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191.
Merten OW 《Cytotechnology》1988,1(2):113-121
Batch cultures of mouse-mouse hybridoma cell lines were carried out and their growth and production kinetics investigated. Three main cell specific production patterns (expressed as pg IgG/cell x hour) were found, which can be used as a classification system for hybridoma cell lines (groups I–III). Cells showing the highest IgG-production at the beginning of the batch culture (during the lag and the onset of the log-phase) were classified as either group I and II. The difference was that cell lines of group II showed a second high cell specific production at the onset of the stationary and death phases. Cell lines of group III had a quite constant production of antibodies during their growth; but IgG secretion completely stopped after the beginning of the stationary phase. The implications of these three production patterns on the design of a production process are discussed. 相似文献
192.
Egg yolk lipoprotein promoted growth of a wide variety of mammalian cell lines, including plasma-cytomas and epithelial cell lines, in serum-free medium. The lipoprotein was active for cell growth when used with insulin, transferrin, ethanolamine and selenite. The most active lipoprotein fraction (YLP-pI7.5) was purified to give a single peak by chromatofocusing and gel filtration, and was homogeneous on a 0.35% agarose gel electrophoretogram. The lipoprotein was characterised as a very low density lipoprotein with a protein content of only 1.3%. This lipoprotein had an optimal concentration of 300 g/ml (4 g protein/ml). It was easily separable from proteinous molecules secreted into the serum-free medium by the cells, since it floated on the surface of the medium after addition of ammonium sulfate, to precipitate protein, and centrifugation. An associated structure of lipid and protein seemed to be still necessary for the lipoprotein to exhibit a growth promoting activity. 相似文献
193.
Twisted ribbons made of polystyrene were used as a packing material for the cultivation of anchorage dependent cells. Normal human fibroblast cells grown on this support in a laboratory scale reactor reached densities of about 5–7×105 cells/ml. The cells adhered strongly to the carrier and no cell detachment was observed upon transfer to serum free medium. The properties of this packing material and its potential use are discussed. 相似文献
194.
Process intensity of fixed bed glass sphere culture systems is increased considerably by replacing solid glass spheres with open pore glass spheres. This technique demonstrates the possibility of having a system capable of both volumetric and cell density scale up and being suitable for substrate attached and suspension cells. The yields achieved for a number of attached cell lines (approximately 107/ml) demonstrate an increase approaching one order of magnitude over solid glass spheres (approximately 106/ml). Also suspension cells were successfully entrapped in the open pore structure with similar yields. 相似文献
195.
Human-human hybridoma SH-76 cells were found to produce a factor that supported the growth of lymphocytic cells at low densities. The factor was purified from serum-free conditioned medium of the hybridoma cells by a successive application of ammonium sulfate precipitation, DEAE-Toyopearl, TSK G3000 SW and DEAE-5PW column chromatograph. The purified factor was a 72K single protein. The factor showed marked growth stimulating effect on lymphocytic cell lines, but had no effect on the growth of human adhesive cancer cell lines. Thus, the factor is a lymphocytic clonal growth factor (LCGF), as found previously in human plasma (Miyata, 1988). The LCGF of SH-76 cells could be produced in growth factor-free RPMI medium and purified easily from the conditioned medium. The factor is inactivated by heating at over 80°C, but is much more stable than the LCGF in human plasma. 相似文献
196.
A rapid fluorometric DNA assay for the measurement of cell density and proliferation in vitro 总被引:4,自引:0,他引:4
Timothy A. McCaffrey Lily A. Agarwal Babette B. Weksler 《In vitro cellular & developmental biology. Plant》1988,24(3):247-252
Summary Many research efforts require the accurate determination of cell density in vitro. However, physical cell counting is inaccurate,
time-intensive and requires removal of the cells from their growth environment, thereby introducing a host of potential artifacts.
The current studies document a very simple method of determining cell density in microtiter wells via DNA-enhanced fluorescence.
Fixed cells are stained with the A-T intercalating DNA stains DAPI or Hoechst 33342 and then fluorescence is quantified in
a plate fluorometer. Fluorescence is shown to be linearly related to cell density as determined by two physical counting methods.
The validity of the method is established in determining serum-stimulated growth of smooth muscle cells and in mitogen-induced
growth of endothelial cells. The fixed cells can be stored for prolonged periods, thus allowing time-course proliferation
assays without interassay variations. The fixed cells are also suitable for determinations of antigens of interest by ELISA.
This method is potentially valuable in many in vitro systems where the quantification of cell density and proliferation is
necessary.
This work supported in part by NIH Cardiovascular Training Grant HL07423 and a grant from the American Federation for Aging
Research to T. M. and HL35724 to B. W.
EDITOR’S STATEMENT The technique described in this paper represents an approach to quantifying cell density in adherent monolayers
of cultured cells in microtiter wells that is rapid and simple and does not require radioisotopes or removal of cells. 相似文献
197.
Allison A. Welder Tina Machu Steven W. Leslie Richard E. Wilcox June Bradlaw Daniel Acosta 《In vitro cellular & developmental biology. Plant》1988,24(8):771-777
Summary Primary mycolardial cell cultures and freshly isolated cardiac cells in suspension resprensent two isolated, whole cell models
for investigating cellular transsarcolemmal45Ca++ exchange in response to a receptor-coupled stimulus. Studies were performed to characterize beta-adrenergic receptor binding,
beta-adrenergic receptor mediated cellular calcium (45Ca++) exchange, and viability in purified primary myocardial cell cultures and freshly isolated cardiac cells in suspension obtained
from 3-to 3-d-old Sprague-Dawley rats. In addition, beta-adrenergic receptor binding was characterized in whole-heart crude
membrane preparations. All three preparations had saturable beta-adrenergic binding sites with the antagonist [125I]iodopindolol ([125I]IPIN). The suspensions had a significantly lower B
max
(42±6 fmol/mg protein) than the membranes and cultures (77±8 and 95±10 fmol/mg protein, respectively). The K
D
of the cultures (218±2.0 pM) was significantly higher than that for the suspensions (107 ±1.3 pM) and membranes (93±1.3 pM). Viability was significantly lower in the suspensions (57%) when compared to 94% viability in myocardial cell cultures after
3 h of incubation in Kreb's Henseleit buffer. Incubation of the cultures with 5.0×10−7
M isoproterenol resulted in a significant increase in45Ca++ exchange as early as 15 s. In contrast,45Ca++ exchange into the suspensions was not increased. Although both primary cell cultures and cardiac cells in suspension possess
saturable beta-adrenergic receptors, only the monolayer cultures exhibited functional beta-adrenergic receptor-mediated45Ca++ exchange. Of the two intact cell models investigated, these data suggest that primary myocardial cell cultures are more suitable
than cell suspensions for investigating beta-adrenergic receptor binding and functions in the postnatal rat heart.
This research was supported by The University of Texas Research Institute, a grant from the Texas Advanced Research Technology
Program awarded to S. W. Leslie and R. E. Wilcox, and contract 223-86-2109 from the Food and Drug Administration. 相似文献
198.
Kiyoshi Akeo Yasuhiko Tanaka Tatsuji Fujiwara 《In vitro cellular & developmental biology. Plant》1988,24(7):705-710
Summary The endocytotic process in cultured human RPE cells was observed after 1 min, 20 min, and 2 h incubation with cationized ferritin.
Within 1 min the ferritin particles were seen to attach to the cell membrane, especially between microvilli. Uncoated and
coated pits could be recognized on the cell membranes, and uncoated and coated endocytotic vesicles were found in the cytoplasm
after 20 min of incubation. These vesicles were surrounded by abundant microfilaments and had no visible membranes. Loss of
membrane may be an initial step in the process of developing into the irregular clumps of ferritin particles found inside
the plasma membrane. With time, more irregular clumps of ferritin, smaller than the particles introduced during incubation,
appeared just beneath the cell membrane. Lysosomes were adjacent to the clumps of ferritin particles associated with microtobules
and finally degraded these particles. The phagolysosomes containing many particles were surrounded by many microtubules. Small
ferritin particles surrounded but had not entered the rough endoplasmic reticulums, and no particles were seen either around
or in the Golgi apparatus.
Presented at the 7th International Congress of Eye Research, Nagoya, Japan, 27 September 1986. 相似文献
199.
A. Birkenfeld Y. Ezra N. Ron D. Navot S. Granovsky J. G. Schenker I. S. Levij I. Vlodavsky 《In vitro cellular & developmental biology. Plant》1988,24(12):1188-1192
Summary The culturing of human endometrium in conventional plastic dishes and media is only partially successful, mainly because a
growth of a heterogeneous population of cells is achieved. Naturally produced extracellular matrix closely resembles the subepithelial
basement membrane and seems to affect both growth and differentiation of cells. These qualities of the extracellular matrix
(ECM) were applied for obtaining endometrial epithelial cultures. Endometrial tissue specimens were plated after slicing on
ECM-coated dishes and kept for up to 8 d. The growth of a confluent homogeneous tissue composed of polygonal epithelial-like
cells was demonstrated. To further characterize these cells, cultures were examined by scanning electron microscopy and transmission
electron microscopy. Scanning electron microscopy revealed flattened polygonal cells covered with microvilli, among which
ciliated cells were observed. By transmission electron microscopy the cells were seen as a monolayer, with some cells overlapping,
closely adherent to the matrix. Microvilli, as well as intracellular vacuoles and glycogen granules were observed. Cell type
specific cytoskeletal markers were demonstrated by antibodies to intermediate filament proteins (keratin and epithelial membrane
antigen). Taken together, the morphologic and immunohistochemical studies indicate that a selective growth of the epithelial
component of endometrial tissue was obtained after plating unprocessed endometrial tissue fragments on ECM-coated culture
dishes.
This work was supported by PHS grant no. CA 30289 to J.V. 相似文献
200.