Effects of different cryopreservation parameters on the differences between trypan blue and fluorescent SYTO 13/GelRed assays.

Post-thaw cell viability assessment is very important in cryopreservation because it is the main assessment method used to optimize cryopreservation protocols for each cell type; hence, having standardized accurate, quick, and reliable assays for post-thaw cell viability measurements is of utmost importance. The trypan blue exclusion assay and nucleic-acid-binding fluorescence-based assays are two different methods for cell viability assessment. Both assays identify cells with damaged membranes by whether they let a compound enter the cell. In this study, these two assays are compared in the context of cryopreservation and the impacts of important cryopreservation parameters on the differences in measurements are investigated. H9c2 myoblasts were cryopreserved with different freezing protocols. Cell membrane integrities were measured immediately after thaw as well as after cryoprotectant removal by a hemocytometer-based trypan blue dye exclusion assay and a dual fluorometric SYTO 13/GelRed assay; and the results were compared. This study quantifies how (i) the absence or presence of different cryoprotectants, (ii) different cell-cryoprotectant incubation conditions, and (iii) the presence or removal of cryoprotectants after thaw affect the differences between these two viability assays.

Quantitative, traceable determination of cell viability using absorbance microscopy.

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.

Myeloma and Hybridoma Cell Counts and Viability Checks.

For most purposes, the number of hybridoma or myeloma cells can be estimated simply by observing the cells under the microscope. When an exact cell count is needed, the number can be determined by using a hemocytometer (improved Neubauer counting chambers are the most commonly used). This is a simple device in which a special coverslip rests on supports that hold it 0.1 mm above the base of the slide. The slide is engraved with a series of lines that form 1 × 1-mm squares. By counting the number of cells within the 0.1-mm3 chamber formed by the 1 × 1-mm square and the height of the coverslip, an accurate quantitation of cells per milliliter can be calculated. To determine the percentage of viable cells within a population, the cell suspension is mixed with a vital dye and observed under the microscope. Vital dyes are excluded from living cells but stain dead cells. The most common dye used for these stains is Trypan Blue.

High-throughput quantification of the effect of DMSO on the viability of lung and breast cancer cells using an easy-to-use spectrophotometric trypan blue-based assay.

One of the main aspects investigated in potential therapeutic compounds is their effect on cells viability and proliferative ability. Although various methods have been developed to investigate these aspects, these methods present with shortcomings in terms of either cost, availability, accuracy, precision, or throughput. This study describes a simple, economic, reproducible, and high-throughput assay to quantify cell death and proliferation. In this assay, adherent cells are fixed, stained with trypan blue, and measured for trypan blue internalization using a spectrophotometric absorbance plate reader. Corresponding cell counts to the absorbance measurements are extrapolated from a standard curve. This assay was used to measure the effect of dimethyl sulfoxide (DMSO) on the viability of breast and lung cancer cells. Decrease in cell count associated with the increase in DMSO percentage and exposure time. The assay's results closely correlated with the conventional trypan blue exclusion assay (Pearson correlation coefficient (r) > 0.99; p < 0.0001), but with higher precision. The assay developed in this study can be used for various applications such as optimization, cell treatment investigations, proliferation, and cytotoxicity studies.

Platinum(II) complexes of imidazophenanthroline-based polypyridine ligands as potential anticancer agents: synthesis, characterization, in vitro cytotoxicity studies and a comparative ab initio, and DFT studies with cisplatin, carboplatin, and oxaliplatin.

The synthesis of the platinum(II) complexes, [Pt(AIP)(bpy)](PF6)2 (1) and [Pt(PIP)(phen)](PF6)2 (2), of anthracene- and pyrene-conjugated imidazophenanthroline ligands and their in vitro cytotoxicity toward the fibroblast cells and the HeLa cell lines are reported. MTT assay demonstrates their cytotoxicity against the HeLa cell lines with the IC50 values of 1.35 and 1.56 µM, respectively, and the cytotoxicity profiles indicate that the HeLa cell lines show more activity than the fibroblast cells. Trypan blue assay highlights significant damage on the HeLa cell lines with a pronounced reduction on their clonogenicity. AO/EB staining shows marked morphologic signs of apoptosis in a dose-dependent manner and the LDH and DNA laddering assays also lend support to the cytotoxicity of the complexes. The molecular docking study reveals that the complexes interact with DNA through hydrogen bonding. The TD-DFT energy-optimized structures of the complexes show that the platinum(II) center has a slightly distorted square-planar geometry. The TD-DFT modelled LUMOs receive major contributions from the platinum d-orbitals, while the HOMOs are delocalized largely on the anthracenyl- and pyrenyl ligands, resulting in the LMCT transition at 352 nm. The structural, bonding, electronic, and optical properties of the complexes 1 and 2 reported in the present work and that of [Pt(AIP)(phen)](PF6)2 (3) and [Pt(PIP)(bpy)](PF6)2 (4), reported by us recently, and the approved drugs cisplatin, carboplatin, and oxaliplatin are described in the light of the optimized geometries, ΔEHOMO-LUMO, polarizability (α), hyperpolarizability (ß), Mulliken negativities, and dipole moments computed from the ab initio and DFT computational studies. The synthesis of Pt(II) complexes of anthracene- and pyrene-appended imidazophenanthroline ligands and their in vitro cytotoxicity against fibroblast cells and HeLa cell lines are reported. The DFT computational study of the complexes and cisplatin, carboplatin, and oxaliplatin are described in search of the ligand design features for the development of new Pt-drugs.

Non-enzymatic conversion of primary oxidation products of Docosahexaenoic acid into less toxic acid molecules.

Docosahexaenoic acid (DHA) is long chain omega-3 fatty acid with known health benefits and clinical significance. However, 4-hydroxy hexenal (HHE), an enzymatic oxidation product of DHA has recently been reported to have health-damaging effects. This conflict raises major concern on the long-term clinical use of these fatty acids. Even though the enzymatic and non-enzymatic conversion of HHE to nontoxic acid molecules is possible by the aldehyde detoxification systems, it has not yet studied. To address this, primary oxidation products of DHA in lipoxidase system were subjected to non-enzymatic conversion at physiological temperature over a period of 1 week. The reaction was monitored using HPLC, IR spectroscopy and biochemical assays (based on the loss of conjugated dienes, lipid peroxides aldehydes). Short term and long term cytotoxicity of the compounds generated at various time points were analyzed. IR and HPLC spectra revealed that the level of aldehydes in the primary oxidation products reduced over time, generating acids and acid derivatives within a week period. In short term and long term cytotoxicity analysis, initial decomposition products were found more toxic than the 1-week decomposition products. Further, when primary oxidation products were subjected to aldehyde dehydrogenase mediated oxidation, it generated products that are also less toxic. The study suggests the possible non-enzymatic conversion of primary oxidation products of DHA to less cytotoxic acid molecules. Exploration of the physiological roles of these acid molecules may explain the biological potential of omega-3 fatty acids.

Evaluation of trypan blue stain in the TC20 automated cell counter as a point-of-care for the enumeration of viable cryptococcal cells in cerebrospinal fluid.

Cerebrospinal fluid (CSF) culture can determine a quantitative viability of Cryptococcus yeasts; however, culture has a long turnaround-time. The TC20 automated cell counter (Bio-Rad) is a benchtop instrument used to count cells in 30 seconds. In vitro studies suggest trypan blue staining can distinguish between viable and dead cryptococcal yeasts. We hypothesized that trypan blue staining with automated cell counting may provide rapid quantification of viable CSF Cryptococcus yeasts. In sum, 96 HIV-infected participants with cryptococcal meningitis were enrolled and provided 194 CSF specimens in Kampala, Uganda. Cryptococcosis was diagnosed by CSF cryptococcal antigen (CRAG). CSF was stained with trypan blue and quantified yeasts with the TC20 cell counter. We compared the log10 transformed cell counter readings with gating of 4-10 µm versus log10 quantitative Cryptococcus cultures/ml. TC20 showed more positive results (95.4%) overall than culture (78.4%) with reference to CSF CRAG. TC20 had higher readings compared to culture in most cases with only a 25% level of agreement between the two methods. TC20 had a poor correlation to culture throughout the 14 days of antifungal therapy. The median of log10 transformed counts were 5.22 (IQR = 4.79-5.44) for the TC20 and 3.99 (IQR = 2.59-5.14) for culture. Overall, a linear regression showed no significant relationship between the TC20 and culture (r = -0.0025; P = .92). TC20 automated cell counting with trypan blue staining was poorly predictive of the quantitative CSF culture and could not be used as a substitute for quantitative culture.

Revisiting in vitro release test for topical gel formulations: The effect of osmotic pressure explored for better bio-relevance.

Release test methods for topical dosage forms including pharmacopeial tests require a large volume of release media, with limited application for high throughput screening. In the present study, we evaluated Transwell assay to miniaturize the release test method for optimization of thermoreversible topical gel formulations. We also explored the osmotic effect on the in vitro release rates from gel formulations to understand the bio-relevance of release media. An extreme vertices type of mixture design in Minitab®16 generated eleven formulations composed of poloxamer 407, poloxamer 188, and phosphate buffered saline (PBS). A quadratic equation adequately described the composition dependence of gelation temperature. Epidermal growth factor (EGF) and trypan blue were used as model drugs for proteins and small molecules, respectively. Cumulative release in PBS containing 30% sucrose exhibited linear correlation with respect to the gel compositions, while PBS without sucrose did not differentiate various compositions. Higher release rates in PBS than in sucrose media are attributable to the osmotic water flow from PBS into the donor phase, and subsequent increase in diffusivity. The time course of in vivo near-infrared fluorescence imaging of topical EGF gels on the wound sites were consistent with the in vitro release profiles measured with PBS as the release media. To the best of our knowledge, this is the first study to propose a release test method suitable for high throughput screening of topical formulations with emphasis on the osmotic pressure effect. Bio-relevant release media composition for a topical formulation would vary depending on its clinical application because the osmotic water flow through the normal skin would be negligible compared to compromised skin.

Evaluation of trypan blue stain in a haemocytometer for rapid detection of cerebrospinal fluid sterility in HIV patients with cryptococcal meningitis.

BACKGROUND: Quantitative culture is the most common method to determine the fungal burden and sterility of cerebrospinal fluid (CSF) among persons with cryptococcal meningitis. A major drawback of cultures is a long turnaround-time. Recent evidence demonstrates that live and dead Cryptococcus yeasts can be distinguished using trypan blue staining. We hypothesized that trypan blue staining combined with haemocytometer counting may provide a rapid estimation of quantitative culture count and detection of CSF sterility. To test this, we evaluated 194 CSF specimens from 96 HIV-infected participants with cryptococcal meningitis in Kampala, Uganda. Cryptococcal meningitis was diagnosed by CSF cryptococcal antigen (CRAG). We stained CSF with trypan blue and quantified yeasts using a haemocytometer. We compared the haemocytometer readings versus quantitative Cryptococcus CSF cultures. RESULTS: Haemocytometer counting with trypan blue staining had a sensitivity of 98% (64/65), while CSF cultures had a sensitivity of 95% (62/65) with reference to CSF CRAG for diagnostic CSF specimens. For samples that were positive in both tests, the haemocytometer had higher readings compared to culture. For diagnostic specimens, the median of log10 transformed counts were 5.59 (n = 64, IQR = 5.09 to 6.05) for haemocytometer and 4.98 (n = 62, IQR = 3.75 to 5.79) for culture; while the overall median counts were 5.35 (n = 189, IQR = 4.78-5.84) for haemocytometer and 3.99 (n = 151, IQR = 2.59-5.14) for cultures. The percentage agreement with culture sterility was 2.4% (1/42). Counts among non-sterile follow-up specimens had a median of 5.38 (n = 86, IQR = 4.74 to 6.03) for haemocytometer and 2.89 (n = 89, IQR = 2.11 to 4.38) for culture. At diagnosis, CSF quantitative cultures correlated with haemocytometer counts (R2 = 0.59, P < 0.001). At 7-14 days, quantitative cultures did not correlate with haemocytometer counts (R2 = 0.43, P = 0.4). CONCLUSION: Despite a positive correlation, the haemocytometer counts with trypan blue staining did not predict the outcome of quantitative cultures in patients receiving antifungal therapy.