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.

High-Throughput SARS-CoV-2 Antiviral Testing Method Using the Celigo Image Cytometer.

The COVID-19 pandemic has created a worldwide public health crisis that has since resulted in 6.8 million reported deaths. The pandemic prompted the immediate response of researchers around the world to engage in rapid vaccine development, surveillance programs, and antiviral testing, which resulted in the delivery of multiple vaccines and repurposed antiviral drug candidates. However, the emergence of new highly transmissible SARS-CoV-2 variants has renewed the desire for discovering new antiviral drug candidates with high efficacy against the emerging variants of concern. Traditional antiviral testing methods employ the plaque-reduction neutralization tests (PRNTs), plaque assays, or RT-PCR analysis, but each assay can be tedious and time-consuming, requiring 2-3 days to complete the initial antiviral assay in biologically relevant cells, and then 3-4 days to visualize and count plaques in Vero cells, or to complete cell extractions and PCR analysis. In recent years, plate-based image cytometers have demonstrated high-throughput vaccine screening methods, which can be adopted for screening potential antiviral drug candidates. In this work, we developed a high-throughput antiviral testing method employing the Celigo Image Cytometer to investigate the efficacy of antiviral drug candidates on SARS-CoV-2 infectivity using a fluorescent reporter virus and their safety by measuring the cytotoxicity effects on the healthy host cell line using fluorescent viability stains. Compared to traditional methods, the assays defined here eliminated on average 3-4 days from our standard processing time for antiviral testing. Moreover, we were able to utilize human cell lines directly that are not typically amenable to PRNT or plaque assays. The Celigo Image Cytometer can provide an efficient and robust method to rapidly identify potential antiviral drugs to effectively combat the rapidly spreading SARS-CoV-2 virus and its variants during the pandemic.

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.

Comparison of chimeric antigen receptor-T cell-mediated cytotoxicity assays with suspension tumor cells using plate-based image cytometry method.

In the recent decade, chimeric antigen receptor (CAR)-T cell therapy has revolutionized strategies for cancer treatments due to its highly effective clinical efficacy and response for B cell malignancies. The success of CAR-T cell therapy has stimulated the increase in the research and development of various CAR constructs to target different tumor types. Therefore, a robust and efficient in vitro potency assay is needed to quickly identify potential CAR gene design from a library of construct candidates. Image cytometry methodologies have been utilized for various CAR-T cell-mediated cytotoxicity assay using different fluorescent labeling methods, mainly due to their ease-of-use, ability to capture cell images for verification, and higher throughput performance. In this work, we employed the Celigo Image Cytometer to evaluate and compare two CAR-T cell-mediated cytotoxicity assays using GFP-expressing or fluorescent dye-labeled myeloma and plasmacytoma cells. The GFP-based method demonstrated higher sensitivity in detecting CAR-T cell-mediated cytotoxicity when compared to the CMFDA/DAPI viability method. We have established the criteria and considerations for the selection of cytotoxicity assays that are fit-for-purpose to ensure the results produced are meaningful for the specific testing conditions.

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.

A novel image-based method for simultaneous counting of Lactobacillus and Saccharomyces in mixed culture fermentation.

Mixed microorganism cultures are prevalent in the food industry. A variety of microbiological mixtures have been used in these unique fermenting processes to create distinctive flavor profiles and potential health benefits. Mixed cultures are typically not well characterized, which may be due to the lack of simple measurement tools. Image-based cytometry systems have been employed to automatically count bacteria or yeast cells. In this work, we aim to develop a novel image cytometry method to distinguish and enumerate mixed cultures of yeast and bacteria in beer products. Cellometer X2 from Nexcelom was used to count of Lactobacillus plantarum and Saccharomyces cerevisiae in mixed cultures using fluorescent dyes and size exclusion image analysis algorithm. Three experiments were performed for validation. (1) Yeast and bacteria monoculture titration, (2) mixed culture with various ratios, and (3) monitoring a Berliner Weisse mixed culture fermentation. All experiments were validated by comparing to manual counting of yeast and bacteria colony formation. They were highly comparable with ANOVA analysis showing p-value > 0.05. Overall, the novel image cytometry method was able to distinguish and count mixed cultures consistently and accurately, which may provide better characterization of mixed culture brewing applications and produce higher quality products.

Automated Assessment of Cancer Drug Efficacy On Breast Tumor Spheroids in Aggrewell™400 Plates Using Image Cytometry.

Tumor spheroid models have proven useful in the study of cancer cell responses to chemotherapeutic compounds by more closely mimicking the 3-dimensional nature of tumors in situ. Their advantages are often offset, however, by protocols that are long, complicated, and expensive. Efforts continue for the development of high-throughput assays that combine the advantages of 3D models with the convenience and simplicity of traditional 2D monolayer methods. Herein, we describe the development of a breast cancer spheroid image cytometry assay using T47D cells in Aggrewell™400 spheroid plates. Using the Celigo® automated imaging system, we developed a method to image and individually track thousands of spheroids within the Aggrewell™400 microwell plate over time. We demonstrate the use of calcein AM and propidium iodide staining to study the effects of known anti-cancer drugs Doxorubicin, Everolimus, Gemcitabine, Metformin, Paclitaxel and Tamoxifen. We use the image cytometry results to quantify the fluorescence of calcein AM and PI as well as spheroid size in a dose dependent manner for each of the drugs. We observe a dose-dependent reduction in spheroid size and find that it correlates well with the viability obtained from the CellTiter96® endpoint assay. The image cytometry method we demonstrate is a convenient and high-throughput drug-response assay for breast cancer spheroids under 400 µm in diameter, and may lay a foundation for investigating other three-dimensional spheroids, organoids, and tissue samples.

High-Throughput Image Cytometry Detection Method for CAR-T Transduction, Cell Proliferation, and Cytotoxicity Assays.

Chimeric antigen receptor (CAR)-T cell therapy has drawn much attention due to its recent clinical success in B-cell malignancies. In general, the CAR-T cell discovery process consists of CAR identification, T-cell activation, transduction, and expansion, as well as assessment of CAR-T cytotoxicity. The current evaluation methods for the CAR-T discovery process can be time-consuming, low-throughput and requires the preparation of multiple sacrificial samples in order to produce kinetic data. In this study, we employed the use of a plate-based image cytometer to monitor anti-CAIX (carbonic anhydrase IX) G36 CAR-T generation and assess its cytotoxic potency of direct and selective killing against CAIX+ SKRC-59 human renal cell carcinoma cells. The transduction efficiency and cytotoxicity results were analyzed using image cytometry and compared directly to flow cytometry and Chromium 51 (51 Cr) release assays, showing that image cytometry was comparable against these conventional methods. Image cytometry method streamlines the assays required during the CAR-T cell discovery process by analyzing a plate of T cells from CAR-T generation to in vitro functional assays with minimum disruption. The proposed method can reduce assay time and uses less cell samples by imaging and analyze the same plate over time without the need to sacrifice any cells. The ability to monitor kinetic data can allow additional insights into the behavior and interaction between CAR-T and target tumor cells. © 2020 International Society for Advancement of Cytometry.

Establishing a reference focal plane using beads for trypan-blue-based viability measurements.

Trypan blue dye exclusion-based cell viability measurements are highly dependent upon image quality and consistency. In order to make measurements repeatable, one must be able to reliably capture images at a consistent focal plane, and with signal-to-noise ratio within appropriate limits to support proper execution of image analysis routines. Imaging chambers and imaging systems used for trypan blue analysis can be inconsistent or can drift over time, leading to a need to assure the acquisition of images prior to automated image analysis. Although cell-based autofocus techniques can be applied, the heterogeneity and complexity of the cell samples can make it difficult to assure the effectiveness, repeatability and accuracy of the routine for each measurement. Instead of auto-focusing on cells in our images, we add control beads to the images, and use them to repeatedly return to a reference focal plane. We use bead image features that have stable profiles across a wide range of focal values and exposure levels. We created a predictive model based on image quality features computed over reference datasets. Because the beads have little variation, we can determine the reference plane from bead image features computed over a single-shot image and can reproducibly return to that reference plane with each sample. The achieved accuracy (over 95%) is within the limits of the actuator repeatability. We demonstrate that a small number of beads (less than 3 beads per image) is needed to achieve this accuracy. We have also developed an open-source Graphical User Interface called Bead Benchmarking-Focus And Intensity Tool (BB-FAIT) to implement these methods for a semi-automated cell viability analyser.

It is critical for the manufacturing and release of living cell-based therapies to determine the viability, the ratio of living cells to the total number of cells (live and dead), in the therapy. Dead cells can be a safety concern for the patient, and dosing is often based on the number of living cells which are the active ingredient of the drug product. Currently, the most common approach to evaluating cell viability is based on the staining of cell samples with the trypan blue marker of cell membrane integrity: a loss in cell membrane integrity with cell death allows the dye into the cell, which can be seen using brightfield microscopy. To classify cells as live/dead, the brightness of the cells is evaluated and cells with bright centres are considered live, while those with dark centres are considered dead. Unfortunately, this approach of staining, imaging and classification is very sensitive to image acquisition settings, including image focus and brightness. This paper introduces a method to establish the required image quality for image viability analysis, providing a tool to return to image acquisition settings that will ensure image quality even when there is variability from sample to sample. In this method, polymeric beads are added to each cell sample prior to cell viability analysis. Using image processing, we extract key features from the beads in the image such as sharpness of the edges of the beads. The image features of the cells can vary significantly from sample to sample and under different cell conditions, but image features of beads have proved to be consistent across samples. We are thus able to collect reference datasets quantifying bead features over a wide range of image acquisition settings (brightness and focus), allowing us to establish a reference focal plan for image acquisition for any cell sample based on bead features. We show that with as few as three beads per image, the reference focal plane can be found from a single acquisition of beads image data over a wide range of image focuses and brightness, allowing users to consistently acquire images for cell viability that meet pre-defined quality requirements.

A rapid single cell sorting verification method using plate-based image cytometry.

Single cell sorting is commonly used for ensuring monoclonality and producing homogenous target cell populations. Current single cell verification methods involve manually confirming the existence of single cells or colonies in a well using a standard light microscope. However, the manual verification method is time-consuming and highly tedious, which prompts a need for an accurate and rapid detection method for verifying single cell sorting capability. Here, we demonstrate a rapid single cell sorting verification method using the Celigo Image Cytometer. Calcein AM-stained Jurkat cells and fluorescent beads are sorted into 96-well half area microplates using the MoFlo Astrios EQ. Whole well bright field and fluorescent images are acquired and analyzed using the image cytometer in less than 8 min. The proposed single cell verification detection method in multi-well microplates can allow for quick optimization of FACS instruments at flow core laboratories, as well as improvement of downstream biological assays by accurately confirming the presence of single cells in each well.

Real-time viability and apoptosis kinetic detection method of 3D multicellular tumor spheroids using the Celigo Image Cytometer.

The development of three-dimensional (3D) multicellular tumor spheroid models for cancer drug discovery research has increased in the recent years. The use of 3D tumor spheroid models may be more representative of the complex in vivo tumor microenvironments in comparison to two-dimensional (2D) assays. Currently, viability of 3D multicellular tumor spheroids has been commonly measured on standard plate-readers using metabolic reagents such as CellTiter-Glo® for end point analysis. Alternatively, high content image cytometers have been used to measure drug effects on spheroid size and viability. Previously, we have demonstrated a novel end point drug screening method for 3D multicellular tumor spheroids using the Celigo Image Cytometer. To better characterize the cancer drug effects, it is important to also measure the kinetic cytotoxic and apoptotic effects on 3D multicellular tumor spheroids. In this work, we demonstrate the use of PI and caspase 3/7 stains to measure viability and apoptosis for 3D multicellular tumor spheroids in real-time. The method was first validated by staining different types of tumor spheroids with PI and caspase 3/7 and monitoring the fluorescent intensities for 16 and 21 days. Next, PI-stained and nonstained control tumor spheroids were digested into single cell suspension to directly measure viability in a 2D assay to determine the potential toxicity of PI. Finally, extensive data analysis was performed on correlating the time-dependent PI and caspase 3/7 fluorescent intensities to the spheroid size and necrotic core formation to determine an optimal starting time point for cancer drug testing. The ability to measure real-time viability and apoptosis is highly important for developing a proper 3D model for screening tumor spheroids, which can allow researchers to determine time-dependent drug effects that usually are not captured by end point assays. This would improve the current tumor spheroid analysis method to potentially better identify more qualified cancer drug candidates for drug discovery research. © 2017 International Society for Advancement of Cytometry.

A novel concentration and viability detection method for Brettanomyces using the Cellometer image cytometry.

Brettanomyces spp. can present unique cell morphologies comprised of excessive pseudohyphae and budding, leading to difficulties in enumerating cells. The current cell counting methods include manual counting of methylene blue-stained yeasts or measuring optical densities using a spectrophotometer. However, manual counting can be time-consuming and has high operator-dependent variations due to subjectivity. Optical density measurement can also introduce uncertainties where instead of individual cells counted, an average of a cell population is measured. In contrast, by utilizing the fluorescence capability of an image cytometer to detect acridine orange and propidium iodide viability dyes, individual cell nuclei can be counted directly in the pseudohyphae chains, which can improve the accuracy and efficiency of cell counting, as well as eliminating the subjectivity from manual counting. In this work, two experiments were performed to demonstrate the capability of Cellometer image cytometer to monitor Brettanomyces concentrations, viabilities, and budding/pseudohyphae percentages. First, a yeast propagation experiment was conducted to optimize software counting parameters for monitoring the growth of Brettanomyces clausenii, Brettanomyces bruxellensis, and Brettanomyces lambicus, which showed increasing cell concentrations, and varying pseudohyphae percentages. The pseudohyphae formed during propagation were counted either as multiple nuclei or a single multi-nuclei organism, where the results of counting the yeast as a single multi-nuclei organism were directly compared to manual counting. Second, a yeast fermentation experiment was conducted to demonstrate that the proposed image cytometric analysis method can monitor the growth pattern of B. lambicus and B. clausenii during beer fermentation. The results from both experiments displayed different growth patterns, viability, and budding/pseudohyphae percentages for each Brettanomyces species. The proposed Cellometer image cytometry method can improve efficiency and eliminate operator-dependent variations of cell counting compared with the traditional methods, which can potentially improve the quality of beverage products employing Brettanomyces yeasts.

Cellometer image cytometry as a complementary tool to flow cytometry for verifying gated cell populations.

Traditionally, many cell-based assays that analyze cell populations and functionalities have been performed using flow cytometry. However, flow cytometers remain relatively expensive and require highly trained operators for routine maintenance and data analysis. Recently, an image cytometry system has been developed by Nexcelom Bioscience (Lawrence, MA, USA) for automated cell concentration and viability measurement using bright-field and fluorescent imaging methods. Image cytometry is analogous to flow cytometry in that gating operations can be performed on the cell population based on size and fluorescent intensity. In addition, the image cytometer is capable of capturing bright-field and fluorescent images, allowing for the measurement of cellular size and fluorescence intensity data. In this study, we labeled a population of cells with an enzymatic vitality stain (calcein-AM) and a cell viability dye (propidium iodide) and compared the data generated by flow and image cytometry. We report that measuring vitality and viability using the image cytometer is as effective as flow cytometric assays and allows for visual confirmation of the sample to exclude cellular debris. Image cytometry offers a direct method for performing fluorescent cell-based assays but also may be used as a complementary tool to flow cytometers for aiding the analysis of more complex samples.

Evaluation of the antitumor effects of c-Myc-Max heterodimerization inhibitor 100258-F4 in ovarian cancer cells.

Epithelial ovarian carcinoma is the most lethal gynecological cancer due to its silent onset and recurrence with resistance to chemotherapy. Overexpression of oncogene c-Myc is one of the most frequently encountered events present in ovarian carcinoma. Disrupting the function of c-Myc and its downstream target genes is a promising strategy for cancer therapy. Our objective was to evaluate the potential effects of small-molecule c-Myc inhibitor, 10058-F4, on ovarian carcinoma cells and the underlying mechanisms by which 10058-F4 exerts its actions. Using MTT assay, colony formation, flow cytometry and Annexin V FITC assays, we found that 10058-F4 significantly inhibited cell proliferation of both SKOV3 and Hey ovarian cancer cells in a dose dependent manner through induction of apoptosis and cell cycle G1 arrest. Treatment with 10058-F4 reduced cellular ATP production and ROS levels in SKOV3 and Hey cells. Consistently, primary cultures of ovarian cancer treated with 10058-F4 showed induction of caspase-3 activity and inhibition of cell proliferation in 15 of 18 cases. The response to 10058-F4 was independent the level of c-Myc protein over-expression in primary cultures of ovarian carcinoma. These novel findings suggest that the growth of ovarian cancer cells is dependent upon c-MYC activity and that targeting c-Myc-Max heterodimerization could be a potential therapeutic strategy for ovarian cancer.

Discriminating multiplexed GFP reporters in primary articular chondrocyte cultures using image cytometry.

Flow cytometry has become a standard tool for defining a heterogeneous cell population based on surface expressed epitopes or GFP reporters that reflect cell types or cellular differentiation. The introduction of image cytometry raised the possibility of adaptation to discriminate GFP reporters used to appreciate cell heterogeneity within the skeletal lineages. The optical filters and LEDs were optimized for the reporters used in transgenic mice expressing various fluorescent proteins. In addition, the need for compensation between eGFP and surrounding reporters due to optical cross-talk was eliminated by selecting the appropriate excitation and emission filters. Bone marrow or articular cartilage cell cultures from GFP and RFP reporter mouse lines were established to demonstrate the equivalency in functionalities of image to flow cytometry analysis. To examine the ability for monitoring primary cell differentiation, articular chondrocyte cell cultures were established from mice that were single or doubly transgenic (Dkk3eGFP and Col2A1GFPcyan), which identify the progression of superficial small articular cell to a mature chondrocyte. The instrument was able to rapidly and accurately discriminate cells that were Dkk3eGFP only, Dkk3eGFP/Col2A1GFPcyan, and Col2A1GFP, which provides a useful tool for studying the impact of culture conditions on lineage expansion and differentiation.

Automated enumeration and viability measurement of canine stromal vascular fraction cells using fluorescence-based image cytometry method.

In recent years, the lipoaspirate collected from adipose tissue has been seen as a valuable source of adipose-derived mesenchymal stem cells for autologous cellular therapy. For multiple applications, adipose-derived mesenchymal stem cells are isolated from the stromal vascular fraction (SVF) of adipose tissue. Because the fresh stromal vascular fraction typically contains a heterogeneous mixture of cells, determining cell concentration and viability is a crucial step in preparing fraction samples for downstream processing. Due to a large amount of cellular debris contained in the SVF sample, as well as counting irregularities standard manual counting can lead to inconsistent results. Advancements in imaging and optics technologies have significantly improved the image-based cytometric analysis method. In this work, we validated the use of fluorescence-based image cytometry for SVF concentration and viability measurement, by comparing to standard flow cytometry and manual hemocytometer. The concentration and viability of freshly collected canine SVF samples are analyzed, and the results highly correlated between all three methods, which validated the image cytometry method for canine SVF analysis, and potentially for SVF from other species.

Development of a high-throughput image cytometric screening method as a research tool for immunophenotypic characterization of patient samples from clinical studies.

Immunophenotyping has been the primary assay for characterization of immune cells from patients undergoing therapeutic treatments in clinical research, which is critical for understanding disease progression and treatment efficacy. Currently, flow cytometry has been the dominant methodology for characterizing surface marker expression for immunological research. Flow cytometry has been proven to be an effective and efficient method for immunophenotyping, however, it requires highly trained users and a large time commitment. Recently, a novel image cytometry system (Cellaca® PLX Image Cytometer, Revvity Health Sciences, Inc., Lawrence, MA) has been developed as a complementary method to flow cytometry for performing rapid and high-throughput immunophenotyping. In this work, we demonstrated an image cytometric screening method to characterize immune cell populations, streamlining the analysis of routine surface marker panels. The T cell, B cell, NK cell, and monocyte populations of 46 primary PBMC samples from subjects enrolled in autoimmune and oncological disease study cohorts were analyzed with two optimized immunophenotyping staining kits: Panel 1 (CD3, CD56, CD14) and Panel 2 (CD3, CD56, CD19). We validated the proposed image cytometry method by comparing the Cellaca® PLX and the AuroraTM flow cytometer (Cytek Biosciences, Fremont, CA). The image cytometry system was employed to generate bright field and fluorescent images, as well as scatter plots for multiple patient PBMC samples. In addition, the image cytometry method can directly determine cell concentrations for downstream assays. The results demonstrated comparable CD3, CD14, CD19, and CD56 cell populations from the primary PBMC samples, which showed an average of 5% differences between flow and image cytometry. The proposed image cytometry method provides a novel research tool to potentially streamline immunophenotyping workflow for characterizing patient samples in clinical studies.

Automated quantification of budding Saccharomyces cerevisiae using a novel image cytometry method.

The measurements of concentration, viability, and budding percentages of Saccharomyces cerevisiae are performed on a routine basis in the brewing and biofuel industries. Generation of these parameters is of great importance in a manufacturing setting, where they can aid in the estimation of product quality, quantity, and fermentation time of the manufacturing process. Specifically, budding percentages can be used to estimate the reproduction rate of yeast populations, which directly correlates with metabolism of polysaccharides and bioethanol production, and can be monitored to maximize production of bioethanol during fermentation. The traditional method involves manual counting using a hemacytometer, but this is time-consuming and prone to human error. In this study, we developed a novel automated method for the quantification of yeast budding percentages using Cellometer image cytometry. The automated method utilizes a dual-fluorescent nucleic acid dye to specifically stain live cells for imaging analysis of unique morphological characteristics of budding yeast. In addition, cell cycle analysis is performed as an alternative method for budding analysis. We were able to show comparable yeast budding percentages between manual and automated counting, as well as cell cycle analysis. The automated image cytometry method is used to analyze and characterize corn mash samples directly from fermenters during standard fermentation. Since concentration, viability, and budding percentages can be obtained simultaneously, the automated method can be integrated into the fermentation quality assurance protocol, which may improve the quality and efficiency of beer and bioethanol production processes.

High-throughput method to analyze the cytotoxicity of CAR-T Cells in a 3D tumor spheroid model using image cytometry.

Solid tumors account for approximately 90% of all adult human cancers. As such, the development of novel cellular therapies has become of increasing importance to target solid tumor malignancies, such as prostate, lung, breast, bladder, colon, and liver cancers. One such cellular therapy relies on the use of chimeric antigen receptor T cells (CAR-T cells). CAR-T cells are engineered to target specific antigens on tumor cells. To date, there are six FDA-approved CAR-T cell therapies that have been utilized for hematologic B cell malignancies. Immune cell trafficking and immunosuppressive factors within the tumor microenvironment increase the relative difficulty in developing a robust CAR-T cell therapy against solid tumors. Therefore, it is critical to develop novel methodologies for high-throughput phenotypic and functional assays using 3D tumor spheroid models to assess CAR-T cell products against solid tumors. In this manuscript, we discuss the use of CAR-T cells targeted towards PSMA, an antigen that is found on prostate cancer tumor cells, the second most common cause of cancer deaths among men worldwide. We demonstrate the use of high-throughput, plate-based image cytometry to characterize CAR-T cell-mediated cytotoxic potency against 3D prostate tumor spheroids. We were able to kinetically evaluate the efficacy and therapeutic value of PSMA CAR-T cells by analyzing the cytotoxicity against prostate tumor spheroids. In addition, the CAR-T cells were fluorescently labeled to visually identify the location of the T cells as cytotoxicity occurs, which may provide more meaningful information for assessing the functionality of the CAR-T cells. The proposed image cytometry method can overcome limitations placed on traditional methodologies to effectively assess cell-mediated 3D tumor spheroid cytotoxicity and efficiently generate time- and dose-dependent results.

A rapid and high-throughput T cell immunophenotyping assay for cellular therapy bioprocess using the Cellaca® PLX image cytometer.

In cellular therapies chimeric antigen receptor (CAR) T or NK cells undergo phenotypic analysis at multiple stages during discovery and development of novel therapies. Patient samples are routinely analyzed via flow cytometry for population identification and distribution of CD3, CD4, and CD8 positive T cells. As an alternative or orthogonal method, image cytometry systems have been used to perform simple cell-based assays in lieu of flow cytometry. Recently, a new image cytometry system, the Cellaca® PLX (Revvity Health Sciences, Inc., Lawrence, MA), was developed for high-throughput cell counting and viability, immunophenotyping, transfection/transduction efficiency, and cell health assays. This novel instrument allows investigators to quickly assess several critical quality attributes (CQAs) such as cell identity, viability, and other relevant biological functions recommended by the International Organization for Standardization using the ISO Cell Characterization documents focused on cellular therapeutic products. In this work, we demonstrate a rapid and high-throughput image cytometry detection method for cellular immunophenotyping and viability using the Cellaca PLX system for samples throughout the cellular therapy workflow. Freshly isolated peripheral blood mononuclear cells (PBMCs) underwent red blood cell (RBC) lysis and CD3 enrichment. Samples were then subsequently stained with Hoechst/CD3/CD4/CD8 or Hoechst/CD3/CD8/RubyDead Dye surface marker kits and measured on the Cellaca PLX and three different flow cytometers for side-by-side comparison and assay validation. Acquisition and analysis of cell viability and cell populations was shown to be faster and more efficient process compared to flow while achieving highly comparable results between the two technology platforms. This data shows that the Cellaca PLX Image Cytometer may provide a rapid alternative or orthogonal method for PBMC immunophenotyping experiments, as well as potentially streamline the workflow to quickly move precious patient samples downstream within the development processes.