In situ microscopic cytometry enables noninvasive viability assessment of animal cells by measuring entropy states

Current state of the art to determine the viability of animal cell suspension cultures is based on sampling and subsequent counting using specific staining assays. We demonstrate for the first time a noninvasive in situ imaging cytometry capable of determining the statistics of a morphologic transition during cell death in suspension cultures. To this end, we measure morphometric inhomogeneity—defined as information entropy—in cell in situ micrographs. We found that the cells are partitioned into two discrete entropy states broadened by phenotypical variability. During the normal course of a culture or by inducing cell death, we observe the transition of cells between these states. As shown by comparison with ex situ diagnostics, the entropy transition happens before or while the cytoplasmatic membrane is loosing its ability to exclude charged dyes. Therefore, measurement of morphometric inhomogeneity constitutes a noninvasive assessment of viability in real time. Biotechnol. Bioeng. 2011;108: 2884–2893. © 2011 Wiley Periodicals, Inc.     

Tumor-basophil interactions in vitro--a scanning and transmission electron microscopic study.

Purified guinea pig basophils, or basophils either specifically degranulated with antigen or nonspecifically degranulated with lectin, were cultured with guinea pig line 1 hepatoma cells for 1 to 24 hr and studied ultrastructurally. As early as 1 hr of culture, degranulated or nongranulated basophils and tumor cells formed close contacts by mutually intertwined elongated cell processes and also in cultures containing degranulated basophils, extruded membrane-free basophil cytoplasmic granules became firmly attached to tumor cells. At later intervals, some tumor cells cultured with basophils exhibited cytostatic and cytopathic changes, including dense mitochondria, centralization of organelles, dilated perinuclear and rough endoplasmic cisternae, cell swelling and cytoplasmic lucency, disrupted cytoplasmic organelle and plasma membranes, nuclear pyknosis and fragmentation. Some tumor cell specialized surface attachments were either disrupted or damaged at points of basophil or basophil granule adhesion. Tumor damage was most extensive in cultures containing degranulated basophils, although only a minority of tumor cells (less than 10%) was affected. Tumor injury was seen much less frequently in the presence of nondegranulated basophils, and was absent in control cultures of tumor alone. The occasional viable tumor cells that phagocytosed basophil granules were apparently unharmed, suggesting that internalization of basophil granules by tumor cells is not cytotoxic.     

Extracellular material of some representatives of the genus Corynebacterium (the electron microscopic aspect)]

At the active developmental phases (up to 2 days) cells of toxigenic and nontoxigenic corynebacteria form extracellular vesicle-like material of two types which can be revealed both on whole cells set off by metal and stained negatively, and in ultrathin sections. Extracellular material of the first type is a derivative of an extensive membranous coat of corynebacteria and is formed as a result of its fragmentation. Vesicles of this type are devoid of electron microscope-dense content, have no tendency to coalescence, and fail to promote cell agglutination. Extracellular material of the second type is primarily formed by local thickening of the surface wall layer limited by its external dense layer and the main massif. These thickenings are filled with a microgranular substance of medium electron optic density; after accumulation this substance is released into the external environment. Vesicles of this type promote cell agglutination. Undoubtedly, extracellular material of the second type has a direct relation to the cell metabolic processes. Extracellular material of both types is encountered in all the cultures grown both on hard and in fluid nutrient media. However, in the latter case the process is apparently much more intensive. There is strict correlation between the morphology of the extracellular material and strain signs of the culture, although material of the second type is found to prevail in the cells from cultures possessing toxigenic activity.     

Freeze-drying of unfixed monolayer cultures for electron microscopic autoradiography

A method has been developed for freezing, drying and embedding of unfixed monolayer cultures for electron microscopic autoradiography (EM ARG). Experimental results showed: a) Aclar 33 C was a more suitable substrate than the plastic of petri dishes, b) cultures pressed rapidly against the polished face of a large copper cylinder chilled in liquid nitrogen had better cellular morphology than did cultures dipped in Freon 12 chilled in liquid nitrogen, and c) cultures embedded in Epon alone had finer extracellular ice spaces and lower background grain densities than did cultures embedded in Epon with 1% silicone. This method has been used to evaluate the effect of fixation on the localization of the neurotransmitter, 3H-gamma-aminobutyric acid (3H-GABA), in neurons of dispersed cell cultures. EM ARG results showed that the neuronal cell bodies and vesicle elements were present in similar numbers in both glutaraldehyde fixed and freeze-dried cultures.     

Freeze-drying of unfixed monolayer cultures for electron microscopic autoradiography

Summary A method has been developed for freezing, drying and embedding of unfixed monolayer cultures for electron microscopic autoradiography (EM ARG). Experimental results showed: a) Aclar 33 C was a more suitable substrate than the plastic of petri dishes, b) cultures pressed rapidly against the polished face of a large copper cylinder chilled in liquid nitrogen had better cellular morphology than did cultures dipped in Freon 12 chilled in liquid nitrogen, and c) cultures embedded in Epon alone had finer extracellular ice spaces and lower background grain densities than did cultures embedded in Epon with 1% silicone.This method has been used to evaluate the effect of fixation on the localization of the neurotransmitter, ³H-gamma-aminobutyric acid (³H-GABA), in neurons of dispersed cell cultures. EM ARG results showed that the neuronal cell bodies and vesicle elements were present in similar numbers in both glutaraldehyde fixed and freeze-dried cultures.     

The effect of serum batch on the in vitro lifespans of cell cultures derived from old and young human donors

In a senescence study, skin fibroblast cultures grown in the presence of a second batch of fetal calf serum (FCS) revealed delayed onsets of cell culture senescence and prolonged in vitro lifespans when compared to cell cultures grown on the initial batch of serum. These statistically significant differences occurred despite the fact that both sera displayed equal growth promoting abilities as measured by cell culture growth curves performed on parallel cultures with the two sera. When cultures grown in either sera were analyzed separately, the onset of cell culture senescence was earlier and in vitro lifespan was shorter in those cultures derived from the old donor group (ages 63–92) when compared to cultures derived from young donors (ages 21–36).     

The behavior of retinal tissue in vitro,light and electron microscopic observations

Summary Retinae from two day old rats were used in this study and the cultures were handled according to standard methods used in this laboratory. In the first few days of cultivation an abundant outgrowth of nerve fibers into the cell-free medium was observed. These fibers later degenerated and by the beginning of the second week they had completely disappeared. In the living cultures, differentiating ganglion cells, bipolar and horizontal neurons could be seen in the main explant in association with various types of glial cells. Rod cells became arranged as epithelial sheets or as clusters of cells which often formed rosettes. The nuclei of these sensory cells possessed a characteristic chromatin pattern by which they always could be differentiated from other cells in the cultures. Cytoplasmic extensions that developed from the free surface of the sensory rod cells were observed within a week following explantation. A limiting membrane separated these extensions from the nucleated part of the rod cells. Morphologic details of the different neuronal cell types could be demonstrated in cultures by Bodian's silver impregnation technique.With the electron microscope, retinal development in culture was observed and compared to the development of the retina of the intact eye. Cilia developed from processes extending from the rod cell free surface. These processes were the rod cell inner segments in which many mitochondria were seen. At the bases of these segments terminal bars developed forming the outer limiting membrane. In the area of the terminal bars microvillous extensions projected between the rod cell inner segments. After twelve days in vitro a bulb-like enlargement containing a lamellar membrane system developed at the end of the cilium. This bulb-like enlargement was a beginning of the rod cell outer segment. The lamellar system did not acquire the symmetry or precise organization during cultivation that was observed in the retina of the intact eye. The distinguishing characteristics of individual neuronal cell types seen in cultivated retinae were the same as those described for their counterparts in the retina in situ, but regular plexiform layers failed to develop. Likewise, there were no indications of typical synapses in the neuropils of the cultures. There were many processes containing vesicles similar to those in presynaptic endings and mitochondria but membrane thickenings were not apparent.The results indicate that the retina cultivated in vitro does not behave as an organized entity. The component cells dissociated more and more with time, and developmental differentiation was observed only at the cellular level.Supported by USPHS Grants 5R01NB03114-06 and 5T01GM00459 from the National Institutes of Health, Bethesda, Maryland.Sincere appreciation is expressed to Mrs. Eleanor Morris for management of the cultures, and to Mr. E. E. Pitsinger, Jr. for his photographic assistance.     

Detection of mycoplasma contamination in cell cultures by a mycoplasma group-specific PCR.

The suitability of a 16S rRNA-based mycoplasma group-specific PCR for the detection of mycoplasma contamination in cell cultures was investigated. A total of 104 cell cultures were tested by using microbiological culture, DNA fluorochrome staining, DNA-rRNA hybridization, and PCR techniques. A comparison of the results obtained with these techniques revealed agreement for 95 cell cultures. Discrepant results, which were interpreted as false negative or false positive on the basis of a comparison with the results obtained with other methods, were observed with nine cell cultures. The microbiological culture technique produced false-negative results for four cell cultures. The hybridization technique produced false-negative results for two cell cultures, and for one of these cell cultures the DNA staining technique also produced a false-negative result. The PCR may have produced false-positive results for one cell culture. Ambiguous results were obtained with the remaining two cell cultures. Furthermore, the presence of contaminating bacteria interfered with the interpretation of the DNA staining results for 16 cell cultures. For the same reason the hybridization signals of nine cell cultures could not be interpreted. Our results demonstrate the drawbacks of each of the detection methods and the suitability of the PCR for the detection of mycoplasmas in cell cultures.     

Light and electron microscopic histochemistry of the monoamines in the human foetal sympathetic ganglion in culture

Synopsis Sympathetic ganglia of 13 to 19-week-old human foetuses were cultured in small pieces with and without nerve growth factor for up to 5 weeksin vitro. The cultures were studied using phase-contrast, fluorescence and electron microscopy. Monoamines were demonstrated with the formaldehyde-induced fluorescence method, with and without pretreatment of the cultures with catecholamines or monoamine oxidase inhibitor.In the long-term cultures, primitive sympathetic cells, sympathicoblasts of types I and II, and young sympathetic neurons showed a fine structure identical to that described earlierin vivo. There were virtually no satellite or Schwann cells in the cultures. The neurons showed a considerable capacity to grow new nerve fibres in culture, even without nerve growth factor. Nerve terminals with accumulations of other nervous structures. Large granular vesicles were regularly found in the sympathicoblasts after glutaraldehyde-osmium tetroxide fixation. After permanganate fixation, dense-cored vesicles typical of adrenergic neurons were not seen, either in the perikarya, or in the processes, although it was possible to demonstrate specific fluorescence. No small intensely fluorescent (SIF) cells were observed.Variable formaldehyde-induced fluorescence was observed in the nerve cell perikarya and nerve fibres. The intensity of the fluorescence increased after treatment of the cultures with monoamine oxidase inhibitor and after incubation with catecholamines.     

Immunocytochemical localization of fibronectin (LETS proteins) on the surface of L6 myoblasts: light and electron microscopic studies.

Fibronectin (LETS protein) is a major cell surface glycoprotein component of a variety of nontransformed, substrate-attached cells in culture. Its presence has been related to increased adhesive properties. Using the peroxidase-antiperoxidase method to localize antibodies to fibronectin, we have observed that the distribution of fibronectin on L6 myoblasts varies with the density of the culture and the differentiative state of the cells. Low density, undifferentiated cultures of L6 myoblasts have a sparse accumulation of fibronectin; the antibody-antigen reaction indicates its presence on cell membranes, especially where several cells are in proximity. Undifferentiated cells in high density cultures have two forms of fibronectin localization-a diffuse staining on the membrane and a dense staining on an extracellular filamentous matrix. This matrix is composed of filaments ranging from 20–25 nm in diameter which occur singly or coalesce to form bundles. The filaments in this matrix are also observed to have dense globules scattered along their length. These filaments, which are at least in part composed of fibronectin, also react with concanavalin A, as do certain plasma membrane components. In contrast to the observations seen in undifferentiated cells, differentiated cells or myotubes have a diffuse membrane staining with antifibronectin antibodies, and the filamentous form is usually absent.     

Changes in protein content per cell during growth of mouse L cells

The relationship between cell density and protein content per cell was examined in monolayer and suspension cultures of mouse L cells. In monolayer cultures, the protein content per cell reached a maximum at 6 h after plating and retained this level for 18 h. Thereafter, the protein content per cell declined gradually during the exponential growth phase and finally returned to the initial level at the stationary phase. The changes were neither due to the effect of trypsinization nor to the exhaustion of the medium. The protein content per cell in a sparse culture was always greater than that in a dense culture for monolayer culture of L cells. In suspension culture the increase of protein content per cell during the lag phase was similar to that found in monolayer cultures. However, the gradual decline of protein content per cell observed during the exponential phase of monolayer cultures was not detected during that of a suspension culture. The results suggest that the decrease of protein content per cell in monolayer cultures may be related to some function of cell plasma membrane which could be inhibited by a cell-to-cell contact.     

A simplified touch tape preparation from tube cultures for microscopic examination of filamentous fungi

Cellophane touch tape preparation provides reproducible results in minimal time when compared to tease mount and slide culture techniques for the identification of fungi from culture plates, but it is difficult to perform from tube cultures. Here, we describe an easy to perform touch tape preparation method that provided a better result from fungal tube culture.     

Immunocytochemistry of Clostridium novyi antigens. Electron microscopic research

The dynamics of the synthesis, transfer and excretion of toxin in C. novyi, growing in a liquid culture medium, have been studied on the level of bacterial ultrastructure by means of immunoferritin techniques modified by the authors. As revealed in this study, the basic mechanism of toxin excretion is realized by the active transfer of toxin through the enveloping structures after its accumulation in the periplasmatic space. In ageing cultures toxin may also be released in the process of bacteriolysis with the degradation of bacterial structures.     

Evidence for contact stimulation of growth of homologous cells in superinoculated cultures of chick embryo cells

The secondary cultures of chick embryo cells were suspended and transferred to homologous cell cultures. Cell adhesion and proliferation were studied in these superinoculated cultures. It was shown that added cells soon adhered to the underlying cell layer which results in a prompt increase in culture density followed by the activation of DNA synthesis and cell division. Stimulation of cell proliferation involved both cell subpopulations composing the superinoculated culture: cells seeded on the built-up cell layer and cells of the layer. The contact nature of added cell mitogenic action on overlaid cell proliferation was evidenced. The cell system described can be used to investigate the adhesive properties of the cell layer apical surface, the relationship between cell growth rate and culture density, and the contact stimulation of cell proliferation.     

Light and electron microscopic localization of D-galactosyl residues in capillary endothelial cells of the canine cerebral cortex

Ricinus communis agglutinin I (RCA-I), a lectin that binds to D-galactosyl residues, intensely stained capillaries in cryostat sections of canine cerebral cortex when evaluated by the avidin-biotin-peroxidase complex method. Of seven lectins tested, only RCA-I gave strong staining of vessels and capillaries with little staining of other cortical cells. Ultrastructural studies using ferritin-, biotin-, and peroxidase-labeled RCA-I indicated that this lectin was bound to the luminal membrane of the cerebral capillary endothelial cell and that lectin receptors were distributed continuously along this membrane. Plasmalemma invaginations that bound RCA-I were also present in endothelial cells. Primary cultures of cerebral capillary endothelial cells grown on plastic or gelatin-coated glass substrates demonstrated staining of the cell membrane and perinuclear structures which appeared to be the Golgi complex and secondary lysosomes. These staining characteristics were retained when the cells were subcultured and were confirmed by ultrastructural studies. In contrast, light microscopy showed that fibronectin was more widely distributed in the cytoplasm, a finding consistent with its occurrence in the endoplasmic reticulum. This work provides support for the concept that lectins may be useful endothelial cell markers in both intact tissue and cell culture.     

TOPICAL PAPER: Utilization of Hairy Root Cultures for Production of Secondary Metabolites

The possibility of producing useful chemicals by plant cell cultures has been studied intensively for the past 30 years. However, problems associated with low product yields and culture instabilities have restricted wider industrial application of plant cell culture. The employment of hairy root culture technology, developed in the past 10 years, offers new opportunities for in vitro production of plant secondary metabolites. In contrast to cell suspension cultures, hairy root cultures are characterized by high biosynthetic capacity and genetic as well as biochemical stability. In this review, the establishment and cultivation of hairy root cultures as well as their properties and application for production of secondary metabolites are discussed.     

Primary cultures of Leydig cells from rat, mouse and pig: advantages of porcine cells for the study of gonadotropin regulation of Leydig cell function

Primary cultures of interstitial cells were prepared from the testis of mice, rats, and pigs. The cells were grown in a defined medium supplemented with low (0.1%) serum and insulin, transferrin and epidermal growth factor. Comparisons of the interstitial cell cultures from the three species were made for plating efficiency, cell survival, maintenance of hCG receptors and maintenance of steroidogenic responsiveness to hCG. The porcine cultures had a higher plating efficiency and higher hCG receptor levels per cell than Leydig cells from either rodent. Additionally, the porcine cells showed an increase in testosterone (T) production with hCG stimulation throughout their lifespan in culture while the rodent cultures showed a decrease in T stimulation with time with no stimulation by day 6 in culture. These data indicate that species differences exist in hCG receptor concentrations per cell, the maintenance of hCG receptors and steroidogenic response in culture. The initial high survival, purity and continued functional response of porcine interstitial cell cultures make them a superior system for the study of gonadotropin regulation of Leydig cell function.     

TES and HEPES buffers in mammalian cell cultures and viral studies: Problem of carbon dioxide requirement

In order to evaluate TES and HEPES as a buffer system for cell culture, the proliferative capacities of cells of several mammalian cell lines in the medium buffered with either of these compounds were examined in cultures in stoppered and open flasks at high and low cell densities. When cultivated in stoppered flasks, cells grew equally well or even better in TES- and HEPES-buffered medium than in NaHCO₃-buffered medium irrespective of cell culture density. In open flasks or Petri dishes in TES- or HEPES-buffered medium, however, the proliferative capacity of cells in low density cultures was limited. The inhibition of cell growth in the latter condition was restored (1) as the cell density of the cultures increased; (2) by feeding continuously the cultures with the gas produced by high density cultures; (3) by introducing a small amount of CO₂ to the environment.These and other evidences presented suggest that, in agreement with the prevailing notions, CO₂ is required by cells as an essential nutrient for growth, and that the desired level of CO₂ in culture can be maintained efficiently by its production by even a small number of cells in culture as long as the culture flasks are stoppered. If flasks are not stoppered, however, the level of CO₂ tension is determined by an equilibrium between the rate of its production by the cells and that of escape from culture to air, resulting in the observed failure in growth of cells in TES- and HEPES-buffered medium at low cell densities unless cultures were further supplemented with added CO₂.     

Kinetic studies of paclitaxel production by Taxus canadensis cultures in batch and semicontinuous with total cell recycle

Suspension cultures of Taxus canadensis were elicited with methyl jasmonate (MJ) under defined headspace ethylene concentrations. Kinetic studies of growth, nutrient consumption, pH variation, and paclitaxel accumulation were conducted in batch cultures and semicontinuous culture with total cell recycle. A dramatic increase of paclitaxel was obtained when the cultures were elicited with 100 microM MJ, but cell growth was thereby arrested. Supplementation of acetyl-CoA and MJ to the culture proved to be another way to improve paclitaxel yields. Using semicontinuous culture with total cell recycle, paclitaxel accumulation was increased by a factor of 4.0 relative to that in the batch culture during 35 days of cultivation.     

Tapping culture--an improved method for cell suspension culture.

A new culture vessel was designed for cell suspension culture. A silicone-covered magnet bar fixed by one end to the side wall of the bottle was held horizontally a short distance from the bottom. A standard type magnetic stirrer was used. In contrast to the conventional horizontal movement of "stirring" in cultures the bar moves vertically with a "tapping" motion. This improvement resulted in less cell injury, higher rate of cell proliferation and formation of fewer bubbles than in the conventional type. Nine cell types were simultaneously cultivated in tapping, stirring and stationary culture. All cell types proliferated more luxuriously in tapping cultures then in stirring cultures. Serial cultivation of cells in tapping cultures was also successful.