Real-time Live Cell Research Articles

Emissive Guanosine Analog Applicable for Real-Time Live Cell Imaging.

A new emissive guanosine analog CF3thG, constructed by a single trifluoromethylation step from the previously reported thG, displays red-shifted absorption and emission spectra compared to its precursor. The impact of solvent type and polarity on the photophysical properties of CF3thG suggests that the electronic effects of the trifluoromethyl group dominate its behavior and demonstrates its susceptibility to microenvironmental polarity changes. In vitro transcription initiations using T7 RNA polymerase, initiated with CF3thG, result in highly emissive 5'-labeled RNA transcripts, demonstrating the tolerance of the enzyme toward the analog. Viability assays with HEK293T cells displayed no detrimental effects at tested concentrations, indicating the safety of the analog for cellular applications. Live cell imaging of the free emissive guanosine analog using confocal microscopy was facilitated by its red-shifted absorption and emission and adequate brightness. Real-time live cell imaging demonstrated the release of the guanosine analog from HEK293T cells at concentration-gradient conditions, which was suppressed by the addition of guanosine.

Decoding Arc transcription: a live-cell study of stimulation patterns and transcriptional output.

Activity-regulated cytoskeleton-associated protein (Arc) plays a crucial role in synaptic plasticity, a process integral to learning and memory. Arc transcription is induced within a few minutes of stimulation, making it a useful marker for neuronal activity. However, the specific neuronal activity patterns that initiate Arc transcription have remained elusive due to the inability to observe mRNA transcription in live cells in real time. Using a genetically encoded RNA indicator (GERI) mouse model that expresses endogenous Arc mRNA tagged with multiple GFPs, we investigated Arc transcriptional activity in response to various electrical field stimulation patterns. The GERI mouse model was generated by crossing the Arc-PBS knock-in mouse, engineered with binding sites in the 3' untranslated region (UTR) of Arc mRNA, and the transgenic mouse expressing the cognate binding protein fused to GFP. In dissociated hippocampal neurons, we found that the pattern of stimulation significantly affects Arc transcription. Specifically, theta-burst stimulation consisting of high-frequency (100 Hz) bursts delivered at 10 Hz frequency induced the highest rate of Arc transcription. Concurrently, the amplitudes of nuclear calcium transients also reached their peak with 10 Hz burst stimulation, indicating a correlation between calcium concentration and transcription. However, our dual-color single-cell imaging revealed that there were no significant differences in calcium amplitudes between Arc-positive and Arc-negative neurons upon 10 Hz burst stimulation, suggesting the involvement of other factors in the induction of Arc transcription. Our live-cell RNA imaging provides a deeper insight into the complex regulation of transcription by activity patterns and calcium signaling pathways.

Recent Developments in Small-Molecule Fluorescent Probes for Cellular Senescence

Cellular senescence is a recently emerged research topic in modern biology. Often described as a double-edged sword, it encompasses numerous essential biological processes, including beneficial effects such as wound healing and embryonic development, as well as detrimental contributions to chronic inflammation and tumor development. Consequently, there is an increasing need to unravel the intricate networks of senescence and develop reliable detection methods to distinguish it from related phenomena. To address these challenges, a variety of detection methods have been developed. In particular, small-molecule fluorescent probes offer distinct advantages such as suitability for real-time live cell monitoring and in vivo imaging, superior tunable properties, and versatile applications. In this review, we explored recent advancements in the development of small-molecule fluorescent probes toward monitoring cellular senescence by targeting various senescence-related biological phenomena. These phenomena include the upregulation of senescence-associated enzymes, perturbation of the subcellular environment, and increased endogenous ROS levels. Moreover, multi-senescence biomarker-targeting approaches are also discussed to improve their sensitivities and specificities for the detection of cellular senescence. With recent advances in senescence probe development, current challenges in this field are also discussed to facilitate further progress.

Parallel illumination for depletion microscopy through acousto-optic spatial light modulation

Several types of super-resolution microscopy techniques, such as Stimulated Emission Depletion (STED), Reversible Saturable Optical Fluorescence Transitions (RESOLFT) or Switching Laser Mode (SLAM) microscopies, employ Laguerre-Gaussian beams (also called vortex or doughnut beams) to obtain fluorescence information within a sub-wavelength region of the specimen under observation, thus breaking the diffraction limit and producing images of greatly improved quality. However, in general, these techniques operate on a point-by-point basis, so scanning the sample in order to build a full, meaningful image, takes time. Parallelization of the illumination is the only way to make these microscopy techniques more suitable for real-time live cell imaging applications. Here, we demonstrate the parallel production of arbitrary arrays of Gaussian and Laguerre-Gaussian lasers foci suitable for super-resolution microscopy, together with the possibility to fast scan through the sample, by means of acousto-optic spatial light modulation, a technique that we have pioneered in the past in several other fields. Parallelized illumination with both Gaussian and doughnut beams will be then used to acquire super-resolution images.

Advances in Organic Fluorescent Probes for Intracellular Zn2+ Detection and Bioimaging.

Zinc ions (Zn2+) play a key role in maintaining and regulating protein structures and functions. To better understand the intracellular Zn2+ homeostasis and signaling role, various fluorescent sensors have been developed that allow the monitoring of Zn2+ concentrations and bioimaging in live cells in real time. This review highlights the recent development of organic fluorescent probes for the detection and imaging of intracellular Zn2+, including the design and construction of the probes, fluorescent response mechanisms, and their applications to intracellular Zn2+ detection and imaging on-site. Finally, the current challenges and prospects are discussed.

Abstract 3248: Metabolic rewiring shifts engineered TCR T cell bioenergetics to enhance cytotoxicity

Abstract Background: Adoptive T cell-based transfer has shown tremendous success in hematological cancers. However, an important obstacle to using cell and gene therapy for solid tumors is the aggressive tumor microenvironment (TME). To significantly improve prognosis of subjects undergoing immunotherapy, it is essential to incorporate unique methods to drive T cell energy production and metabolism towards empowering T cells to dismantle the suppressive TME. This can promote anti-tumor effects and persistence of the transfused engineered T cells. In the present study we present a simple metabolic preconditioning process that improves T cell performance and bioenergetics. Methods: Using the Agilent xCELLigence RTCA eSight, and Seahorse XF Analyzer we assessed the killing efficiency, and bioenergetics of engineered T-cells after pretreating with Arginine. Briefly, engineered TCR T-cells against MART-1 were generated by transducing CD3+ T-cells (Hemacare, Seattle, WA) with retrovirus SAMEN-DMF5 with a CD34 marker gene, against MART-1. The TCR T cells were pre-conditioned in 6mM Arginine for 7 days, followed by a killing assay using MART-1 expressing melanoma cell line as target cells (624.38) at the E:T ratio of 5:1. Target melanoma cells, 624.38 cells, were engineered to express a red-fluorescent nuclear protein. The comparison was made with transduced T-cells grown in RPMI (no added amino acid supplementation, and non-transduced T cells. The cytotoxicity assay was assessed using real-time impedance and live cell imaging data collected on xCELLigence RTCA eSight. Concurrently, the metabolic profiling of the engineered T-cells was examined by the XF96 Analyzer. Results: Supplementing the growth medium with 6 mM Arginine significantly increased the potency of MART-1 engineered T cells (up to 6-fold) and bioenergetic studies revealed increased spare respiratory capacity, basal respiration, and ATP production via mitochondrial respiration. Conclusions: Arginine pre-conditioning improved TCR T cell potency through metabolic rewiring favoring mitochondrial respiration. For Research Use Only. Not for use in diagnostic procedures RA45236.3483333333 Citation Format: Rashmi Pillai, Xiaoyu Zhang, Yama Abassi. Metabolic rewiring shifts engineered TCR T cell bioenergetics to enhance cytotoxicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3248.

Visualizing mitochondrial heme flow through GAPDH in living cells and its regulation by NO

Iron protoporphyrin IX (heme) is a redox-active cofactor that is bound in mammalian cells by GAPDH and allocated by a process influenced by physiologic levels of NO. This impacts the activity of many heme proteins including indoleamine dioxygenase-1 (IDO1), a redox enzyme involved in immune response and tumor growth. To gain further understanding we created a tetra-Cys human GAPDH reporter construct (TC-hGAPDH) which after labeling could indicate its heme binding by fluorescence quenching. When purified or expressed in a human cell line, TC-hGAPDH had properties like native GAPDH and heme binding quenched its fluorescence by 45–65%, allowing it to report on GAPDH binding of mitochondrially-generated heme in live cells in real time. In cells with active mitochondrial heme synthesis, low-level NO exposure increased heme allocation to IDO1 while keeping the TC-hGAPDH heme level constant due to replenishment by mitochondria. When mitochondrial heme synthesis was blocked, low NO caused a near complete transfer of the existing heme in TC-hGAPDH to IDO1 in a process that required IDO1 be able to bind the heme and have an active hsp90 present. Higher NO exposure had the opposite effect and caused IDO1 heme to transfer back to TC-hGAPDH. This demonstrated: (i) flow of mitochondrial heme through GAPDH is tightly coupled to target delivery, (ii) NO up- or down-regulates IDO1 activity by promoting a conserved heme exchange with GAPDH that goes in either direction according to the NO exposure level. The ability to drive a concentration-dependent, reversible protein heme exchange is unprecedented and reveals a new role for NO in biology.

Ubiquitination of Major Histocompatibility Complex II Changes Its Immunological Recognition Structure

Ubiquitination is a process that dictates the lifespan of major histocompatibility complex class II (MHC II)/peptide complexes on antigen-presenting cells. This process is tightly controlled by the levels of ubiquitin ligases, and disruptions in the turnover of MHC II can lead to the improper development of CD4+ T cells within the thymus and hinder the formation of regulatory T cells in the peripheral tissue. To investigate the underlying mechanisms, we utilized dendritic cells lacking the Membrane-associated RING-CH (MARCH) I ubiquitin ligase. We discovered that the overexpression of MARCH I decreases the interaction with LAG-3. Moreover, the MHC II molecules tethered with ubiquitin also showed diminished binding to LAG-3. We employed Diffracted X-ray Blinking (DXB), a technique used for single-molecule X-ray imaging, to observe the protein movements on live cells in real time. Our observations indicated that the normal MHC II molecules moved more rapidly across the cell surface compared to those on the MARCH I-deficient dendritic cells or MHC II KR mutants, which is likely a result of ubiquitination. These findings suggest that the signaling from ubiquitinated MHC II to the T cell receptor differs from the non-ubiquitinated forms. It appears that ubiquitinated MHC II might not be quickly internalized, but rather presents antigens to the T cells, leading to a range of significant immunological responses.

Real-Time Assessment of Mitochondrial Function in Cytotrophoblast and Syncytialized Trophoblast Cells Using the Seahorse XFe24 Extracellular Flux Analyzer.

Physiological processes utilize variable amounts of energy required for optimal growth, development, and survival. This energy is supplied by total intracellular adenosine triphosphate (ATP) and is mainly generated by mitochondrial oxidative phosphorylation and to a lesser extent via glycolysis. Here, we provide a detailed protocol for obtaining measurements of energy metabolism using the Seahorse XFe24 Extracellular Flux Analyzer. Specifically, this assay measures mitochondrial oxidative phosphorylation based on oxygen consumption rate (OCR) and glycolysis by analyzing the extracellular acidification rate (ECAR) via real-time live cell analysis. Using trophoblast cell lines, this protocol focuses on analyzing mitochondrial respiration for both cytotrophoblasts and syncytiotrophoblasts.

A non-iterative frame-reduced structured illumination microscopy using checkerboard modulation

Structured illumination microscopy (SIM) is a tremendously potential super-resolution microscopy technique requiring nine raw images to enhance two-fold lateral resolution. For SIM, it is a challenging dilemma to achieve super-resolution imaging with a reduced number of images and fast data processing speed at the same time. Here, we demonstrate a new SIM reconstruction framework that uses only four raw images without requiring extensive iterative computation. The four-frame method is achieved by applying checkerboard pattern illumination modulation to supersede the sinusoidal fringe illumination. It is worth noting that the proposed scheme prominently shortens image acquisition time, combined with a remarkably higher image reconstruction rate compared to current frame-reduced methods. Meanwhile, the reconstruction process is non-iterative and unlimited by the field of view size. Consequently, our work is expected to bring about breakthroughs in facilitating the improvement of real-time live cell observation.

Arvanil induces ferroptosis of hepatocellular carcinoma by binding to MICU1.

Hepatocellular carcinoma (HCC) is a primary liver cancer with a high mortality rate that requires research and improved treatment strategies. Chemotherapy is still one of the main methods of HCC treatment, but it may lead to drug resistance and damage to normal organs. Capsaicin, a naturally occurring active ingredient in chili peppers, has demonstrated anticancer properties in a variety of malignant tumor cell lines. However, the anti-cancer mechanism of capsaicin needs to be further explored in HCC. In this study, we utilized Arvanil, a non-stimulating synthetic capsaicin analog, in place of capsaicin. We found that Arvanil induced high mitochondrial calcium flow, which contributed to a decrease in mitochondrial membrane permeability transition pore (mPTP) opening and oxidative phosphorylation levels, ultimately triggering cellular ferroptosis by live cells in real time with a high content screening (HCS) platform and confocal microscopy. It was further confirmed by vina molecular docking and point mutation experiments that Arvanil directly binds to two amino acid sites of mitochondrial calcium uptake protein 1 (MICU1), namely Ser47 and Phe128, to trigger this process, which in turn inhibits the growth of HCC cells. In addition, it was confirmed that Arvanil enhances cisplatin chemosensitivity by inducing HCC cellular ferroptosis in vivo. In conclusion, our study suggests that Arvanil induces ferroptosis in HCC cells and is a candidate drug for the treatment of HCC.

Covalently bridge metals to biomass carbon for sensing DA and FA with high specificity and sensitivity

Due to the high catalytic activity and electrical conductivity, “transition metal-nonmetal” compounds have broad application prospects in the construction of electrochemical sensors. However, it is still a challenge to solve the problem of catalyst deactivation and slow electron transfer due to nanoparticle agglomeration's blockage of active sites. Here, we prepare a series of covalently bonded “C-M-O” bridge bonds on biomass carbon for the first time. The bridge bonds have a straight-like “electron path”, which shortens the electron transfer path and improves the electrical conductivity, and catalytic activity. The bridge bonds exhibit excellent catalytic activity even after being subjected to prolonged mechanical external forces. Besides, the bridge bonds can integrate a biosensor with MIP and exhibit excellent electrocatalytic performance, including high sensitivity, low detection limit, excellent stability, superior interference immunity, and the ability to accurately monitor DA and FA released from real samples and live cells in real time. The strategy of forming “C-M-O” bridge bonds based on covalently bonded supports provides a new perspective for solving the agglomeration of nanocatalysts blocking active site deactivation and improving electron transfer rate.

MAxSIM: multi-angle-crossing structured illumination microscopy with height-controlled mirror for 3D topological mapping of live cells

Mapping 3D plasma membrane topology in live cells can bring unprecedented insights into cell biology. Widefield-based super-resolution methods such as 3D-structured illumination microscopy (3D-SIM) can achieve twice the axial ( ~ 300 nm) and lateral ( ~ 100 nm) resolution of widefield microscopy in real time in live cells. However, twice-resolution enhancement cannot sufficiently visualize nanoscale fine structures of the plasma membrane. Axial interferometry methods including fluorescence light interference contrast microscopy and its derivatives (e.g., scanning angle interference microscopy) can determine nanoscale axial locations of proteins on and near the plasma membrane. Thus, by combining super-resolution lateral imaging of 2D-SIM with axial interferometry, we developed multi-angle-crossing structured illumination microscopy (MAxSIM) to generate multiple incident angles by fast, optoelectronic creation of diffraction patterns. Axial localization accuracy can be enhanced by placing cells on a bottom glass substrate, locating a custom height-controlled mirror (HCM) at a fixed axial position above the glass substrate, and optimizing the height reconstruction algorithm for noisy experimental data. The HCM also enables imaging of both the apical and basal surfaces of a cell. MAxSIM with HCM offers high-fidelity nanoscale 3D topological mapping of cell plasma membranes with near-real-time ( ~ 0.5 Hz) imaging of live cells and 3D single-molecule tracking.

Deep-learning accelerated super-resolution radial fluctuations (SRRF) enables real-time live cell imaging

The Super-resolution radial fluctuations (SRRF) algorithm analyzes radial and temporal fluorescence intensity fluctuations in an image sequence, which typically includes tens or hundreds of raw images to generate one super-resolution image. At present, most of the SRRF applications rely on large amounts of raw images or tedious post-processing and computation, thus are not feasible for real-time live cell super-resolution imaging. Here, we developed a novel deep learning accelerated SRRF method, which significantly reduced the requirement of raw images for super-resolution reconstruction. Our results showed that by using only 5 low signal-to-noise ratio (SNR) images, we were able to achieve super-resolution SRRF reconstruction to a comparable resolution as the traditional method. We demonstrated that by integration of GPU computing and the sliding window reconstruction method, the dynamic contraction of microtubules and the interactions between microtubules and clathrin-coated pits (CCPs) can be visualized in real-time with super-resolution. In summary, we established the deep learning accelerated SRRF method, which permits real-time, long-term and multi-color live cell super-resolution imaging, and we anticipate it will have vast biomedical applications.

Long-term, real-time and label-free live cell image processing and analysis based on a combined algorithm of CellPose and watershed segmentation

Developing a rapid and quantitative method to accurately evaluate the physiological abilities of living cells is critical for tumor control. Many experiments have been conducted in the field of biology in an attempt to measure the proliferation and movement abilities of cells, but existing methods cannot provide real-time and objective data for label-free cells. The quantitative imaging technique, including an automatic segmentation algorithm for individual label-free cells, has been a breakthrough in this regard. In this study, we develop a combined automatic image processing algorithm of CellPose and watershed segmentation for the long-term and real-time imaging of label-free cells. This method shows strong reliability in cell identification regardless of cell densities, allowing us to obtain accurate information about the number and proliferation ability of the target cells. Additionally, our results also suggest that this method is a reliable way to assess real-time data on drug cytotoxicity, cell morphology, and cell movement ability.

Shear stress induces autophagy in Schlemm's canal cells via primary cilia-mediated SMAD2/3 signaling pathway.

The Schlemm's canal (SC) is a circular, lymphatic-like vessel located at the limbus of the eye that participates in the regulation of aqueous humor drainage to control intraocular pressure (IOP). Circumferential flow of aqueous humor within the SC lumen generates shear stress, which regulates SC cell behaviour. Using biochemical analysis and real-time live cell imaging techniques, we have investigated the activation of autophagy in SC cells by shear stress. We report, for the first time, the primary cilium (PC)-dependent activation of autophagy in SC cells in response to shear stress. Moreover, we identified PC-dependent shear stress-induced autophagy to be positively regulated by phosphorylation of SMAD2 in its linker and C-terminal regions. Additionally, SMAD2/3 signaling was found to transcriptionally activate LC3B, ATG5 and ATG7 in SC cells. Intriguingly, concomitant to SMAD2-dependent activation of autophagy, we also report here the activation of mTOR pathway, a classical autophagy inhibitor, in SC cells by shear stress. mTOR activation was found to also be dependent on the PC. Moreover, pharmacological inhibition of class I PI3K increased phosphorylation of SMAD2 at the linker and activated autophagy. Together, our data indicates an interplay between PI3K and SMAD2/3 signaling pathways in the regulation of PC-dependent shear stress-induced autophagy in SC cells.

Real-time evaluation of the biocompatibility of calcium silicate-based endodontic cements: An in vitro study.

An ideal filling material should hermetically seal the communication pathways between the canal system and surrounding tissues. Therefore, during the last few years, the development of obturation materials and techniques to create optimal conditions for the proper healing of apical tissues has been a focus of interest. The effects of calcium silicate-based cements (CSCs) on periodontal ligament cells have been investigated, and promising results have been obtained. To date, there are no reports in the literature that have evaluated the biocompatibility of CSCs using a real-time live cell system. Therefore, this study aimed to evaluate the real-time biocompatibility of CSCs with human periodontal ligament cells (hPDLCs). hPDLC were cultured with testing media of endodontic cements for 5 days: TotalFill-BC Sealer, BioRoot RCS, Tubli-Seal, AH Plus, MTA ProRoot, Biodentine, and TotalFill-BC RRM Fast Set Putty. Cell proliferation, viability, and morphology were quantified using real-time live cell microscopy with the IncuCyte S3 system. Data were analyzed using the one-way repeated measures (RM) analysis of variancemultiple comparison test (p < .05). Compared to the control group, cell proliferation in the presence of all cements was significantly affected at 24 h (p < .05). ProRoot MTA and Biodentine lead to an increase in cell proliferation; there were no significant differences with the control group at 120 h. In contrast, Tubli-Seal and TotalFill-BC Sealer inhibited cell growth in real-time and significantly increased cell death compared to all groups. hPDLC co-cultured with sealer and repair cements showed a spindle-shaped morphology except with cements Tubli-Seal and TotalFill-BC Sealer where smaller and rounder cells were obtained. The biocompatibility of the endodontic repair cements performed better than the sealer cements, highlighting the cell proliferation of the ProRoot MTA and Biodentine in real-time. However, the calcium silicate-based TotalFill-BC Sealer presented a high percentage of cell death throughout the experiment similar to that obtained.

Mitotic spindle disassembly in human cells relies on CRIPT having hierarchical redox signals.

Swift and complete spindle disassembly in late mitosis is essential for cell survival, yet how it happens is largely unknown in mammalian cells. Here we used real-time live cell microscopy and biochemical assays to show that the primordial dwarfism (PD)-related cysteine-rich protein CRIPT dictates the spindle disassembly in a redox-dependent manner in human cells. This previously reported cytoplasmic protein was found to have a confined nuclear localization with a nucleolar concentration during interphase but was distributed to spindles and underwent redox modifications to form disulfide bonds in CXXC pairs during mitosis. Then, it directly interacted with, and might transfer a redox response to, tubulin subunits via a putative redox exchange among cysteine residues to induce microtubule depolymerization. Expression of CRIPT proteins with mutations of these cysteine residues blocked spindle disassembly, generating two cell types with long-lasting metaphase spindles or spindle remnants. Live-cell recordings of a disease-relevant mutant (CRIPTC3Y) revealed that microtubule depolymerization at spindle ends during anaphase and the entire spindle dissolution during telophase might share a common CRIPT-bearing redox-controlled mechanism.

Integration of leave-one-out method and real-time live cell reporter array system to assess the toxicity of mixtures

The ever-increasing number of chemicals and complex mixtures demands a time-saving and cost-effective platform for environmental risk assessment. However, there is limit promising tool for evaluating the contribution of each component to the total toxicity effects of the mixture. Here, four widely distributed environmental pollutants with different mode-of-actions, i.e., cadmium chloride (Cd), nitrofurazone (NFZ), triclosan (TCS), and tris(2-chloroethyl) phosphate (TCEP), were selected as components of artificial mixture. Integration of leave-one-out method and high-dimensional live cell array system was used to explore relative contribution of each component from the mixture. A quaternary mixture (All_4_chems) and four ternary mixtures (Leave_Cd, Leave_NFZ, Leave_TCS and Leave_TCEP) were investigated by Escherichia coli (E. coli) live cell array system with 90 environmental stress genes modified by green fluorescent protein (GFP) expressing reporter vectors. E. coli cytotoxicity tests demonstrated that TCS has antagonism effect with other three chemicals (Cd, NFZ and TCEP), while it was additive effect in other three binary combinations. A total of 26, 23, 13, 31 and 23 genes were significantly altered with fold-change greater than 2 over the 4 h exposure by All_4_chems, Leave_Cd, Leave_NFZ, Leave_TCS and Leave_TCEP, respectively. Clustering analysis based on time-series gene expression patterns and transcriptional effect level index (TELI) showed that Leave_TCEP has similar profiles with All_4_chems, demonstrating TCEP has the least contribution among four components to the quaternary mixture. Leave_NFZ has the least number of significantly altered genes, implying NFZ has the largest toxicity effect contribution to the quaternary mixture. The relative contribution in different pathways indicated that Cd has the most contribution to the mixture in redox stress, while TCS has the least contribution in DNA stress pathway. Collectively, our results demonstrated the utility of high-dimensional toxicogenomics data and leave-one-out method in prioritizing the relative contribution of each component in mixture.

Abstract 3084: Label-free, real-time live cell assays for 3D organoids embedded in Matrigel®

Abstract Organoid technologies are increasingly being used as in-vitro models of human development and disease as they exhibit structural, morphogenetic, and functional properties that recapitulate in vivo pathophysiology. To successfully use these models across a variety of research disciplines and applications, approaches that reduce variability and technology pipelines to image &amp; quantify these complex cell models are required. Here, we demonstrate simple, robust workflows for monitoring and automatically quantifying features, such as morphology, growth and death of organoids using real time live cell analysis. To quantitatively optimize and characterize organoid cultures in-vitro, mouse hepatic, intestinal and pancreatic organoids were embedded in Matrigel® domes (50% or 100%) in 24-well plates and imaged over time in an Incucyte® Live Cell System. Organoid growth, differentiation, and maturation was measured using Incucyte’ s automated Organoid Software Analysis Module, which tracks changes in size and morphology. Integrated metrics enabled objective determination of cell-type specific growth conditions and passaging regimes. To illustrate the utility of the Incucyte® Organoid Analysis Software Module to track organoid growth and death in 96-well plates, intestinal and hepatic organoid fragments were embedded in Matrigel® (50%) for 3 days prior to treatment with protein kinase inhibitor staurosporine (1 µM, STP). Vehicle treated organoids increased in size (10-fold; intestinal or 3-fold; hepatic) over time while marked reduction was observed in the presence of STP. Using label-free size and morphology metrics we could distinguish between cytotoxic and cytostatic mechanisms of action (MoA) of known chemotherapeutic compounds. STP, cisplatin (CIS, DNA synthesis inhibitor) or fluorouracil (5-FU, thymidylate synthetase inhibitor) exhibited concentration dependent inhibition of hepatic organoid growth, yielding IC50 values of 3 nM for STP, 9.7 µM for CIS and 0.78 µM for 5-FU. Whilst attenuation of size was observed across all compounds, increases in eccentricity and darkness indictive of 3D structure disruption and cell death respectively were only observed in CIS and STP-treated organoids (cytotoxic MoA). Differences between the size and morphology readouts illustrated the cytostatic mechanism of 5-FU. Use of this approach was extended to visualize and quantify CFTR function. Following forskolin stimulation, a concentration-dependent increase in intestinal organoid size was observed. In the presence of CFTR inhibitor CFTRinh-172 the maximal response was reduced by &amp;gt;50% (~150% at 10 µM) illustrating that swelling was CFTR-dependent. These data demonstrate the capability to kinetically visualize and quantify distinct organoid morphologies, assess drug-induced cellular changes label-free and illustrates the amenability of this approach across a range of disease areas. Citation Format: Tim R. Jackson, Miniver Oliver, Daniel Appledorn, Tim Dale, Kalpana Barnes. Label-free, real-time live cell assays for 3D organoids embedded in Matrigel® [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3084.