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Quantitative, traceable determination of cell viability using absorbance microscopy.

ABSTRACT: Cell viability, an essential measurement for cell therapy products, lacks traceability. One of the most common cell viability tests is trypan blue dye exclusion where blue-stained cells are counted via brightfield imaging. Typically, live and dead cells are classified based on their pixel intensities which may vary arbitrarily making it difficult to compare results. Herein, a traceable absorbance microscopy method to determine the intracellular uptake of trypan blue is demonstrated. The intensity pixels of the brightfield images are converted to absorbance images which are used to calculate moles of trypan blue per cell. Trypan blue cell viability measurements, where trypan blue content in each cell is quantified, enable traceable live-dead classifications. To implement the absorbance microscopy method, we developed an open-source AbsorbanceQ application that generates quantitative absorbance images. The validation of absorbance microscopy is demonstrated using neutral density filters. Results from four different microscopes demonstrate a mean absolute deviation of 3% from the expected optical density values. When assessing trypan blue-stained Jurkat cells, the difference in intracellular uptake of trypan blue in heat-shock-killed cells using two different microscopes is 3.8%. Cells killed with formaldehyde take up ~50% less trypan blue as compared to the heat-shock-killed cells, suggesting that the killing mechanism affects trypan blue uptake. In a test mixture of approximately 50% live and 50% dead cells, 53% of cells were identified as dead (±6% standard deviation). Finally, to mimic batches of low-viability cells that may be encountered during a cell manufacturing process, viability was assessed for cells that were 1) overgrown in the cell culture incubator for five days or 2) incubated in DPBS at room temperature for five days. Instead of making live-dead classifications using arbitrary intensity values, absorbance imaging yields traceable units of moles that can be compared, which is useful for assuring quality for biomanufacturing processes.

SUBMITTER: Babakhanova G

PROVIDER: S-EPMC8769294 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundAlthough quantitative single-cell analysis is frequently applied in animal systems, e.g. to identify novel drugs, similar applications on plant single cells are largely missing. We have exploited the applicability of high-throughput microscopic image analysis on plant single cells using tobacco leaf protoplasts, cell-wall free single cells isolated by lytic digestion. Protoplasts regenerate their cell wall within several days after isolation and have the potential to expand and proliferate, generating microcalli and finally whole plants after the application of suitable regeneration conditions.ResultsHigh-throughput automated microscopy coupled with the development of image processing pipelines allowed to quantify various developmental properties of thousands of protoplasts during the initial days following cultivation by immobilization in multi-well-plates. The focus on early protoplast responses allowed to study cell expansion prior to the initiation of proliferation and without the effects of shape-compromising cell walls. We compared growth parameters of wild-type tobacco cells with cells expressing the antiapoptotic protein Bcl2-associated athanogene 4 from Arabidopsis (AtBAG4).ConclusionsAtBAG4-expressing protoplasts showed a higher proportion of cells responding with positive area increases than the wild type and showed increased growth rates as well as increased proliferation rates upon continued cultivation. These features are associated with reported observations on a BAG4-mediated increased resilience to various stress responses and improved cellular survival rates following transformation approaches. Moreover, our single-cell expansion results suggest a BAG4-mediated, cell-independent increase of potassium channel abundance which was hitherto reported for guard cells only. The possibility to explain plant phenotypes with single-cell properties, extracted with the single-cell processing and analysis pipeline developed, allows to envision novel biotechnological screening strategies able to determine improved plant properties via single-cell analysis.

Project description:A dietary supplement, kelp contains a significant amount of arsenic that is mostly arsenosugars. The determination of arsenosugars is difficult due to a lack of arsenosugar calibration standard, because arsenosugar compounds are not commercially available. This work reports the determination of arsenicals in a kelp extract with traceability to the International System of Units (SI). The hydrophilic fraction of arsenicals was reproducibly extracted from a candidate Standard Reference Material (SRM) 3232 Kelp Powder (Thallus Laminariae) in development at the National Institute of Standards and Technology (NIST). Arsenosugars and dimethylarsinic acid (DMA) were separated into fractions using analytical liquid chromatography (LC) cation and anion columns. The arsenic in the fractions was determined using instrumental neutron activation analysis (INAA). Cation exchange separation was used for INAA determination of arsenosugar 3-[5'-deoxy-5'-(dimethylarsinoyl)-β-ribofuranosyloxy]propylene glycol (As(328)) for the first time, while DMA, arsenosugars 3-[5'-deoxy-5'-(dimethylarsinoyl)-β-ribofuranosyloxy]-2-hydroxypropyl 2,3-dihydroxypropyl hydrogen phosphate (As(482)), and 3-[5'-deoxy-5'-(dimethylarsinoyl)-β-ribofuranosyloxy]-2-hydroxypropanesulfonic acid (As(392)) were determined following anion exchange separation. The contents of DMA, As(328), As(482), and As(392) were 0.41 mg kg-1 ± 0.09 mg kg-1, 1.10 mg kg-1 ± 0.25 mg kg-1, 5.23 mg kg-1 ± 0.46 mg kg-1, and 13.17 mg kg-1 ± 0.67 mg kg-1, respectively. Separately, components of arsenic species in the kelp extract including DMA, As(328), and inorganic arsenic were determined using LC-inductively coupled plasma mass spectrometry. Results of DMA and As(328) were 0.485 mg kg-1 ± 0.024 mg kg-1 and 1.14 mg kg-1 ± 0.03 mg kg-1, respectively, which were in good agreement with those determined by INAA in fractions of LC eluent. The most toxic species, AsIII and AsV were found to be < 0.07 mg kg-1 and 0.231 mg kg-1 ± 0.018 mg kg-1, respectively. Results were traceable to SI.

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Quantitative, traceable determination of cell viability using absorbance microscopy.

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