Cellaca® PLX image cytometer as an alternative for immunophenotyping, GFP/RFP transfection efficiencies, and apoptosis analysis.

Cell and gene therapy is a fast-growing field for cancer therapeutics requiring reliable instrumentation and technologies. Key parameters essential for satisfying Chemistry Manufacturing and Controls criteria standards are routinely performed using flow cytometry. Recently, image cytometry was developed for cell characterization and cell-based assays but had not yet demonstrated sufficient sensitivity for surface marker detection. We developed the Cellaca® PLX image cytometry system and the respective methodologies required for immunophenotyping, GFP and RFP transfection/transduction efficiencies, and cell health analyses for routine cell characterization. All samples tested were compared directly to results from the CytoFLEX flow cytometer. PBMCs were stained with T-cell surface markers for immunophenotyping, and results show highly comparable CD3, CD4, and CD8 populations (within 5 %). GFP- or RFP-expressing cell lines were analyzed for transfection/transduction efficiencies, and the percentage positive cells and respective viabilities were equivalent on both systems. Staurosporine-treated Jurkat cells were stained for apoptotic markers, where annexin V and caspase-3 positive cells were within 5 % comparing both instruments. The proposed system may provide a complementary tool for performing routine cell-based experiments with improved efficiency and sensitivity compared to prior image cytometers, which may be significantly valuable to the cell and gene therapy field.

A Multiplex Assay to Simultaneously Monitor Apoptosis and Necrosis Using the Cellaca® PLX Image Cytometer.

Apoptosis is the programmed cell death pathway that is critical for maintaining homeostasis, in which cancer cells can evade to ensure survival. For pharmaceutical drug discovery, it is important to characterize and compare different cancer therapeutics (i.e., small molecules, antibody drugs, cell therapies) that can initiate the process of apoptosis, enabling the identification of potential therapeutic candidates. In this work, we developed and demonstrated a multiplex detection method for monitoring apoptosis and necrosis with Annexin V, Caspase-3, and Propidium Iodide (PI) using the Cellaca® PLX Image Cytometer (Revvity Health Sciences, Inc., Lawrence, MA). First, apoptosis was induced in Jurkat and K562 cell lines with staurosporine over the course of 24 h, where apoptosis and necrosis were assessed at 0, 1, 1.5, 2, 4, 20, and 24 h timepoints. Samples were stained with Hoechst 33342 (total dye), Annexin V-APC (early-stage apoptosis), Caspase-3 488 (late-stage apoptosis), and PI (necrosis) at each timepoint and evaluated using image cytometry. Results showed that apoptotic factors and cascades were successfully detected along the pathway from early- to late-stage apoptosis, and ultimately necrosis. A clear trend was observed analyzing apoptotic and necrotic populations during the first 1.5 h, showing differences of up to ~15% in single Annexin V+ and Caspase-3+ populations in treated Jurkat cells, however, a significant increase in double positive apoptotic/necrotic cells for Annexin V+PI+ and Capase-3+PI+ was not observed until 20 h. Upon further analysis between apoptotic populations only, Annexin V+ only populations were higher than Caspase-3+ only populations by up to ~20% between 0 and 1.5 h. Conversely, K562 cells did not exhibit a notable change in apoptotic and necrotic populations due to low sensitivity to staurosporine. The proposed image cytometric detection method may provide an effective and efficient tool for rapid and reliable simultaneous detection of early- late-stage apoptosis, and necrosis. Therefore, allowing researchers to better characterize and screen potential cancer therapeutic drug candidates for their treatment efficacy in a higher throughput manner.

Practical Characterization Strategies for Comparison, Qualification, and Selection of Cell Viability Detection Methods for Cellular Therapeutic Product Development and Manufacturing.

Cellular therapy development and manufacturing has focused on providing novel therapeutic cell-based products for various diseases. The International Organization for Standardization (ISO) has provided guidance on critical quality attributes (CQAs) that shall be considered when testing and releasing cellular therapeutic products. Cell count and viability measurements are two of the CQAs that are determined during development, manufacturing, testing, and product release. The ISO Cell Counting Standard Part 1 and 2 addressed the needs for improving the quality of cell counting results. However, there is currently no guidance on the qualification and selection of a fit-for-purpose cell viability detection method. In this work, we present strategies for the characterization and comparison of AO/PI and AO/DAPI staining methods using the heat-killed (HK) and low temperature/nutrient-deprived (LT/ND) cell death models to evaluate the comparability of cell viability measurements and identify potential causes of differences. We compared the AO/PI and AO/DAPI staining methods using HK and LT/ND-generated dead cells, investigated the staining time effects on cell viability measurements, and determined their viability linearity with different mixtures of live and dead cells. Furthermore, we validated AO/PI and AO/DAPI cell viability measurement with a long-term cell proliferation assay. Finally, we demonstrate a practical example of cell viability measurement comparison using AO/PI and AO/DAPI on antibiotic-selected transduced Jurkat and THP-1 cells to select a fit-for-purpose method for functional genomics screening. The proposed strategies may potentially enable scientists to properly characterize, compare, and select cell viability detection methods that are critical for cellular therapeutic product development and manufacturing.

Optical Coherence Tomography for Brain Imaging and Developmental Biology.

Optical coherence tomography (OCT) is a promising research tool for brain imaging and developmental biology. Serving as a three-dimensional optical biopsy technique, OCT provides volumetric reconstruction of brain tissues and embryonic structures with micrometer resolution and video rate imaging speed. Functional OCT enables label-free monitoring of hemodynamic and metabolic changes in the brain in vitro and in vivo in animal models. Due to its non-invasiveness nature, OCT enables longitudinal imaging of developing specimens in vivo without potential damage from surgical operation, tissue fixation and processing, and staining with exogenous contrast agents. In this paper, various OCT applications in brain imaging and developmental biology are reviewed, with a particular focus on imaging heart development. In addition, we report findings on the effects of a circadian gene (Clock) and high-fat-diet on heart development in Drosophila melanogaster. These findings contribute to our understanding of the fundamental mechanisms connecting circadian genes and obesity to heart development and cardiac diseases.

Full-range space-division multiplexing optical coherence tomography angiography.

In this study, we demonstrated a full-range space-division multiplexing optical coherence tomography (FR-SDM-OCT) system. Utilizing the galvanometer-based phase modulation full-range technique, the total imaging range of FR-SDM-OCT can be extended to >20 mm in tissue, with a digitizer sampling rate of 500 MS/s and a laser sweeping rate of 100 kHz. Complex conjugate terms were suppressed in FR-SDM-OCT images with a measured rejection ratio of up to ∼46 dB at ∼1.4 mm depth and ∼30 dB at ∼19.4 mm depth. The feasibility of FR-SDM-OCT was validated by imaging Scotch tapes and human fingernails. Furthermore, we demonstrated the feasibility of FR-SDM-OCT angiography (FR-SDM-OCTA) to perform simultaneous acquisition of human fingernail angiograms from four positions, with a total field-of-view of ∼1.7 mm × âˆ¼7.5 mm. Employing the full-range technique in SDM-OCT can effectively alleviate hardware requirements to achieve the long depth measurement range, which is required by SDM-OCT to separate multiple images at different sample locations. FR-SDM-OCTA creates new opportunities to apply SDM-OCT to obtain wide-field angiography of in vivo tissue samples free of labeling.

Wide-field Ophthalmic Space-Division Multiplexing Optical Coherence Tomography.

High-speed ophthalmic optical coherence tomography systems are of interest because they allow rapid, motion-free, and wide-field retinal imaging. Space-division multiplexing optical coherence tomography (SDM-OCT) is a high-speed imaging technology which takes advantage of the long coherence length of microelectromechanical vertical cavity surface emitting laser (MEMs VCSEL) sources to multiplex multiple images along a single imaging depth. We demonstrate wide-field retinal OCT imaging, acquired at an effective A-scan rate of 800,000 A-scans/sec with volumetric images covering up to 12.5 mm × 7.4 mm on the retina acquired in less than 1 second. A clinical feasibility study was conducted to compare the ophthalmic SDM-OCT with commercial OCT systems, illustrating the high-speed capability of SDM-OCT in a clinical setting.

LncRNA MIR100HG promotes cancer cell proliferation, migration and invasion in laryngeal squamous cell carcinoma through the downregulation of miR-204-5p.

Purpose: LncRNA MIR100HG promotes several types of malignancies, while its involvement in other human diseases is unknown. Patients and methods: Our study included 70 patients with LSCC who were diagnosed and treated in the First Affiliated Hospital and College of Clinical Medicine of Henan University of Science and Technology from January 2016 to July 2018. qRT-PCR, cell transfection, in vitro cell proliferation assay, cell migration and invasion assay were applied for the research. Results: In the present study we found that MIR100HG was upregulated, while miR-204-5p was downregulated in tumor tissues than in adjacent healthy tissues of laryngeal squamous cell carcinoma (LSCC) patients. Expression of MIR100HG was significantly affected by AJCC stage. A significant and inverse correlation between MIR100HG and miR-204-5p was found in tumor tissues but not in adjacent healthy tissues of LSCC patients. Overexpression of MIR100HG resulted in the downregulation of miR-204-5p in LSCC cells, while miR-204-5p overexpression failed to significantly affect MIR100HG expression. Overexpression of MIR100HG led to promoted, while miR-204-5p, overexpression led to inhibited proliferation, migration and invasion of LSCC cells. In addition, miR-204-5p overexpression attenuated the enhancing effects of MIR100HG overexpression on cancer cell proliferation, migration and invasion. Conclusion: Therefore, lncRNA MIR100HG promoted cancer cell proliferation, migration and invasion in LSCC possibly through the downregulation of miR-204-5p.

Bi-layer blood vessel mimicking microfluidic platform for antitumor drug screening based on co-culturing 3D tumor spheroids and endothelial layers.

Two-dimensional (2D) cell culture is not ideal for traditional drug screening, because 2D culture does not accurately mimic the physiological microenvironment of tumor cells. Thus, a drug-screening system which more closely mimics the microenvironment of in vivo tumors is necessary. Here, we present a biomimicking bilayer microfluidic device that can facilitate antitumor drug screening. The microfluidic device consists of two polydimethylsiloxane (PDMS) pieces with channels which are separated by a semipermeable membrane to allow water, oxygen, and nutrition supply, while preventing cell migration. The channels embedded on the two PDMS pieces overlap each other over a long distance to ensure a larger exchange area to mimic the blood vessel-tumor model. High concentrations of endothelial cells (EC) are first seeded onto the membrane through the apical channel, and after a two-day culture, a confluent EC monolayer forms. Tumor spheroid-laden Matrigel is then seeded into the basal channel. After the Matrigel is cured, the device is ready for drug testing. Paclitaxel is used as the model drug for testing. Confocal microscopy and ImageJ are used to assess the efficacy of different concentrations of paclitaxel, and optical coherence tomography (OCT) is employed to determine the tumor volumetric change after the drug treatment. The results indicate that the proposed bilayer microfluidic device in combination with confocal and OCT optical characterization provide an efficient platform for antitumor drug testing.

Longitudinal Morphological and Physiological Monitoring of Three-dimensional Tumor Spheroids Using Optical Coherence Tomography.

Tumor spheroids have been developed as a three-dimensional (3D) cell culture model in cancer research and anti-cancer drug discovery. However, currently, high-throughput imaging modalities utilizing bright field or fluorescence detection, are unable to resolve the overall 3D structure of the tumor spheroid due to limited light penetration, diffusion of fluorescent dyes and depth-resolvability. Recently, our lab demonstrated the use of optical coherence tomography (OCT), a label-free and non-destructive 3D imaging modality, to perform longitudinal characterization of multicellular tumor spheroids in a 96-well plate. OCT was capable of obtaining 3D morphological and physiological information of tumor spheroids growing up to about 600 µm in height. In this article, we demonstrate a high-throughput OCT (HT-OCT) imaging system that scans the whole multi-well plate and obtains 3D OCT data of tumor spheroids automatically. We describe the details of the HT-OCT system and construction guidelines in the protocol. From the 3D OCT data, one can visualize the overall structure of the spheroid with 3D rendered and orthogonal slices, characterize the longitudinal growth curve of the tumor spheroid based on the morphological information of size and volume, and monitor the growth of the dead-cell regions in the tumor spheroid based on optical intrinsic attenuation contrast. We show that HT-OCT can be used as a high-throughput imaging modality for drug screening as well as characterizing biofabricated samples.

Integrating optical coherence tomography with gravimetric and video analysis (OCT-Gravimetry-Video method) for studying the drying process of polystyrene latex system.

Latex, an aqueous dispersion of sub-micron polymer particles, is widely used as polymer binder in waterborne coatings and adhesives. Drying of a latex is inhomogeneous, during which the spatial distribution of particles is non-uniform and changes with time, usually resulting in a compromise of the integrity of a dried film. To study drying inhomogeneity of latex, we developed a system integrating optical coherence tomography (OCT) with gravimetric and video analysis (OCT-Gravimetry-Video method) to non-destructively monitor the drying process of non-film-forming latexes consisting of hard polystyrene spheres over time. OCT structural and speckle images of the latex's internal structure show the packing process of particles, the detachment of latex and the formation of apparent shear bands in cross-sectional views. Video recordings show the formation of cracks and the propagation of the drying boundary in the horizontal direction. The drying curve, measured by gravimetry, shows the drying rate and the water content of the latex at each drying stage. Furthermore, we find that the particle size affects packing and cracking phenomena remarkably. The OCT-Gravimetry-Video method serves as a general and robust approach to investigate the drying process of waterborne latex system. This method can be employed for fundamental studies of colloids and for evaluations of industrial latex products.

Facile Tumor Spheroids Formation in Large Quantity with Controllable Size and High Uniformity.

A facile method for generation of tumor spheroids in large quantity with controllable size and high uniformity is presented. HCT-116 cells are used as a model cell line. Individual tumor cells are sparsely seeded onto petri-dishes. After a few days of growth, separated cellular islets are formed and then detached by dispase while maintaining their sheet shape. These detached cell sheets are transferred to dispase-doped media under orbital shaking conditions. Assisted by the shear flow under shaking and inhibition of cell-to-extracellular matrix junctions by dispase, the cell sheets curl up and eventually tumor spheroids are formed. The average size of the spheroids can be controlled by tuning the cell sheet culturing period and spheroid shaking period. The uniformity can be controlled by a set of sieves which were home-made using stainless steel meshes. Since this method is based on simple petri-dish cell culturing and shaking, it is rather facile for forming tumor spheroids with no theoretical quantity limit. This method has been used to form HeLa, A431 and U87 MG tumor spheroids and application of the formed tumor spheroids in drug screening is also demonstrated. The viability, 3D structure, and necrosis of the spheroids are characterized.

Wide-field high-speed space-division multiplexing optical coherence tomography using an integrated photonic device.

Space-division multiplexing optical coherence tomography (SDM-OCT) is a recently developed parallel OCT imaging method in order to achieve multi-fold speed improvement. However, the assembly of fiber optics components used in the first prototype system was labor-intensive and susceptible to errors. Here, we demonstrate a high-speed SDM-OCT system using an integrated photonic chip that can be reliably manufactured with high precisions and low per-unit cost. A three-layer cascade of 1 × 2 splitters was integrated in the photonic chip to split the incident light into 8 parallel imaging channels with ~3.7 mm optical delay in air between each channel. High-speed imaging (~1s/volume) of porcine eyes ex vivo and wide-field imaging (~18.0 × 14.3 mm2) of human fingers in vivo were demonstrated with the chip-based SDM-OCT system.

Optical Coherence Tomography Detects Necrotic Regions and Volumetrically Quantifies Multicellular Tumor Spheroids.

Three-dimensional (3D) tumor spheroid models have gained increased recognition as important tools in cancer research and anticancer drug development. However, currently available imaging approaches used in high-throughput screening drug discovery platforms, for example, bright-field, phase contrast, and fluorescence microscopies, are unable to resolve 3D structures deep inside (>50 µm) tumor spheroids. In this study, we established a label-free, noninvasive optical coherence tomography (OCT) imaging platform to characterize 3D morphologic and physiologic information of multicellular tumor spheroids (MCTS) growing from approximately 250 to 600 µm in height over 21 days. In particular, tumor spheroids of two cell lines, glioblastoma (U-87MG) and colorectal carcinoma (HCT116), exhibited distinctive evolutions in their geometric shapes at late growth stages. Volumes of MCTS were accurately quantified using a voxel-based approach without presumptions of their geometries. In contrast, conventional diameter-based volume calculations assuming perfect spherical shape resulted in large quantification errors. Furthermore, we successfully detected necrotic regions within these tumor spheroids based on increased intrinsic optical attenuation, suggesting a promising alternative of label-free viability tests in tumor spheroids. Therefore, OCT can serve as a promising imaging modality to characterize morphologic and physiologic features of MCTS, showing great potential for high-throughput drug screening. Cancer Res; 77(21); 6011-20. ©2017 AACR.

Deposition and drying dynamics of liquid crystal droplets.

Drop drying and deposition phenomena reveal a rich interplay of fundamental science and engineering, give rise to fascinating everyday effects (coffee rings), and influence technologies ranging from printing to genotyping. Here we investigate evaporation dynamics, morphology, and deposition patterns of drying lyotropic chromonic liquid crystal droplets. These drops differ from typical evaporating colloidal drops primarily due to their concentration-dependent isotropic, nematic, and columnar phases. Phase separation occurs during evaporation, and in the process creates surface tension gradients and significant density and viscosity variation within the droplet. As a result, the drying multiphase drops exhibit different convective currents, drop morphologies, and deposition patterns (coffee-rings).