Rapid determination of general cell status, cell viability, and optimal harvest time in eukaryotic cell cultures by impedance flow cytometry

The determination of cell viability is essential to many areas of life sciences and biotechnology. Typically, cell viability measurements are based on the optical analysis of stained cells, which requires additional labeling steps and is hard to implement online. Frequency-dependent impedance flow cytometry (IFC) provides a label-free, fast, and reliable alternative to determine cell viability at the single cell level based on the Coulter principle. Here, we describe the application of IFC to eukaryotic cell cultures and compare the results to commonly used staining methods. Yeast cell parameters were assessed in normal and heat-inactivated cells as well as in alcoholic fermentation and long-term batch cultures providing a precise and fast determination of the cell viability and further quantitative measures of the cell culture status. As an important new application, we have investigated recombinant protein production in the widely used baculovirus insect cell expression system. The IFC analysis revealed the presence of a subpopulation of cells, which correlates with the protein expression yield, but it is not detectable with conventional optical cell counters. We tentatively identify this subpopulation as cells in the late phase of infection. Their detection can serve as a predictor for the optimal time point of harvest. The IFC technique should be generally applicable to many eukaryotic cell cultures in suspension, possibly also implemented online

Impedance Flow Cytometry: A Novel Technique in Pollen Analysis

<div><p>Introduction</p><p>An efficient and reliable method to estimate plant cell viability, especially of pollen, is important for plant breeding research and plant production processes. Pollen quality is determined by classical methods, like staining techniques or <i>in vitro</i> pollen germination, each having disadvantages with respect to reliability, analysis speed, and species dependency. Analysing single cells based on their dielectric properties by impedance flow cytometry (IFC) has developed into a common method for cellular characterisation in microbiology and medicine during the last decade. The aim of this study is to demonstrate the potential of IFC in plant cell analysis with the focus on pollen.</p><p>Method</p><p>Developing and mature pollen grains were analysed during their passage through a microfluidic chip to which radio frequencies of 0.5 to 12 MHz were applied. The acquired data provided information about the developmental stage, viability, and germination capacity. The biological relevance of the acquired IFC data was confirmed by classical staining methods, inactivation controls, as well as pollen germination assays.</p><p>Results</p><p>Different stages of developing pollen, dead, viable and germinating pollen populations could be detected and quantified by IFC. Pollen viability analysis by classical FDA staining showed a high correlation with IFC data. In parallel, pollen with active germination potential could be discriminated from the dead and the viable but non-germinating population.</p><p>Conclusion</p><p>The presented data demonstrate that IFC is an efficient, label-free, reliable and non-destructive technique to analyse pollen quality in a species-independent manner.</p></div

Low Intensity and Frequency Pulsed Electromagnetic Fields Selectively Impair Breast Cancer Cell Viability

<div><p>Introduction</p><p>A common drawback of many anticancer therapies is non-specificity in action of killing. We investigated the potential of ultra-low intensity and frequency pulsed electromagnetic fields (PEMFs) to kill breast cancer cells. Our criteria to accept this technology as a potentially valid therapeutic approach were: <b>1</b>) cytotoxicity to breast cancer cells and; <b>2</b>) that the designed fields proved innocuous to healthy cell classes that would be exposed to the PEMFs during clinical treatment.</p><p>Methods</p><p>MCF7 breast cancer cells and their normal counterparts, MCF10 cells, were exposed to PEMFs and cytotoxic indices measured in order to design PEMF paradigms that best kill breast cancer cells. The PEMF parameters tested were: <b>1</b>) frequencies ranging from 20 to 50 Hz; <b>2</b>) intensities ranging from 2 mT to 5 mT and; <b>3</b>) exposure durations ranging from 30 to 90 minutes per day for up to three days to determine the optimum parameters for selective cancer cell killing.</p><p>Results</p><p>We observed a discrete window of vulnerability of MCF7 cells to PEMFs of 20 Hz frequency, 3 mT magnitude and exposure duration of 60 minutes per day. The cell damage accrued in response to PEMFs increased with time and gained significance after three days of consecutive daily exposure. By contrast, the PEMFs parameters determined to be most cytotoxic to breast cancer MCF-7 cells were not damaging to normal MCF-10 cells.</p><p>Conclusion</p><p>Based on our data it appears that PEMF-based anticancer strategies may represent a new therapeutic approach to treat breast cancer without affecting normal tissues in a manner that is non-invasive and can be potentially combined with existing anti-cancer treatments.</p></div

MCF7 and MCF10 cell metabolic status analyzed by IFC at 9 MHz.

<p>(<b>A</b>) Dot plots generated from MCF7 cells after exposure to PEMFs of 2, 3 or 5 mTs and in control (non-exposed) samples and analyzed at a scan frequency of 9 MHz. Exposed samples exhibited a larger right-side population, particularly after exposure to 3 mT PEMFs. (<b>B</b>) Dot plots of MCF7 cells after exposure to 30, 60 or 90 minutes of PEMFs (3 mT, 20 Hz) per day for 3 days; the right-side population was preferentially enhanced in response to 60 minutes exposures. (<b>C</b>) Histograms depicting the percentage increase in the size of the right population normalized to controls after exposure to 2, 3 or 5 mT PEMFs for 60 minutes. Each value is the average of 4 independent experiments (1 replicate/experiment, n = 4) (± SD). P-values, left to right: 0.00879, 0.0017 and 0.07033. (<b>D</b>) Size of right population as a function of exposure duration and normalized to each respective control (unexposed) sample; the right-side population was preferentially enhanced in response to 60 minutes exposures. Each value is the average of 4 independent experiments (1 replicate/experiment, n = 4) (± SD). P-values, left to right: 0.6786, 0.0017 and 1. (<b>E</b>) Dot plots generated from MCF10 cells exposed to 3 mT PEMFs (20 Hz) for 60 minutes/day for three days and in control (unexposed) samples, revealing essentially no change in response to treatment. The dot plots shown were generated from cells of the same experimental date and are representative of cells responses observed in all of the independent experiments with identical conditions. Also see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072944#pone.0072944.s008" target="_blank">Figure S8</a>, for the spread of individual measurements.</p

Time course in the development of cell death in response to PEMF exposure.

<p>Histograms showing the total number of cells (dark grey) and the total number of dead cells (trypan blue positive, light grey) after 1, 2 or 3 days of daily PEMF exposure (<b>B</b>, <b>D</b>) or in unexposed (control) cultures (<b>A</b>, <b>C</b>). (<b>A</b>, <b>C</b>) Unexposed cultures exhibited a steady increase in bulk cell number during 3 days in culture. (<b>B</b>) Exposure to 3 mT PEMFs for 60 min/day abrogated the typical monotonic increase in total cell number (dark grey) observed in unexposed samples (<b>A</b>) concomitant with an increase in the amount of trypan blue positive cells (light grey) that increased in significance with consecutive daily exposures to PEMFs. The total number of cells in treated samples showed a 40% (+/– 6%) decrease relative to control, whereas trypan blue positive cells increased by 20% (+/– 13%), (total cells in control sample – total cell in treated sample)/total cells in control sample) and (dead cells in control sample – dead cell in treated sample)/dead cells in control sample), respectively. (<b>D</b>) Exposure to 3 mT PEMFs for 90 min/day slowed the increase in total cell number (dark grey) typical of control samples in combination with an increase in the amount of trypan blue positive cells (light grey) that increased in significance with consecutive daily exposures to PEMFs. The total amount of cells in treated sample showed a 20% (+/– 4%) decrease relative to control, whereas trypan blue positive cells increased by 36% (+/– 10%), (total cells in control sample – total cell in treated sample)/total cells in control sample) and (dead cells in control sample – dead cell in treated sample)/dead cells in control sample), respectively. All the values represent the averages of 4 independent experiments with 3 replicates/experiment (n = 12) for the 60-min/day time points and 2 replicates/experiments (n = 8) for 90-min/day time points. P-values, left to right: 0.3246, 0.02032, 0.00004 for 60min/day of exposure and 0.2595, 0.02953, 0.00015 for 90 min/day of exposure.</p

Assessment of PEMF-induced apoptosis by Annexin V assay.

<p>MCF7 (cancer) and MCF10 (non-tumorigenic) cells were treated with the PEMF paradigms producing the greatest amount of cell death in MCF7 (3 mT for 60 min/day for 3 consecutive days). (<b>A</b>) Dot plots generated by FCM analyses of MCF7 cells show greater increases in the proportions of cells in early (Annexin V+/PI-) and later stages of apoptosis (Annexin V+/PI+) in treated samples (left) relative to control (unexposed) samples (right). (<b>B</b>) MCF10 (non-tumorigenic) cells appear to be unharmed by PEMFs as underscored by the similar amounts of viable cells in treated (89%) versus unexposed (80%) cultures.</p

Box plots depicting the increase in cell death after 1, 2 or 3 days of consecutive PEMF treatment.

<p>(<b>A</b>) 3 mT PEMFs for 60 min/day impaired MCF7 cancer cell viability sufficiently to cause a time-dependent accumulation of compromised cells over the time course of 1 to 3 days. The most significant degree of cell impairment was seen after 3 days (4 independent experiments with 3 replicates/experiment (n = 12)) (p-values, left to right: 0.3246, 0.02032, 0.00004) (also see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072944#pone-0072944-t001" target="_blank">Table 1</a> for the mean, high value, low value and average absolute deviation from median). (<b>B</b>) MCF7 cancer cells treated with 3 mT PEMFs for 90 min/day for 1, 2 or 3 days. Overall, 90 min/day of exposure produced less cytotoxicity than 60 min/day. Data were generated from 4 independent experiments with 2 replicates/experiment (n = 8) (p-values, left to right: 0.2595, 0.02953, 0.00015) (also see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072944#pone-0072944-t002" target="_blank">table 2</a> for the mean, high value, low value and average absolute deviation from median).</p

Time course of apoptosis induction by PEMFs in MCF7 cells determined by FCM.

<p>(<b>A</b>) Overlay of MCF7 cells treated with 3 mT PEMFs for 60 min/day for 1, 2 or 3 consecutive days. PEMF-induced DNA damage accrued with time yet, only obtained significance after 3 consecutive days of exposure. (<b>B</b>) Overlay of MCF7 cells exposed to 3 mT PEMFs for 90 min/day for 1, 2 or 3 consecutive days. As in <b>A</b> statistical significance was only achieved after three days. Paralleling our trypan blue (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072944#pone-0072944-g001" target="_blank">figures 1B</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072944#pone-0072944-g002" target="_blank"><b>2</b></a><b>A-D</b> and <b>3 A-B</b>) and FCM (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072944#pone-0072944-g004" target="_blank">figure <b>4</b></a><b>A-D</b>) results, 90 min/day of exposure to PEMFs (3 mT) was less cytotoxic than 60 min/day. (<b>C</b>) Percentage of MCF7 apoptotic cells (relative to control) detected by flow cytometry after exposure to 3 mT PEMFs for 60 minutes per day for 1 day up to 3 days. Values represent the averages of 3, 3 and 5 independent experiments for 1, 2 or 3 days exposure, respectively (1 replicate/experiment (total n = 3, 3, 5, respectively)) (average ± SD); P values, left to right: 0.1, 0.1 and 0.02857. (<b>D</b>) Percentage of MCF7 apoptotic cells after exposure to 3 mT PEMFs for 90 minutes/day for 1, 2 or 3 consecutive days. Values represent the averages of 3, 3 and 5 independent experiments for 1, 2 or 3 days of exposure, respectively (single replicates (total n = 3, 3, 5, respectively)) (average ± SD); P values, left to right: 0.1, 0.1 and 0.02857.</p

Post-PEMF apoptosis determination by impedance flow cytometry (IFC) at 0.5 MHz.

<p>(<b>A</b>) Dot plots generated from MCF7 cell exposed to 2, 3 or 5 mT amplitude PEMFs for 60 minutes per day for three days. The histograms above and to the right of each dot plot show the apoptotic cell subpopulation shaded in black. MCF-7 cancer cells treated with 3 mT PEMFs exhibited the greatest separation between viable (right) and non-viable (left) cell populations as well as a higher overall percentage of dead cells. (<b>B</b>) Viability of MCF7 cells after exposure to 3 mT (20 Hz) for 30, 60 or 90 minutes per day for three days. (<b>C</b>) Percentage of MCF7 apoptotic cells detected by IFC in response to 2, 3 or 5 mT PEMFs normalized to its respective control. Each value represents the average of 4 independent experiments (1 replicate/experiment, n = 4) (± SD); P values, left to right: 0.4818, 0.0004552 and 0.1818. (<b>D</b>) Percentage of MCF7 dead cells in each treated sample normalized to its respective control in response to 30, 60 or 90 minutes exposures to PEMFs. Each value represents the average of 4 independent experiments (1 replicate/experiment, n = 4) (± SD). P-values, left to right: 0.1905, 0.0004552 and 0.3929. (<b>E</b>) MCF10 cells treated with PEMFs (3 mT, 20 Hz) for 60 minutes/day for three days. The dot plots shown were generated from cells of the same experimental date and are representative of cells responses observed in all of the independent experiments with identical conditions. Two different replicates obtained from 2 independents experiments were chosen for the 3 mT, 60 minute condition for figure <b>A</b> and <b>B.</b> Also see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072944#pone.0072944.s008" target="_blank">Figure S8</a> for the spread of individual measurements.</p

Dead cells/total cells in MCF7 cells after 3 mT PEMFs treatment for 60 min/day for 3 days.

<p>Values refer to the box plots of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072944#pone-0072944-g003" target="_blank">figure 3A</a> showing the amount of dead cells/total cells in treated samples compared to relative control samples. Data were generated from 4 independent experiments (3 replicates/experiments, n =  12).</p