Separation of viable and nonviable animal cell using dielectrophoretic filter

Selective separation of cells using dielectrophoresis (DEP) has recently been studied and methods have been proposed. However, these methods are not applicable to large‐scale separation because they cannot be performed efficiently. In DEP separation, the DEP force is effective only when it is applied close to the electrodes. Utilizing a DEP filter is a solution for large‐scale separation. In this article, the separation efficiency for viable and nonviable cells in a DEP filter was examined. The effects of an applied AC electric field frequency and the gradient of the squared electric field intensity on a DEP velocity for the viable and nonviable animal cells (3‐2H3 cell) were discussed. The frequency response of the DEP velocity differed between the viable and the nonviable cells. We deducted an empirical equation that can be used as guiding principle for the DEP separation. The results indicate that the viable and the nonviable cells were separated using the DEP filter, and the best operating conditions such as the applied voltage and the flow rate were discussed. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

FORMATION AND ORIGIN OF BASAL LAMINA AND ANCHORING FIBRILS IN ADULT HUMAN SKIN

The purpose of this investigation was to study the formation and origin of basal lamina and anchoring fibrils in adult human skin. Epidermis and dermis were separated by "cold trypsinization." Viable epidermis and viable, inverted dermis were recombined and grafted to the chorioallantoic membrane of embryonated chicken eggs for varying periods up to 10 days. Basal lamina and anchoring fibrils were absent from the freshly trypsinized epidermis before grafting although hemidesmosomes and tonofilaments of the basal cells remained intact. Basal lamina and anchoring fibrils were absent from freshly cut, inverted surface of the dermis. Beginning 3 days after grafting, basal lamina was noted to form immediately subjacent to hemidesmosomes of epidermal basal cells at the epidermal-dermal interface. From the fifth to the seventh day after grafting, basal lamina became progressively more dense and extended to become continuous in many areas at the epidermal-dermal interface. Anchoring fibrils appeared first in grafts consisting of epidermis and viable dermis at five day cultivation and became progressively more numerous thereafter. In order to determine the epidermal versus dermal origin of basal lamina and anchoring fibrils, dermis was rendered nonviable by repeated freezing and thawing 10 times followed by recombination with viable epidermis. Formation of basal lamina occurred as readily in these recombinants of epidermis with freeze-thawed, nonviable dermis as with viable dermis, indicating that dermal viability was not essential for synthesis of basal lamina. This observation supports the concept of epidermal origin for basal lamina. Anchoring fibrils did not form in recombinants containing freeze-thawed dermis, indicating that dermal viability was required for anchoring fibrils formation. This observation supports the concept of dermal origin of anchoring fibrils.     

Inclined sedimentation for selective retention of viable hybridomas in a continuous suspension bioreactor.

The continuous separation of nonviable hybridoma cells from viable hybridoma cells by using a narrow rectangular channel that is inclined from the vertical has been investigated experimentally. The effectiveness of the settler in selectively retaining viable hybridomas in the bioreactor while permitting the removal of nonviable hybridomas has been shown to depend on the flow rate through the settler. Intermediate flow rates through the settler have been found to provide the highest removal of nonviable hybridomas relative to viable hybridoma retention. At high dilution rates through the chemostat, over 95% of the viable cells could be partitioned to the bottom of the settler while over 50% of the nonviable cells are removed through the top of the settler. This successful separation is due to the significantly larger size of the viable hybridomas than the nonviable ones. A continuous perfusion experiment was performed in which an external inclined settler was used to retain virtually all of the viable hybridomas in the culture, while selectively removing from the culture approximately 20% of the nonviable cells that entered the settler. A stable viable cell concentration of 1.0 x 10(7) cells/mL was achieved, as was an antibody productivity of over 50 micrograms/(mL.day). These represent 3- and 6-fold increases, respectively, over the values obtained from a chemostat culture without cell retention.     

Effects of the ccd function of the F plasmid on bacterial growth.

The ccd segment of the mini F plasmid containing the ccdA and ccdB genes controls the coordination between plasmid proliferation and cell physiology and fate. When the DNA replication of a thermosensitive-replication plasmid carrying the ccd segment of mini F is blocked, plasmid DNA molecules are progressively diluted through cell division until the copy number reaches 1 per cell. From this time on, there is little increase in the number of viable cells, although cells continue to divide, resulting in a mixed population of viable cells (mostly plasmid containing), nonviable but residually dividing cells, and nonviable nondividing cells. Results are presented suggesting that plasmid-containing cells are viable and continue to divide, whereas plasmid-free segregants are nonviable and form filaments after a few residual divisions, with DNA synthesis reduced or arrested in the filaments. Although the ccd functions are known to induce the SOS response when plasmid replication is blocked, the production of nonviable plasmid-free segregants is independent of the SOS cell division inhibition mechanism determined by the sfiA and sfiC genes.     

Pyrimidine dimer excision in surviving and nonsurviving cells of ultraviolet-irradiated cultures of Escherichia coli.

We compared dimer excision in viable and nonviable cells fractions separated from Escherichia coli B/r cultures exposed to ultraviolet (UV) irradiation. For cells grown on minimal medium with glycerol as a carbon source, both fractions from the irradiated (20 J/m2, 5% survival) culture excised 60 to 70% of the thymine dimers from prelabeled DNA within 120 min. This percentage was, within experimental error, the same as that obtained from unseparated cultures. When isolated viable and nonviable populations were given a second UV exposure (20 J/m2) both types of cells were again able to excise dimers. The UV survival curve for the isolated viable population indicates that these cells are no more sensitive to radiation than exponentially growing cells not previously exposed to UV. The extent of dimer excision after UV irradiation was also the same in viable and nonviable cells separated from cultures grown on a glucose minimal medium in which both populations excised about 85% of the dimers within 120 min. These results show that the extent of removal of pyrimidine dimer from deoxyribonucleic acid is not precisely correlated with survival of repair-competent bacterial cells after exposure to UV light.     

Analysis of mammalian viable cell biomass based on cellular ATP

Analysis of cellular ATP as a means of measuring viable biomass loading was investigated in hybridoma cell culture. ATP analysis by the luciferin-luciferase assay was compared with trypan blue-stained hemocytometer counts. The cell-specific ATP content varied between 2 and 6 fmol per viable cell over a batch culture. ATP levels were highest during exponential growth, and decreased during the stationary and decline phases. Electronic counting and volume measurements were performed to assay the viable cell biomass. Cell sorting, using fluorescein diacetate, was used to separate viable and nonviable cells in cultures with between 35% and 90% viable cells. Viable cells contained over 2 orders of magnitude greater cell-specific ATP than nonviable cells. Cell-specific ATP correlated directly with the viable cell volume rather than viable cell numbers. Over the range of batch culture conditions, ATP analysis should provide a more accurate measurement of hybridoma viable biomass than hemocytometer counts.     

Biofilm structure and cell vitality in a laboratory model of subgingival plaque

The accumulation of dental plaque below the gingival margin (i.e. subgingival plaque) is responsible for the most prevalent microbe-induced diseases of humans--the periodontal diseases. Access to this plaque is difficult, making studies of its structure in vivo very difficult. We have, therefore, used a constant-depth film fermenter to grow microcosm subgingival dental plaques under conditions similar to those existing in vivo to enable us to study certain aspects of its structure. Confocal laser scanning microscopy revealed that the biofilms consisted of pillar-like stacks of bacteria separated by water channels. In terms of their structure, these microcosm dental plaques reached a pseudo steady-state after 4 days. Individual optical sections generally showed the outer layers of the biofilm stacks to contain a high proportion of nonviable cells surrounding an inner core of predominantly viable cells with "veins" of nonviable bacteria penetrating from the outer layers through into the inner core. Such a structure differs from that classically described for biofilms growing in an aerobic atmosphere where the arrangement of viable and nonviable bacteria is usually reversed. The reasons for the preponderance of nonviable bacteria in the outer layer of the bacterial stacks remain to be established.     

A permanent cell viability assay using alcian blue.

The alcian blue dye exclusion method for glutaraldehyde-fixed cells has been utilized with "centrifugal cytology" to prepare permanent records of the viability of individual cells present in suspensions. The viability of spleen cell suspensions separated by linear bovine serum albumin density gradient centrifugation has been measured with this method. Combined light and scanning electron microscopy of nonviable and viable cells demonstrated membrane alterations in alcian blue-stained nonviable cells, while viable cells were spherical and displayed uniform surface features.     

Rapid viability assessment of spores of several fungi by an ionic intensified fluorescein diacetate method

Fluorescein diacetate (FDA) was applied to the viability assessment of spores of Aspergillus niger, Rhizopus stolonifer, Fusarium oxysporum, and Penicillium citrinum. The fluorescence of individual cells was quantitated with a charge coupled device (CCD) detector. When staining was carried out in a phosphate buffer solution (10 mM, pH 7.0), weak or no fluorescence was emitted from viable spores of A. niger and R. stolonifer, which made it difficult to distinguish between viable (nontreated) and nonviable (heat treated at 90°C for 30 min) spores. The addition of NaCl, KCl, or MgCl₂ to the staining solution caused an increase in the fluorescence intensity of A. niger viable spores, from which nonviable spores could be distinguished. The same effect of NaCl was observed in staining the spores of other species.     

A Permanent Cell Viability Assay Using Alcian Blue

The alcian blue dye exclusion method for glutaraldehyde-fixed cells has been utilized with “centrifugal cytology” to prepare permanent records of the viability of individual cells present in suspensions. The viability of spleen cell suspensions separated by linear bovine serum albumin density gradient centrifugation has been measured with this method. Combined light and scanning electron microscopy of nonviable and viable cells demonstrated membrane alterations in alcian blue-stained nonviable cells, while viable cells were spherical and displayed uniform surface features.     

Dead cell discrimination with 7-amino-actinomycin D in combination with dual color immunofluorescence in single laser flow cytometry.

Identification of nonviable cells in immunofluorescently stained cell populations is essential for obtaining accurate data. Fluorescent non-vital DNA dyes, particularly propidium iodide (PI), have been used routinely in flow cytometry for discrimination of dead cells from viable cells on the basis of fluorescence. We describe here the use of an alternative DNA dye, 7-amino-actinomycin D (7-AAD), which can replace PI for the exclusion of nonviable cells. As an example, we present in this paper the utilization of 7-AAD on various leukemic cell lines for dead cell exclusion whenever the viable cell population could not be discriminated reliably from nonviable cells on the light scatter histogram; 7-AAD is suitable for dead cell discrimination in lengthy experiments because it is efficiently excluded by intact cells and has a high DNA binding constant. In addition, the dye is valuable in combination with phycoerythrin (PE)-fluorescence dual-color flow cytometry on a single argon laser instrument, since its emission in the far red can easily be separated from the emission of PE; 7-AAD was used on fluoresceinisothiocyanate (FITC) and PE surface-labeled human thymocytes for characterization of the dying subpopulation of cells which is undergoing programmed cell death. In this heterogeneous cell preparation, the spectral properties of the dye permitted the classification of viable and nonviable cell subpopulations by multiparameter analysis.     

Determining the spatial distribution of viable and nonviable bacteria in hydrated microcosm dental plaques by viability profiling

AIMS: The aim of this study was to use confocal laser scanning microscopy (CLSM) to examine the spatial distribution of both viable and nonviable bacteria within microcosm dental plaques grown in vitro. Previous in vivo studies have reported upon the distribution of viable bacteria only. METHODS AND RESULTS: Oral biofilms were grown on hydroxyapatite (HA) discs in a constant-depth film fermenter (CDFF) from a saliva inoculum. The biofilms were stained with the BacLight LIVE/DEAD system and examined by CLSM. Fluorescence intensity profiles through the depth of the biofilm showed an offset between the maximum viable intensity and the maximum nonviable intensity. Topographical differences between the surface properties of the viable and nonviable biofilm virtual surfaces were also measured. CONCLUSIONS: The profile of fluorescence intensity from viable and nonviable staining suggested that the upper layers of the biofilm contain proportionally more viable bacteria than the lower regions of the biofilm. SIGNIFICANCE AND IMPACT OF STUDY: Viability profiling records the transition from predominantly viable to nonviable bacteria through biofilms suggesting that this technique may be of use for quantifying the effects of antimicrobial compounds upon biofilms. The distribution of viable bacteria was similar to that found in dental plaque in vivo suggesting that the CDFF produces in vitro biofilms which are comparable to their in vivo counterparts in terms of the spatial distribution of viable bacteria.     

Isolation of the nonviable cells produced during normal growth of recombination-deficient strains of Escherichia coli K-12

During normal growth, cultures of recombination-deficient (Rec(-)) strains contain a population of cells that do not form colonies. Such cells are not present in a culture of an isogenic Rec(+) strain. We present a procedure for isolating and studying this defective population of cells. Exposure of a growing Rec(-) or Rec(+) culture to low levels of penicillin causes the dividing cells to elongate. The size of the nonviable cells present in the Rec(-) cultures is unaffected. The nonviable cells are then separated from the elongated cells by velocity sedimentation. This isolation technique provides a convenient way of analyzing the composition, biosynthetic capacity, and enzymatic function of the nonviable cells before isolation. In this paper we present data showing that before fractionation the nonviable cells in the Rec(-) culture are defective in their ability to synthesize beta-galactosidase, whereas the Rec(-) viable cells behave like the Rec(+) cells in this regard. This observation confirms the existence of at least two classes of cells in liquid cultures of Rec(-) strains grown under normal conditions. That class of cells which is unable to synthesize beta-galactosidase is the same class that cannot form colonies when plated on solid medium.     

Using propidium monoazide to distinguish between viable and nonviable bacteria, MS2 and murine norovirus

Aims: The ability to distinguish between viable and/or infectious micro-organisms and inactivated cells is extremely important for correctly performing microbial risk assessments. In this study, we evaluated whether propidium monoazide (PMA)-qPCR could distinguish between viable and nonviable bacteria and viruses. Methods and Results: A PMA-qPCR combined assay was applied to viable and inactivated bacteria (Escherichia coli and Bacillus subtilis) and viruses (MS2 and murine norovirus [MNV]). PMA, a DNA-intercalating agent, in combination with PCR was better able to distinguish between viable and nonviable bacteria and viruses than conventional PCR. Conclusions: These results suggest that a combined PMA-qPCR assay can be used to measure the viability of bacterial cells and bacteriophage MS2, but not MNV. Significance and Impact of the Study: PMA-qPCR could potentially be used to measure the viability of some micro-organisms, including virus. However, a thorough evaluation should be performed prior to measuring the viability of micro-organisms by PMA-qPCR in a quantitative way.     

Automated image analysis of live/dead staining of the fungus Aureobasidium pullulans on microscope slides and leaf surfaces

An image analysis program and protocol for the identification and enumeration of live versus dead cells of the yeast-like fungus Aureobasidium pullulans was developed for both populations on microscope slides and leaf surfaces. Live cells took up CellTracker Blue, while nonviable cells stained with DEAD Red. Image analysis macro programs running under Optimas software were used to acquire images and to differentiate and enumerate viable from nonviable cells. The software was capable of discriminating green as a third parameter for identification and quantification of green fluorescent protein-expressing cells in a wild-type population.     

Fluorescence Staining for the Determination of Cell Viability

Primuline was shown to differentiate between viable and nonviable yeast cells taken from certain environments; the technique may be applicable to higher cells.     

Use of a photolabeling technique to identify nonviable cells in fixed homologous or heterologous cell populations

Flow cytometric determination of viable versus nonviable cells in fixed samples can be accomplished by utilizing the irreversible binding of photoactivated ethidium monoazide (EMA). EMA is a positively charged molecule which is excluded by cells with intact membranes (viable cells), included by cells with damaged membranes, and can be photochemically crosslinked to nucleic acids using visible light. EMA fluorescence can be excited using a standard argon laser operating at 488 nm and is able to be distinguished from fluorescein and phycoerythrin. Fixation is important when analyzing cells from a potentially infectious origin. EMA is photochemically crosslinked and therefore unable to leak out of cells when removed from the extracellular media, unlike propidium iodide (PI) or other viability stains, which were heretofore commonly used. We demonstrate the usefulness of EMA in combination with fluoresceinated and phycoerythrin labeled monoclonal antibodies in immunophenotyping. The photoaffinity labeling technique allows for a quick and efficient means of identifying nonviable cells which cannot be distinguished on the basis of light-scattering properties.     

A SIMPLE METHOD FOR THE ISOLATION AND ENUMERATION OF SPARSE POPULATIONS OF CYANOBACTERIA IN ESTUARINE WATERS1

A simple, sensitive assay method for the isolation and enumeration of sparse populations of cyanobacteria in an estuarine system is described. The method, based on the standard membrane-filter plate count technique, differentiates between viable and nonviable cells. It was found that an estuarine water-based agar medium was the most suitable medium for isolation of cyanobacteria. Because of the restricted nature of colony development, isolation of individual species is easily accomplished.