Electronic Cell Volume Research Articles

Single cell analysis applied to antibody fragment production with Bacillus megaterium: development of advanced physiology and bioprocess state estimation tools

BackgroundSingle cell analysis for bioprocess monitoring is an important tool to gain deeper insights into particular cell behavior and population dynamics of production processes and can be very useful for discrimination of the real bottleneck between product biosynthesis and secretion, respectively.ResultsHere different dyes for viability estimation considering membrane potential (DiOC2(3), DiBAC4(3), DiOC6(3)) and cell integrity (DiBAC4(3)/PI, Syto9/PI) were successfully evaluated for Bacillus megaterium cell characterization. It was possible to establish an appropriate assay to measure the production intensities of single cells revealing certain product secretion dynamics. Methods were tested regarding their sensitivity by evaluating fluorescence surface density and fluorescent specific concentration in relation to the electronic cell volume. The assays established were applied at different stages of a bioprocess where the antibody fragment D1.3 scFv production and secretion by B. megaterium was studied.ConclusionsIt was possible to distinguish between live, metabolic active, depolarized, dormant, and dead cells and to discriminate between high and low productive cells. The methods were shown to be suitable tools for process monitoring at single cell level allowing a better process understanding, increasing robustness and forming a firm basis for physiology-based analysis and optimization with the general application for bioprocess development.

Electronic Cell Volume and ALDH Expression of Hematopoietic Stem Cells Obtained by Apheresis

Small cell volume or diameter (3–7μm) appears to be one of the characteristics that may be used to identify early stem cells. Hematopoietic stem cells may be characterized by the expression of cell surface markers such as CD34, CD117 and CD133. In addition, stem cells such as “SP cells” can be recognized by the efflux of dyes such as Hoechst 33342 and presence of aldehyde dehydrogenase (ALDH). In prior studies we used the Beckman Coulter® Cell Lab Quanta(™) SC MPL analyzer to determine the electronic cell volume, diameter and marker expression (CD34, CD90, CD117 and CD133) of cells from peripheral blood apheresis (HPC, Apheresis) samples (Shariatmadar et al. Cytometry B , 74B:182–188, 2008; Sharma et al. Cytometry A , 73A:160–167, 2008). In the present study, we used this analyzer to determine cell volume, diameter and marker expression of ALDHpositive stem cells from HPC, Apheresis samples of 44 patients with hematologic malignancies mobilized with granulocyte colony-stimulating factor. The mean electronic volume of the ALDHpositive cells was 286μm3 (SD±27) corresponding to a diameter of 8.2μm. The mean percentage of ALDHpositive cells analyzed was 0.51% (SD± 0.004). CD34 expression was seen in 0.13% (SD±0.001) of the ALDHpositive cells. The mean electronic volume of the ALDH and CD34 positive cells was 270μm3 (SD± 33) corresponding to a diameter of 8.0μm. CD133, CD117 and CD90 expression was seen in 0.28% (SD± 0.01), 0.17 (SD± 0.05) and 0.04% (SD± 0.03) of the ALDHpositive cells. The mean electronic volume of the ALDHpositive cells with CD133 expression was 275μm3 (SD±29) corresponding to a diameter of 8.1μm while that of the ALDHpositive cells with CD117 and CD90 expression was 284μm3 (SD± 32) and 265μm3 (SD± 35), corresponding to a diameter of 8.15 and 7.9μm.Stem Cell MarkerElectronic Volume (μm3)Diameter (μm)CD90 (Thy-1)300μm3 (SD± 38.5)8.3μm (95% CI=8.1–8.4)CD117 (c-Kit)349μm 3 (SD± 47.4)8.7μm (95% CI=8.5–8.8)CD133 (Sca-1)322μm3 (SD± 44.5)8.5μm (95 % CI= 8.3–8.6)ALDH+286μm3 (SD± 27.0)8.2μm (95 % CI= 8.1–8.3)ALDH+CD34+270μm3 (SD± 33.0)8.0μm (95 % CI= 7.8–8.2)ALDH+ CD90+265μm3 (SD± 35.0)7.9μm (95 % CI= 7.8–8.1)ALDH +CD117+284μm 3 (SD± 32.0)8.15μm (95 % CI= 7.9–8.2)ALDH +CD133+275μm3 (SD± 29.0)8.1μm (95 % CI= 8.0–8.3)The use of electronic cell volume in conjunction with side scatter might provide a useful method for characterization of stem/progenitor cell populations with specific marker expression. In our study, hematopoietic stem cells expressing ALDH and bearing early stem cell markers were relatively larger than those previously reported. Future studies will aim to characterize the long term repopulating capability of this fraction of stem cells in in-vivo models.

Electronic volume of CD34 positive cells from peripheral blood apheresis samples

The authors have used a flow analyzer to measure electronic cellular volume of peripheral blood hematopoietic stem/progenitor cells obtained by granulocyte-colony stimulating factor (G-CSF) mobilization and apheresis (HPC-A) of patients with hematological malignancies. Fifty three apheresis samples stained with CD45-fluorescein isothiocyanate (FITC) and CD34-R-phycoerythrin (PE)-labeled antibodies after erythrocyte lysis with BD FACS Lysing Solution were analyzed for electronic cell volume and two-color FITC and PE fluorescence. Lymphocytes, monocytes and granulocytes in the HPC-A samples had a mean electronic volume of 414, 797, and 670 microm(3), respectively corresponding to cell diameter of 9.25, 11.5, and 10.85 microm. In 53 HPC-A samples analyzed, the mean electronic volume of the CD34 positive mononuclear cells was 407 microm(3) while the CD45 positive cells had mean volume of 453 microm(3). CD34 positive stem/progenitor cells have a smaller volume and diameter than CD45 positive mononuclear cells in HPC-A samples. In the present study the CD34 stem/progenitor cells had a considerably larger diameter than that of stem cells previously reported in the literature. With the availability of electronic cell volume as a parameter in flow cytometric analysis, further studies can be carried out to correlate stem cell volume with specific phenotypic marker expression.

Antisense oligodeoxynucleotide to the cystic fibrosis transmembrane conductance regulator inhibits cyclic AMP-activated but not calcium-activated cell volume reduction in a human pancreatic duct cell line.

Cystic fibrosis (CF) is characterized by a defect in cAMP-regulated chloride channels in epithelial cells. The CF gene product CF transmembrane conductance regulator (CFTR) is expressed in the apical membrane of pancreatic duct cells, and mutant CFTR accounts for the pathology in the CF pancreas. PANC 1, a pancreatic duct cell line, has not been considered a good model for studying CFTR and pancreatic chloride transport because CFTR mRNA and protein are undetectable using standard methods. Using electronic cell sizing and cell volume reduction under isotonic conditions, PANC 1 cells were found to possess both cAMP and calcium-activated chloride conductances. Using CFTR antisense oligodeoxynucleotides, the cAMP-activated conductance could be specifically inhibited in a concentration- and time-dependent manner. These findings demonstrate that PANC 1 cells express CFTR and a CFTR-independent calcium-activated chloride channel. With electronic cell sizing and CFTR antisense oligodeoxynucleotides, PANC 1 cells can provide an ideal system for the study of pancreatic duct cell physiology and pathophysiology with respect to the role of CFTR in the pancreas. These findings also suggest that antisense oligodeoxynucleotides may provide a more sensitive yet highly specific means of detecting low levels of expression of CFTR than currently available.

Improved multilaser/multiparameter flow cytometer for analysis and sorting of cells and particles

An improved multilaser instrument has been developed for quantitative analysis and separation of biological cells and particles. Argon ion, krypton ion, and dye lasers are employed as excitation sources to sequentially illuminate cells labeled with multiple fluorochromes as they pass through an improved flow chamber that incorporates an electronic cell-volume sensor and an optical measurement region. Detectors located on the axis of each excitation beam are used to measure axial light loss and forward light scatter. Multicolor fluorescence is measured using a five-channel detector located orthogonal to the laser beam(s)-cell stream intersection(s). Sequential measurements are made on a cell-by-cell basis to provide pulse height, area, and width signals that are made coincident in time by analog delay modules to increase data throughput. Analog electronics are used to compute real-time ratios, sums, and differences of signals. Up to eight signals are acquired and displayed as single-parameter frequency distribution histograms and bivariate diagrams using a microcomputer. Processed signals also activate cell sorting according to computer-controlled preselected parametric criteria. The unique measurement capabilities and other new features designed into this instrument represent marked technological improvements over our previous system.

Analysis of cytosolic ionized calcium variation in polymorphonuclear leukocytes using flow cytometry and Indo‐1 AM

The present report describes the increase in cytosolic-free calcium levels (Ca2+i) induced by chemoattractants in polymorphonuclear leukocytes (PMN) using the new fluorescent Ca2+ chelator Indo-1 AM. Increases in cytosolic Ca2+ were measured by flow cytometry. With this approach, 98% of PMN were found to respond to F-Met-Leu-Phe (FMLP) and leukotriene B4 (LTB4) stimulation. Although both substances induced a rapid and relatively homogeneous rise in Ca2+i, only FMLP gave a sustained Ca2+i rise, whereas that induced by LTB4 appeared transient. In addition, the combined use of wide-angle light scatter and electronic cell volume measurement allowed analysis of Ca2+i variations in the total peripheral blood cell population. The supernatant of a human fibrous histiocytoma cell line (GCT) was able to increase Ca2+ release in PMN. This activity may be ascribed to a new granulocytic activation factor, as neither human recombinant interleukin-1 alpha (IL-1 alpha), granulocyte-colony stimulating factor (G-CSF), nor granulocyte-macrophage-colony stimulating factor (GM-CSF), which were present in this supernatant, were able to induce a Ca2+i rise in PMN.

Cytotoxic effect of heterologous and autologous serum factor on guinea pig thymocytes assayed by electronic cell volume distribution analysis.

The cytotoxic effect of serum on guinea pig thymocytes was studied by dye exclusion and by analysis of cell volume distribution curves obtained by means of a computer-assisted electronic cell volume distribution analyzer. Guinea pig, rabbit and human sera were shown to be cytotoxic for guinea pig thymocytes (incubation for 1 h at 37 degrees C or overnight at 4 degrees C). A similar, but less potent, activity was found in autologous guinea pig serum (4 degrees C). The activity was dialysable and lyophilizable but was destroyed by heating at 56 degrees C for 30 min. It was recovered after ammonium sulphate precipitation followed by dialysis. The effect of this dialysate was dose-dependent. A tentative definition of viable and dead cells was made on the basis of 'electronic' cell volume. The following results indicate that by this definition electronic cell volume distribution allowed detection of the cytotoxic serum factor: (1) sera and dialysates produced marked distortion of cell volume distribution curves, not seen in controls treated with heat-inactivated preparations; (2) the dose-response relationship was similar when dye exclusion and cell volume distribution was used as assay: (3) both methods detected similar differences in sensitivity of different thymocyte subpopulations to serum; (4) the electronic method could be used on separate thymocyte subpopulations although they differed in cell volume. This rapid, objective and reproducible method for studying the cytotoxic effect of sera may be useful during attempts to purify and identify the active factor(s).

Kinetic study of Kurloff cells in guinea pig thymus.

One single injection of estradiol to male guinea pigs resulted in the appearance of Kurloff cells in the thymus and spleen in a maximal number after 2-3 weeks. In both organs, Kurloff cells of three categories were observed with different size and number of the inclusion(s). In the thymus, cells with small and medium-sized inclusions were present almost exclusively among low-density thymocytes. Cells with one large inclusion were initially present among low-density cells, but with time an increasing proportion were found among high-density thymocytes. This indicates that the different categories of Kurloff cells represent maturational stages and follow the normal differentiation step of thymocytes from low to high density. According to electronic cell volume distribution analysis, estradiol treatment was associated with a shift in cell volume towards larger cells, and this shift was correlated in time with the appearance of Kurloff cells with large inclusions. In thymus sections there was evidence for a massive and selective migration of Kurloff cells via interlobular lymph vessels. No difference in localization of Kurloff cells was noted at various times after estradiol treatment. Attempts to induce Kurloff cell formation from bone marrow, spleen or thymus cells during 2 weeks in vitro were unsuccessful, also when culturing the cells in serum from estradiol-treated animals. This negative result, the long lag phase of estradiol-induced Kurloff cell formation in vivo, and the reported lack of estradiol receptor in Kurloff cells indicate that the Kurloff cell induction by estradiol may be indirect.

Analysis of cephalosporin-induced changes in the volume distribution of Escherichia coli cultures by an electronic counter-channelanalyser.

The demonstration of morphological alterations of the bacterial cell together with bactericidal kinetics are of value for the description of the antibacterial activity of beta-lactam antibiotics. One of the indicators of antibiotic effect on bacterial morphology is the change in bacterial cell volume. This can be demonstrated graphically and numerically by means of electronic cell counting and volume distribution analysis using the Coulter Counter-Channelanalyser system connected to a computer. After registration of the distribution of the relative volume of the Escherichia coli population, the mean cell volume was calculated. The latter parameter increased more than 6 fold with increasing cefotaxim-exposure times. The onset of the delayed (2 h after cefotaxim administration) bacteriolytic effect was reorganized by the appearance of small cell breakdown particles and quantified by counting. In order to demonstrate the therapeutic process graphically the distribution curves were transformed to a common scale. Since a complete storage of the data per sample is performed normally within one minute, the Coulter Counter-Channelanalyser technique can be carried out simultaneously with the bactericidal kinetic experiment. The data demonstrate the speed and intensity of the morphological cell alterations induced by the beta-lactam antibiotic.

Design of flow chamber with electronic cell volume capability and light detection optics for multilaser flow cytometry

A multibeam optical detection system has been developed with a high optical efficiency, achieved through a reduction in the number of optical interfaces employed in the system. This reduction is made possible by a combination of employing simple lenses, gluing the objective lens directly upon the face of the flow cuvette and the extraction of only one fluorescence signal from each laser beam. A modified flow chamber is also described that includes fluidic resistance elements for the elimination of most of the electric shielding normally associated with electronic cell volume measurements.

The biology of tumor growth in the non-Hodgkin's lymphomas. A dual parameter flow cytometry study of 220 cases.

Dual parameter flow cytometry studies (cell DNA content and electronic cell volume) were performed in 220 cases of non-Hodgkin's lymphoma. All cases were characterized as B or T cell malignancies, based on immunologic surface marker characteristics. Aneuploidy by flow cytometry was more common among the B cell lymphomas than among the T cell lymphomas, and was most common among the large B cell lymphomas and B cell lymphomas of intermediate size. Ploidy index distributions showed a prominent hyperdiploid peak, as well as tumor cell populations with near-tetraploid DNA contents. In serial studies, a decrease in ploidy index was observed in association with clinical and histologic transformation in one case. The highest S fractions were observed among the large and intermediate B cell lymphomas and among the aggressive T cell lymphomas. In clinical samples consisting of mixtures of diploid and aneuploid populations, the data on the aneuploid components could often be separated from other components of the mixture in multiparameter studies on the basis of the larger electronic cell volumes of the aneuploid cells. In each case, the aneuploid large cell component almost invariably had a higher S fraction than the residual component(s) of the mixture. Overall, the data are consistent with a model of clonal selection and clonal evolution in the lymphomas in which early cytogenetic abnormalities that involve little or no change in total cell DNA content are followed by cell tetraploidization that is associated with cytogenetic instability and chromosome loss over the course of time.

The mutual effect of hydrogen ion concentration and osmotic pressure on the shape of the human erythrocyte as determined by light scattering and by electronic cell volume measurement.

The measurement of the fluctuations of the scattered light were the source of information on the flatness of erythrocytes. Data on cell volume, together with the measure of scattered light fluctuations, defined the cell shape. The measurements were repeated in a wide range of osmotic pressures and pH values in order to affect both the hemoglobin and cytoskeleton. Major volume changes were detected at low osmotic pressures and pH. The erythrocyte volume was determined by electronic volume measurement. The cell flatness is maximal at physiological conditions.

Simultaneous three color and electronic cell volume analysis with a single UV excitation source.

We have adapted an Ortho ICP-22 flow cytometer (Ortho Instruments, Westwood MA) for the simultaneous measurement of three independent fluorochromes and cell volume. This has been accomplished by the addition of a third photomultiplier tube and the development of a new electronic cell volume (ECV) flow cell. Cells are first analyzed as they pass through the 100 U ECV aperture and are then excited approximately 15 musec later by the 365 nm mercury are beam reflected by a 400 nm dicroic mirror. Independent blue, green and red signals can be associated by a delay circuit to the ECV signal from the same cell. We have developed this system as an aid in the analysis of tumor cell and macrophage heterogeneity and differentiation. The choice of stain combinations to be used is extremely flexible and permits the analysis of a wide range of enzyme activities in conjunction with DNA/RNA and phagocytic probes. Data presented indicates the value of this approach in identifying the presence of plasminogen activator-like activity in both tumor and inflammatory cells within a malignant effusion as well as the quantitative expression of a number of markers of macrophage differentiation. Although the described techniques have been developed on a mercury arc instrument, they can be used equally well with cell sorters.

Studies on Thymocyte Subpopulations in Guinea Pigs

Thymocytes were separated according to increasing buoyant density into the three subpopulations la (25% of recovered cells), lb (20%) and II (55%), and according to binding to peanut agglutinin (PNA) into PNA<sup>+</sup> (65 %) and PNA<sup>–</sup> cells (35 %). The frequency of PNA<sup>+</sup> was 56% in la, 60% in lb and 66% in population II. Electronic cell volume determinations disclosed mean volumes of 160 fl for la, 130 fl for lb and 100 fl for population II. PNA<sup>+</sup> and PNA<sup>–</sup> cells were very similar as regards cell volume. Thus, PNA<sup>+</sup> and PNA<sup>–</sup> cells are remarkably uniformly distributed among cell categories of different density and cell volume. The rapidly cycling thymocytes, regarded as the most immature cells in the thymus, and the target cells for a thymocyte growth factor both belonged to the PNA<sup>+</sup> cells of population la. The mitogen-responsive thymocytes also belonged to population la, but were PNA<sup>-</sup>. The largest subpopulation of thymocytes, apparently corresponding to the small, non-cycling cortical cells, were recovered as PNA<sup>+</sup> cells of population II.

Relationship of somatic cell count and cell volume analysis of goat's milk to intramammary infection with coagulase-negative staphylococci.

The prevalence of intramammary infection in 4 commercial goat herds was studied in conjunction with electronic somatic cell count and volume analysis determined using a Coutler Counter and volume analyser. Neither streptococci nor mycoplasma were isolated from any half and the prevalence of intramammary infection with Staphylococcus aureus ranged from 0 to 3% between herds. For coagulase-negative staphylococci the range from infected halves was 36-71%. There was no significant difference between the mean total microscopic somatic cell count for halves infected with coagulase-negative staphylococci and those free from infection. A similar trend was observed for electronic somatic cell counts although the mean electronic cell count was greater than the mean total microscopic count on the 2 occasions that they were compared. The correlation coefficients between the 2 cell counting methods were 0.86 and 0.94. Between herds there were significant differences in mean electronic somatic cell count, with herd means ranging from 438 x 103 to 1684 x 103 cells/ml. In 2 of the 4 herds studied, milk samples from halves infected with coagulase-negative staphylococci had a significantly higher prevalence of cell volume distributions with a modal cell volume between 65 mu 3 and 100 mu 3. This was attributed to a higher proportion of polymorphonuclear neutrophils. Use of electronic somatic cell count and cell volume analysis were considered of little value in predicting infection caused by coagulase-negative staphylococci as there was a high proportion of false negative and false positive predictions.

Analysis of somatic cell volume distribution as an aid to the diagnosis mastitis.

Electronic somatic cell counts (ESCC) and relative cell volume distributions (RCVD) in milk samples were obtained simultaneously on a Coulter Counter Model TA and their potential assessed, either singly or in combination, for identifying mastitic quarters and cows in 2 herds. Using log (ESCC) alone the probability P m of misclassifying quarters infected with major pathogens was 6.7% for herd 1 and 18.1% for herd 2. The inclusion of RCVD variables with log (ESCC) did not substantially improve the chance of detecting quarters infected with major pathogens. However, the RCVD variables did improve the chance of identifying quarters infected by major and minor pathogens compared to the use of log (ESCC) alone, the P m figures being reduced by about one third. When testing composite quarter samples, where milk from infected quarters was diluted with that from uninfected quarters, the inclusion of RCVD variables decreased the P m compared to the use of log (ESCC) alone. In hered 1 the improvement was from 26.1 to 3.0%, while in herd 2 the effect was less marked, the P m being reduced from 28.6 to 21.6%. However, as the RCVD assessment is provided simultaneously with ESCC, the additional information is obtained at minimal cost.

Electrolytic degradation of DNA fluorochromes during flow cytometric measurement of electronic cell volume.

Changes in flow cytometric measurement of DNA content can result from electrolytic chemical degradation of mithramycin, ethidium bromide, and propidium iodide during simultaneous measurement of electronic cell volume. Bench electrolysis also degrades these fluorochromes without changing the quantum yields, even when they are complexed to DNA. In the flow cytometer, electrolytic production of chlorine at the anode is the probable cause of this degradation, since exposure of these fluorochromes to chlorine gas produces the same effect. It is therefore advisable to measure the DNA content distribution alone before simultaneously measuring the DNA content and the electronic cell volume. If unavoidable effects on the DNA distribution are present, narrow forward-angle light scatter should be used as the cell size indicator during dual parameter measurements. Modifying instrument design by reversing electrode polarity might eliminate this problem.

The automated multiparameter analyzer for cells (AMAC) IIA, a true bridge circuit Coulter-type electronic cell volume transducer.

A true bridge Coulter effect (electronic cell volume) transducer has been developed. All resistances of this bridge are now the result of current flow through saline channels. Contamination by electrode products including gas bubbles has been completely eliminated since both power electrodes are now remote from the flow chamber. Since the orifice is in series with an approximately 10 K ohm resistance generated by a gel-filled capillary and a displacement rheostat, it floats electrically, at virtual ground. The other side of the bridge consists of a fluid side-wire. Removing the power electrode from the orifice outlet makes possible downward flow and the use of a single outer sheath, and eliminates noise generated by gas bubbles which could possibly be trapped. It should now be possible to combine this design with that of the AMAC III square orifice, to produce an electro-optical sorter where all parameters are measured simultaneously. This true bridge circuit possesses the further advantage that noise due both to the power supply and to overvoltage at the power electrodes is common-mode rejected, and any drift due to changes in electrode polarization is eliminated. Preliminary experiments confirm results with the AMAC II that hemoglobinopathies can be recognized by the increased coefficient of variation (CV) of the erythrocyte spectra.

Topochemical reactions of rutile related structures with lithium

The topochemical lithiation of rutile related MO 2 with n-BuLi, and in nonaqueous lithium electrochemical cells is reported. This series illustrates the importance of electronic conductivity and cell volume to substantial lithium incorporation. The Li xMO 2 compounds are metastable and decompose at temperatures less than 250°C.

Development of instrumentation and fluorochromes for automated multiparameter analysis of cells.

We have developed and interfaced to a computer an automated instrument (the AMAC III) which is designed to observe simultaneously several physical parameters of cells. Typical parameters include electronic cell volume (Coulter effect), RF amplitude (opacity), multiwavelength fluorescence of cytological stains, and cell light-scattering. The use of a new ultraviolet laser combined with a holographic grating spectrograph promises to increase the number of fluorescing species that can be detected simultaneously. This number can be further increased by use of special rare-earth-based fluorochromes, that emit well-defined, spectrally distinct peaks.