infection of various transformed derivatives of mouse 3T3 cells resulted in the formation of predominantly pseudovirions, whereas infection of mouse 3T3 cells produced mainly polyoma virus. The effect that transformation of the host cell had on polyoma pseudovirus formation was further demonstrated by using phenotypic revertants isolated from some of the transformed cell lines. The revertants were characterized by their morphology, saturation densities, and colony-forming ability in methylcellulose suspension. By these criteria they were distinct from their transformed parents and similar to 3T3 cells. After infection, the revertants produced predominantly polyoma virus and few pseudovirus. Thus, for the cell lines used in this study, phenotypic transformation enhanced the formationof polyoma pseudovirions.     

Rollinia mucosa cell suspension cultures: Establishment and growth conditions

Different types and concentrations of plant growth regulators were tested in order to obtain the best callus and cell suspension culture growth conditions of Rollinia mucosa (Jacq.) Baill. (Annonaceae). Picloram was shown to be the most efficient for induction and production of friable calluses, independent of the concentration used. Cellular morphology and viability, fresh and dry weights, pH and medium sugar concentration were determined for cell suspension cultures. Dissimilation curves were used for the characterization of the growth of cell suspension cultures. Picloram provided the most rapid growth and produced the highest biomass, with little variation in morphology (differentiated cells). It also provided the highest dissimilation, when compared with cell suspension cultures maintained in media with 2,4-D or NAA + BA + GA₃. Stable cell suspension cultures can be established in MS medium supplemented with 20.8 μM picloram. This revised version was published online in August 2006 with corrections to the Cover Date.     

An immunogold-silver staining method for detection of cell surface antigens in cell smears

We developed an indirect immunogold-silver staining method for detection of leukocyte cell surface antigens in cell smears. Air-dried and fixed cytocentrifuge preparations or smears of peripheral blood leukocytes were incubated with monoclonal antibodies (MAb) and colloidal gold-labeled secondary antibodies. The preparations were post-fixed and silver enhancement was performed. The smears were counterstained with May-Grunwald-Giemsa and examined in brightfield light microscopy. The morphology of the cells was well preserved. Leukocytes reacting with the MAb showed black granules on their surface membranes. The intense immunostaining and the low background allowed a rapid enumeration of the positive cells. The labeling could be detected with high sensitivity by epipolarization microscopy. This immunogold-silver staining method was used to quantify T- and B-lymphocytes and natural killer cells in buffy coat smears of normal adult blood. These lymphocyte subsets correlated well with those obtained in smears with the alkaline phosphatase-anti-alkaline phosphatase (APAAP) method and with those found by labeling of mononuclear cells in suspension with immunogold-silver staining. This immunogold-silver staining method forms a good alternative to immunoenzyme methods for study of hematologic cells. In addition, it could be a general procedure for detection of cell surface antigens in all kinds of cell smears.     

Flow cytometric assessment of cell viability: a multifaceted analysis

Flow cytometry offers the possibility to simultaneously analyze, on a cell by cell basis, different parameters related to cell viability i.e. cell size, morphology and incorporation of dyes. Different types of analysis: light absorption of unstained/stained cells, forward angle light scattering (FALS), right angle light scattering (RALS) or both, cell fluorescence based on dye retention or dye exclusion (due to erythrosin B, ethidium bromide, fluorescein diacetate, rhodamine 123) were tested and compared, with the classical Trypan blue exclusion test, for their effectiveness in the determination of cell viability. Two types of cells in monolayer cultures (L929, SIRC) and a freshly isolated suspension of mouse splenocytes were used. For each dye, the optimal dose, incubation time and conditions for analysis were determined. Viability indications by different techniques for the three type of cell line and their reliability as compared with Trypan blue were analyzed.     

Identification and in situ localization of the "thymic nurse cell" in man

The observation of the "thymic nurse cell" (TNC), a reticuloepithelial cell with intracytoplasmic lymphocytes, in suspension of murine thymic tissue prompted us to investigate the existence of this cell in cell suspension, as well as in tissue sections of the human thymus. TNC-like cells were enriched in suspension by enzymatic disintegration of thymic tissue and 1 X G sedimentation over 50% fetal calf serum gradients. TNC-like cells were negative for lysosomal enzymes: in this respect, as well as in light microscopic morphology, the cells were different from tissue macrophages with intracytoplasmic lymphocytes. In electron microscopy, TNC-like cells showed reticuloepithelial characteristics. In 1-micron tissue sections, clusters of lymphocytes with a possible reticuloepithelial nucleus were observed close to blood capillaries in the cortical area. Ultrastructural analysis confirmed the epithelial nature of this cell, as well as its location adjacent to blood capillaries. We concluded that there is in situ existence of TNC in man. This observation enables studies on the role of TNC in intrathymic T cell maturation.     

In vitro cell culture of the Ararat cochineal insect, Porphyrophora hamelii Brandt]

The possibility of cultivating the Ararat cochineal cells in the nutrient media developed for cell cultures of the other insect species was investigated. The initial cochineal cell culture was obtained in suspension. The cell culture on the substrate (glass) dies within 7 days. 2 peaks of proliferative activity was observed in the suspension culture. The increase of cell ploidy and their death were found between these two peaks. The small cells survived and kept their capacity for proliferation. The hemolymph did not stimulate cell proliferation but enabled their better attachment to the substrate. The use of different media did not reveal marked differences in cell behaviour. The pigment granules disappeared during the first week of cultivation.     

Heterogeneity amongst splenic stromal cell lines which support dendritic cell hematopoiesis

Summary Long-term cultures (LTC) producing dendritic cells (DC) have been previously established from spleen. LTC support the development of nonadherent cells comprising small DC progenitors and immature DC. Similarly, the splenic stroma STX3, derived from a LTC which ceased DC production, can support DC development from precursors in overlaid bone marrow. The STX3 stroma is an immortalised mixed population of endothelial cells and elongated spindle-shaped cells, thought to be fibroblasts. The stromal cell components of STX3 have been studied here. A panel of 102 cell lines was established by single-cell sorting. A range of clone morphology, including cobblestone cells and elongated spindle-shaped cells, was reflective of heterogeneity in STX3. However, similar expression levels for the endothelial genes ACVRL1/ALK1, COL18A1, and MCAM in 13 splenic stromal cell lines suggested that both cell types had endothelial origin. The hematopoietic support function of stromal clones was tested in coculture assays with a bone marrow cell overlay. Splenic stromal cell lines with different morphology were both supporters and nonsupporters of hematopoiesis, in terms of foci formation or release of suspension cells. Cloning of STX3 led to the isolation of a panel of splenic endothelial cell lines heterogeneous in terms of morphology and hematopoietic support function.     

Agitation,aeration and perfusion modules for cell culture bioreactors

For an optimized bioreactor design which is adapted to the cultivation of sensitive animal cells different modular bioreactor components for gentle agitation, sufficient aeration and long-term perfusion were developed and investigated with respect to their suitability from laboratory to production scale. Aeration systems have been designed for both shear sensitive cells and cells which tolerate bubbles. The systems are based on either membranes for bubble-free aeration or stainless steel sparger systems. They were characterized by determination of their oxygen transfer capacity and optimized in cultivation processes of different cell lines under process conditions such as batch and perfusion mode.Different impellers for suspension cells and cells grown on carriers were investigated for their suitability to ensure homogeneous gentle mixing. A large pitch blade impeller as well as a novel 3-blade segment impeller are appropriate for homogeneous mixing at low shear rates. Especially with the 3-blade segment impeller fluid mechanical stress can be reduced at a given stirrer speed which is advantageous for the cultivation of cells attached to microcarriers or extremely shear sensitive suspension cells. However, our results indicate that shear sensitivity of animal cells has been generally overestimated.Continuous perfusion of both suspension cell cultures and cells cultivated on microcarriers could be successfully performed over extended periods of time using stainless steel spinfilters with appropriate pore sizes and systems based on microporous hydrophilic membranes. Spinfilters are suitable cell retention systems for technical scale bioreactors allowing continuous perfusion cultures of suspension cells (pore size 10 to 20 m) as well as anchorage dependent cells grown on microcarriers (pore size 75 m) over six weeks to 3 months.Applying the developed modules for agitation, aeration and perfusion process adapted bioreactor set-ups can be realized which ensure optimum growth and product formation conditions in order to maximize cell and product yields.     

Oscillations in cell morphology and redox state

Fluctuations in the intensity of light scattered and absorbed by cells in suspension have been analysed by smoothing, periodogram and power spectrum methods to reveal oscillations attributed to changes in cell morphology and the redox state of NADH and FAD (periods 10 s to 30 min). The rhythms are themselves periodically modulated in amplitude at a similar frequency and exhibit burst characteristics. The low frequency scatter dynamics are provisionally attributed to oscillations in gross morphology and the high frequency variation to changes at the cell surface. Agents, such as insulin and transferrin, affect the dynamics. The scatter results suggest that rhythmic changes in cell morphology associated with locomotion are largely inherent in the cell and not due to periodic attachment and detachment from a surface.     

Disintegration of dried yeast cells and its effect on protein extractability,sedimentation property,and viscosity of the cell suspension

The morphology of dried Candida lipolytica yeast suspended in aqueous solutions (H₂O, 0.4% NaOH, 2N HCl, and 6N HCl) and organic solvents (95% alcohol and acetone) was studied using a scanning electron microscope (SEM) and an optical microscope. The effect of high-pressure homogenization on cell-wall structure and cell clumps was also determined. The protein extractability, sedimentation property, and viscosity of cells subjected to different mechanical and chemical treatments were also investigaged. The dried yeast cells were in a spherical agglomeration consisting of 100s of closely bound cells. The clump was resistant to water, aqueous 2N HCl solution at 25°C, 95% alcohol and acetone, but vulnerable to 6N HCl, aqueous 0.4% NaOH solution, and homogenization. The homogenization of the cell suspension not only broke the clump but also cracked the cell-wall structure. The aqueous alkaline solution could have weakened the cell wall and increased the solubility of the protein released through the cracks in the cell wall. The destruction of the agglomeration and the cell-wall structure increased the hydration of the cell and thereby increased the stability of the suspension. The sedimentation and the viscosity of the cell suspension corresponded to the morphological changes and the extractability of protein in the cell suspensions with different treatments.     

Image-based analysis of cell-specific productivity for plant cell suspension cultures

More and more plant cell suspension cultures are regarded as an attractive alternative to mammalian cells as host organism for production of complex recombinant proteins. The most important advantages of the production platform are low costs, easy scalability and enhanced safety by complete lack of animal components in the cultivation media. In order to characterize, understand and control such systems accurately, it is important to determine the cell-specific productivity (Qp) of plant cell-based production platforms. Compared to many microbial and mammalian cells the morphology of plant cells is nonhomogeneous and the cells tend to form aggregates, therefore commercial cell counting systems are too unreliable to determine cell numbers in plant suspension cultures. We addressed this limitation by developing a novel cell counting method based on a combination of cell-staining and automated confocal fluorescence microscopy. This method allowed us, for the first time, to determine the cell-specific productivity of transgenic tobacco (Nicotiana tabacum cv. Bright Yellow-2) cell suspension cultures producing the human antibody M12. In the future this method will be a useful tool in the development of optimized plant cell-based production processes.     

Plant cell suspension culture rheology

The results of rheological measurements on 10 different plant cell suspension cultures are presented. Nicotiana tabacum (tobacco) suspension cultures grown in serial batch subculture display high viscosity and power law rheology. This "undesirable" rheology is shown to be a result of elongated cell morphology. The rheology of Papaver somniferum (poppy) cell suspensions is quite different; poppy suspensions behave as Newtonian fluids and have relatively low viscosity (less than 15 cP) at fresh cell densities up to 250 g/L. This flow behavior can be attributed to a lack of elongation in batch-grown poppy cells. A simple correlation for the viscosity as a function of cell density is developed for poppy suspensions up to 300 g fresh weight (FW)/L. It is shown that tobacco cells do not elongate when grown in semicontinuous culture (daily media replacement). These semicontinuously cultured cells have rheological behavior that is indistinguishable from that of poppy, further confirming the dependence of rheology on plant cell morphology. The rheology of a wide variety of other plant suspensions at 200 g FW/L is presented. Most cell suspensions, including soybean, cotton, bindweed, and potato, display low viscosities similar to poppy suspensions. Only carrot and atriplex exhibit slight pseudoplastic behavior which corresponded to a slight degree of cellular elongation for these cultures. This demonstrates that complex rheology associated with elongated cell morphology is much less common than low-viscosity Newtonian behavior. High viscosity in plant cell culture is therefore not an intrinsic characteristic of plant cells but, instead, is a result of the ability to grow cultures to extremely high cell densities due to low biological oxygen demand. (c) 1993 John Wiley & Sons, Inc.     

Isolation of single cell suspensions from the rat mammary gland: Separation,characterization, and primary culture of various cell populations

Summary Isolation and characterization of a single cell suspension from the rat mammary gland was achieved by combining selective enzymatic digestion and the mechanical agitation of a Stomacher laboratory blender with immunohistological identification of cell-specific markers. Utilizing this procedure we were able to isolate single cell suspensions of high yield (10 to 15×10⁶ cells/rat) and viability (>98%) with a concurrent decrease in isolation time and the amount of proteolytic enzymes required. Five distinct cell fractions were isolated from the mammary gland cell suspension after banding on discontinuous Percoll gradients. These populations were characterized both before and after primary cell culture by a combination of histological, immunohistological, and autoradiographic techniques. Fractions two and three were found to be enriched for mammary epithelial cells, as identified by their high binding of antikeratin antibodies. These populations also exhibited a minimal degree of binding to actin, myosin, and fibronectin antibodies. Fraction three also exhibited a high labeling index as measured by autoradiography following in vivo administration of [methyl-³H]thymidine. The remaining fractions were found to contain higher percentages of myoepithelial cells or other mammary cell types. Inasmuch as there is a direct correlation between mammary gland cell types and susceptibility to mammary gland carcinomas, further studies of these cell populations may provide new insights into the mechanisms underlying mammary gland carcinogenesis. This work was supported by grant R809580 from the U. S. Environmental Protection Agency, Office of Research Grants and Centers, Washington, D. C.     

The dynamics of surface acoustic wave‐driven scaffold cell seeding

Flow visualization using fluorescent microparticles and cell viability investigations are carried out to examine the mechanisms by which cells are seeded into scaffolds driven by surface acoustic waves. The former consists of observing both the external flow prior to the entry of the suspension into the scaffold and the internal flow within the scaffold pores. The latter involves micro‐CT (computed tomography) scans of the particle distributions within the seeded scaffolds and visual and quantitative methods to examine the morphology and proliferation ability of the irradiated cells. The results of these investigations elucidate the mechanisms by which particles are seeded, and hence provide valuable information that form the basis for optimizing this recently discovered method for rapid, efficient, and uniform scaffold cell seeding. Yeast cells are observed to maintain their size and morphology as well as their proliferation ability over 14 days after they are irradiated. The mammalian primary osteoblast cells tested also show little difference in their viability when exposed to the surface acoustic wave irradiation compared to a control set. Together, these provide initial feasibility results that demonstrate the surface acoustic wave technology as a viable seeding method without risk of denaturing the cells. Biotechnol. Bioeng. 2009;103: 387–401. © 2009 Wiley Periodicals, Inc.     

Depletion of intracellular potassium disrupts coated pits and reversibly inhibits cell polarization during fibroblast spreading

To learn more about the possible role of the coated pits endocytic pathway in cell adhesion, we studied attachment and spreading of fibroblasts whose coated pits were disrupted by depletion of intercellular potassium. Fibroblasts incubated in suspension in potassium-free medium lost 80% of their intracellular potassium within 10 min and showed disrupted coated pits based on fluorescence staining of clathrin. Potassium-depleted cells attached and spread on fibronectin-coated substrata over the same time course (15 min-2 h) as control cells. Unlike controls, however, potassium-depleted fibroblasts attained a radial morphology with circumferentially organized actin filament bundles and were unable to make the transition to a polarized morphology with stress fibers. In the radially spread fibroblasts, fibronectin receptors and vinculin colocalized in focal adhesion sites and appeared to be membrane insertion points for circumferentially arranged actin filament bundles, but these sites were much smaller than the focal adhesion plaques in polarized cells. The effects of potassium depletion on cell adhesion were reversible. Within 1 h after switching K(+)-depleted fibroblasts to medium containing KCl, cells developed a polarized morphology with actin stress fibers inserting into focal adhesion plaques. Coated pits also reformed on the cell surface during this time. Because formation of focal adhesion plaques preceded reappearance of clathrin-coated pits at the cell margins, it seems unlikely that coated pits play a direct role in adhesion plaque assembly. Polarization of fibroblasts upon addition of KCl was inhibited by ouabain showing that intracellular potassium was required for activity. Polarization also was inhibited when potassium-depleted cells were switched to potassium-containing medium under hypertonic or acidified conditions, both of which have been shown to inhibit receptor- mediated endocytosis. Our results suggest that the coated pit endocytic pathway is not required for initial attachment, spreading, and formation of focal adhesions by fibroblasts, but may play a role in cell polarization.