Time-lapse Microscopic Imaging Research Articles

Macrophage specificity: Imaging and characterization of interaction between macrophages and fluorescent organosilica nanoparticles depending on surface structure and size.

Macrophages have key functions in tissue homeostasis and also diseases. Macrophages show marked heterogeneity in response to environmental stimuli. The heterogeneity of macrophages was characterized by using morphology, surface marker, single cell sequence, and so on. We are investigating macrophage specificity using our nanomedical technology. We are developing novel materials, functional organosilica nanoparticles, and performed imaging to evaluate the diversity and specificity of mouse macrophage uptake. The organosilica nanoparticles surface-functionalized with polyethylene glycol (PEGylated) were applied to time-lapse fluorescent microscopic imaging and flow cytometry analyses. Single-cell imaging and analysis revealed that PEGylated nanoparticles revealed a stealth function against most macrophages. Notably, we found that the “PEG-resistant macrophages” that could take up PEGylated nanoparticles at the same level as bare nanoparticles. In addition, size-dependent uptake of macrophages was also observed in various tissues. The size-dependent uptake of functional organosilica nanoparticles by macrophages in the mouse various organs in vivo. These results indicated imaging and characterization of interaction between macrophages and fluorescent organosilica nanoparticles depending on surface structure and size were very useful to evaluate the heterogeneity and possible specificity of macrophage.

MICAL2PV suppresses the formation of tunneling nanotubes and modulates mitochondrial trafficking.

Tunneling nanotubes (TNTs) are actin‐rich structures that connect two or more cells and mediate cargo exchange between spatially separated cells. TNTs transport signaling molecules, vesicles, organelles, and even pathogens. However, the molecular mechanisms regulating TNT formation remain unclear and little is known about the endogenous mechanisms suppressing TNT formation in lung cancer cells. Here, we report that MICAL2PV, a splicing isoform of the neuronal guidance gene MICAL2, is a novel TNT regulator that suppresses TNT formation and modulates mitochondrial distribution. MICAL2PV interacts with mitochondrial Rho GTPase Miro2 and regulates subcellular mitochondrial trafficking. Moreover, down‐regulation of MICAL2PV enhances survival of cells treated with chemotherapeutical drugs. The monooxygenase (MO) domain of MICAL2PV is required for its activity to inhibit TNT formation by depolymerizing F‐actin. Our data demonstrate a previously unrecognized function of MICAL2 in TNT formation and mitochondrial trafficking. Furthermore, our study uncovers a role of the MICAL2PV‐Miro2 axis in mitochondrial trafficking, providing a mechanistic explanation for MICAL2PV activity in suppressing TNT formation and in modulating mitochondrial subcellular distribution.

A novel machine learning based approach for iPS progenitor cell identification.

Identification of induced pluripotent stem (iPS) progenitor cells, the iPS forming cells in early stage of reprogramming, could provide valuable information for studying the origin and underlying mechanism of iPS cells. However, it is very difficult to identify experimentally since there are no biomarkers known for early progenitor cells, and only about 6 days after reprogramming initiation, iPS cells can be experimentally determined via fluorescent probes. What is more, the ratio of progenitor cells during early reprograming period is below 5%, which is too low to capture experimentally in the early stage. In this paper, we propose a novel computational approach for the identification of iPS progenitor cells based on machine learning and microscopic image analysis. Firstly, we record the reprogramming process using a live cell imaging system after 48 hours of infection with retroviruses expressing Oct4, Sox2 and Klf4, later iPS progenitor cells and normal murine embryonic fibroblasts (MEFs) within 3 to 5 days after infection are labeled by retrospectively tracing the time-lapse microscopic image. We then calculate 11 types of cell morphological and motion features such as area, speed, etc., and select best time windows for modeling and perform feature selection. Finally, a prediction model using XGBoost is built based on the selected six types of features and best time windows. Our model allows several missing values/frames in the sample datasets, thus it is applicable to a wide range of scenarios. Cross-validation, holdout validation and independent test experiments show that the minimum precision is above 52%, that is, the ratio of predicted progenitor cells within 3 to 5 days after viral infection is above 52%. The results also confirm that the morphology and motion pattern of iPS progenitor cells is different from that of normal MEFs, which helps with the machine learning methods for iPS progenitor cell identification.

Automated quantification study of human cardiomyocyte synchronization using holographic imaging.

This paper investigates the rhythm strip and parameters of synchronization of human induced pluripotent stem cell (iPS) derived cardiomyocytes. The synchronization is evaluated from quantitative phase images of beating cardiomyocytes which are obtained using the time-lapse digital holographic imaging method. By quantitatively monitoring the dry mass redistribution, digital holography provides the physical contraction-relaxation signal caused by autonomous cardiac action potential. In order to analyze the synchronicity at the cell-to-cell level, we extracted single cardiac muscle cells, which contain the nuclei, from the phase images of cardiomyocytes containing multiple cells resulting from the fusion of k-means clustering and watershed segmentation algorithms. We demonstrate that mature cardiomyocyte cell synchronization can be automatically evaluated by time-lapse microscopic holographic imaging. Our proposed method can be applied for studies on cardiomyocyte disorders and drug safety testing systems.

Correlated random walks of human embryonic stem cells in vitro

We perform a detailed analysis of the migratory motion of human embryonic stem cells in two-dimensions, both when isolated and in close proximity to another cell, recorded with time-lapse microscopic imaging. We show that isolated cells tend to perform an unusual locally anisotropic walk, moving backwards and forwards along a preferred local direction correlated over a timescale of around 50 min and aligned with the axis of the cell elongation. Increasing elongation of the cell shape is associated with increased instantaneous migration speed. We also show that two cells in close proximity tend to move in the same direction, with the average separation of m or less and the correlation length of around 25 μm, a typical cell diameter. These results can be used as a basis for the mathematical modelling of the formation of clonal hESC colonies.

Time-Lapse Imaging for the Detection of Chromosomal Abnormalities in Primate Preimplantation Embryos.

The use of time-lapse microscopic imaging has proven to be a powerful tool for the study of mitotic divisions and other cellular processes across diverse species and cell types. Although time-lapse monitoring (TLM) of human preimplantation development was first introduced to the in vitro fertilization (IVF) community several decades ago, it was not until relatively recently that TLM systems were commercialized for clinical embryology purposes. Traditionally, human IVF embryos are assessed by successful progression and morphology under a stereomicroscope at distinct time points prior to selection for transfer. Due to the high frequency of aneuploidy, embryos may also be biopsied at the cleavage or blastocyst stage for preimplantation genetic screening (PGS) of whole and/or partial chromosomal abnormalities. However, embryo biopsy is invasive and can hinder subsequent development, and there are additional concerns over chromosomal mosaicism and resolution with PGS. Moreover, embryos are typically outside of the incubator in suboptimal culture conditions for extended periods of time during these procedures. With TLM systems, embryos remain in the stable microenvironment of an incubator and are simultaneously imaged for noninvasive embryo evaluation using a fraction of the light exposure as compared to a stereomicroscope. Each image is then compiled into a time-lapse movie, the information from which can be extrapolated to correlate morphological, spatial, and temporal parameters with embryo quality and copy number status. Here, we describe the various TLM systems available for clinical and/or research use in detail and provide step-by-step instructions on how the measurement of specific timing intervals and certain morphological criteria can be implemented into IVF protocols to enhance embryo assessment and avoid the selection of aneuploid embryos. We also discuss the biological significance of processes unique to mitotically dividing embryos and the likelihood that complex chromosomal events such as chromothripsis occur during preimplantation development in humans and other mammals, particularly nonhuman primates.

Time-lapse imaging of cell death in cell culture and whole living organisms using turn-on deep-red fluorescent probes.

Cell death is a central process in developmental biology and also an important indicator of disease status and treatment efficacy. Two related fluorescent probes are described that are molecular conjugates of one or two zinc dipicolylamine (ZnDPA) coordination complexes with an appended solvatochromic benzothiazolium squaraine dye. The probes were designed to target the anionic phospholipid, phosphatidylserine (PS), that is exposed on the surface of dead and dying cells. A series of spectrometric and microscopy studies using liposomes and red blood cell ghosts as models showed that the probe with two ZnDPA targeting units produced higher affinity, stronger fluorescence "turn-on" effect, and better image contrast than the probe with one ZnDPA. Both fluorescent probes enabled "no-wash" time-lapse microscopic imaging of mammalian cell death within a culture. The probe with two ZnDPA units was used for non-invasive time-lapse imaging of cell death during the development of Xenopus laevis (frog) embryos. In vivo fluorescence micrographs revealed probe accumulation within the embryo tail, head and spine regions that were undergoing regression and apoptosis during growth and maturation. These new fluorescent probes are likely to be useful for time-resolved, non-invasive in vivo imaging of cell death process in range of living organisms. From a broader perspective, it should be possible to utilize the negative solvatochromism exhibited by benzothiazolium squaraine dyes for development of various "turn-on" deep-red fluorescent probes and materials that target cell surface biomarkers for in vitro and in vivo imaging.

Revelation of Different Nanoparticle-Uptake Behavior in Two Standard Cell Lines NIH/3T3 and A549 by Flow Cytometry and Time-Lapse Imaging.

The uptake of nanomaterials into different cell types is a central pharmacological issue for the determination of nanotoxicity as well as for the development of drug delivery strategies. Most responses of the cells depend on their intracellular interactions with nanoparticles (NPs). Uptake behavior can be precisely investigated in vitro, with sensitive high throughput methods such as flow cytometry. In this study, we investigated two different standard cell lines, human lung carcinoma (A549) and mouse fibroblast (NIH/3T3) cells, regarding their uptake behavior of titanium dioxide NPs. Cells were incubated with different concentrations of TiO2 NPs and samples were taken at certain time points to compare the uptake kinetics of both cell lines. Samples were analyzed with the help of flow cytometry by studying changes in the side and forward scattering signal. To additionally enable a detection via fluorescence, NPs were labeled with the fluorescent dye fluorescein isothiocyanate (FITC) and propidium iodide (PI). We found that NIH/3T3 cells take up the studied NPs more efficiently than A549 cells. These findings were supported by time-lapse microscopic imaging of the cells incubated with TiO2 NPs. Our results confirm that the uptake behavior of individual cell types has to be considered before interpreting any results of nanomaterial studies.

Dynamics of Snake-like Swarming Behavior of Vibrio alginolyticus

Swarming represents a special case of bacterial behavior where motile bacteria migrate rapidly and collectively on surfaces. Swarming and swimming motility of bacteria has been studied well for rigid, self-propelled rods. In this study we report a strain of Vibrio alginolyticus, a species that exhibits similar collective motility but a fundamentally different cell morphology with highly flexible snake-like swarming cells. Investigating swarming dynamics requires high-resolution imaging of single cells with coverage over a large area: thousands of square microns. Researchers previously have employed various methods of motion analysis but largely for rod-like bacteria. We employ temporal variance analysis of a short time-lapse microscopic image series to capture the motion dynamics of swarming Vibrio alginolyticus at cellular resolution over hundreds of microns. Temporal variance is a simple and broadly applicable method for analyzing bacterial swarming behavior in two and three dimensions with both high-resolution and wide-spatial coverage. This study provides detailed insights into the swarming architecture and dynamics of Vibrio alginolyticus isolate B522 on carrageenan agar that may lay the foundation for swarming studies of snake-like, nonrod-shaped motile cell types.

Compact multi-band fluorescent microscope with an electrically tunable lens for autofocusing.

Autofocusing is a routine technique in redressing focus drift that occurs in time-lapse microscopic image acquisition. To date, most automatic microscopes are designed on the distance detection scheme to fulfill the autofocusing operation, which may suffer from the low contrast of the reflected signal due to the refractive index mismatch at the water/glass interface. To achieve high autofocusing speed with minimal motion artifacts, we developed a compact multi-band fluorescent microscope with an electrically tunable lens (ETL) device for autofocusing. A modified searching algorithm based on equidistant scanning and curve fitting is proposed, which no longer requires a single-peak focus curve and then efficiently restrains the impact of external disturbance. This technique enables us to achieve an autofocusing time of down to 170 ms and the reproductivity of over 97%. The imaging head of the microscope has dimensions of 12 cm × 12 cm × 6 cm. This portable instrument can easily fit inside standard incubators for real-time imaging of living specimens.

Enhanced anti-tumor efficacy and safety profile of tumor microenvironment-responsive oncolytic adenovirus nanocomplex by systemic administration

Oncolytic adenovirus (Ad) holds great promise as a potential gene therapy for cancer. However, intravenously administered Ad may encounter difficulties due to unfavorable host responses, non-specific interactions, and the heterogeneity of the tumor cell population. As an approach to combine the advantages of oncolytic Ad and synthetic polymers and to address the associated difficulties, Ad was physically complexed with a pH-sensitive block copolymer, methoxy poly(ethylene glycol)-b-poly(l-histidine) (mPEG-b-pHis). The in vitro transduction efficiency at an acidic extracellular pH was remarkably enhanced in cancer cells when treated with the Ad expressing green fluorescent protein (GFP) coated with mPEG-b-pHis (c-dE1/GFP) as compared to that of naked Ad (n-dE1/GFP). Time-lapse total internal reflection fluorescence microscopic imaging revealed a significantly enhanced cellular uptake rate of c-dE1/GFP at acidic tumor pH when compared with that at neutral pH or naked cognate Ad (n-dE1/GFP). In addition, c-dE1/GFP remained relatively stable in human serum-containing media, and considerably reduced both the innate and adaptive immune response against Ad. Moreover, the therapeutic efficacy and survival benefit of mPEG-b-pHis-complexed oncolytic Ad (c-H5mT/Luc) by systemic treatment was significantly enhanced compared to that with naked oncolytic Ad (n-H5mT/Luc) in both coxsackie and adenovirus receptor-positive and -negative tumors. Whole-body bioluminescence imaging showed 7.3-fold higher luciferase expression at the tumor site and 23.0-fold less luciferase expression in liver tissue for c-H5mT/Luc relative to that for naked oncolytic Ad (n-H5mT/Luc). Considering the heterogeneity of tumor tissue, these results are important for guiding the development of more potent and specific treatment of devastating metastatic cancers using this viral system. Statement of significanceAlthough adenoviral systems have shown considerable promise and undergone extensive evaluation attempts to specifically target Ad vectors to cancer cells have met limited success. This shortcoming is due to the strong immune response stimulated by Ad and the hepatotoxicity of the viral particles. To overcome restricted vector issues, we generated Ad/mPEG-b-pHis for tumor microenvironment-targeting hybrid vector systems, an oncolytic Ad coated with a pH-responsive polymer, mPEG-b-pHis. The Ad/mPEG-b-pHis exhibited pH-dependent transduction efficiency and cancer-cell killing effects. Moreover, systemic administration of oncolytic Ad/mPEG-b-pHis led to marked suppression of tumor growth and tumor-specific viral replication. Ad successfully avoided the innate and adaptive immune responses and liver accumulation with the help of mPEG-b-pHis on its surface.

Identification of polyethylene glycol-resistant macrophages on stealth imaging in vitro using fluorescent organosilica nanoparticles.

An in vitro imaging system to evaluate the stealth function of nanoparticles against mouse macrophages was established using fluorescent organosilica nanoparticles. Surface-functionalized organosilica nanoparticles with polyethylene glycol (PEG) were prepared by a one-step process, resulting in a brush-type PEG layer. A simultaneous dual-particle administration approach enabled us to evaluate the stealth function of nanoparticles with respect to single cells using time-lapse fluorescent microscopic imaging and flow cytometry analyses. Single-cell imaging and analysis revealed various patterns and kinetics of bare and PEGylated nanoparticle uptake. The PEGylated nanoparticles revealed a stealth function against most macrophages (PEG-sensitive macrophages); however, a stealth function against certain macrophages (PEG-insensitive macrophages) was not observed. We identified and characterized the PEG-resistant macrophages that could take up PEGylated nanoparticles at the same level as bare nanoparticles.

Protective effect of N-acetylcysteine against nicardipine hydrochloride-induced autophagic cell death of human vascular endothelial cells.

Nicardipine hydrochloride (NIC) injection has been widely used for emergency treatment of abnormally high blood pressure. However, NIC injection often causes severe peripheral vascular injury. The purpose of the present study was to reduce the NIC-induced cell injury in human vascular endothelial cells by use of clinical agents. The mechanism of NIC-induced cell injury was evaluated by time-lapse microscopic imaging, autophagosome staining with monodansylcadaverine, immunostaining of light chain 3 isoform B (LC-3B) and assessment of cell viability after exposure to NIC with or without an inhibitor of autophagosome formation (3-methyladenine, 3-MA). Results from autophagosome labeling and immunostaining of LC-3B revealed an increase of autophagosomes and LC-3B in NIC-treated cells. NIC-mediated reduction of cell viability was inhibited by 3-methyladenine. Moreover, we found that N-acetylcysteine (NAC) reduced NIC-induced cell injury in human vascular endothelial cells. These findings suggest that NIC causes severe peripheral venous irritation via induction of autophagic cell death and that inhibition of autophagy with NAC could contribute to the reduction of NIC-induced vascular injury.

Tumor-stromal cross talk: direct cell-to-cell transfer of oncogenic microRNAs via tunneling nanotubes

Tunneling nanotubes (TnTs) represent a novel mechanism by which intercellular components such as proteins, Golgi vesicles, and mitochondria can be transferred from cell to cell in the complex tumor microenvironment. Here, we report data showing that microRNAs (miRNAs) are transferred through TnTs in osteosarcoma (OS) and ovarian cancer as invitro model systems. miRNA array analysis demonstrated significant upregulation of miR-19a in OS tumors resected from human patients, and differential expression of miR-199a in ovarian cancer cell lines resistant or sensitive to platinum chemotherapy. K7M2 murine OS cells were transfected with miR-19a and cultured with nontransfected K7M2 cells in low-serum, hyperglycemic medium for up to 72hours to induce TnT formation. miRNA transfer via TnTs was detected by time-lapse microscopic imaging. miR-19a was also transported via TnTs connecting transfected K7M2 cells and nontransfected stromal MC3T3 murine osteoblast cells. Similar findings were observed in studies of TnT-mediated transport of miR-199a among SKOV3 ovarian cancer cells and nonmalignant immortalized ovarian epithelial cells. To quantify TnT-mediated transport of miRNAs, we used modified Boyden chambers to separate miR-19a-transfected K7M2 cells (top chamber) and DiI-labeled MC3T3 cells (bottom chamber) compared with open culture of these cells. Fluorescence-activated cell sorting analysis of cells collected after 48hours of culture indicated that miR-19a-positive MC3T3 cells were 3-fold higher in open culture; this finding suggests that miR-19a transfer occurred via TnTs, exclusive of other forms of cell-cell communication. These studies demonstrate that TnTs mediate direct transfer of genetic material between tumor and stromal cells.

Tumor exosomes induce tunneling nanotubes in lipid raft-enriched regions of human mesothelioma cells

Tunneling nanotubes (TnTs) are long, non-adherent, actin-based cellular extensions that act as conduits for transport of cellular cargo between connected cells. The mechanisms of nanotube formation and the effects of the tumor microenvironment and cellular signals on TnT formation are unknown. In the present study, we explored exosomes as potential mediators of TnT formation in mesothelioma and the potential relationship of lipid rafts to TnT formation. Mesothelioma cells co-cultured with exogenous mesothelioma-derived exosomes formed more TnTs than cells cultured without exosomes within 24–48h; and this effect was most prominent in media conditions (low-serum, hyperglycemic medium) that support TnT formation (1.3–1.9-fold difference). Fluorescence and electron microscopy confirmed the purity of isolated exosomes and revealed that they localized predominantly at the base of and within TnTs, in addition to the extracellular environment. Time-lapse microscopic imaging demonstrated uptake of tumor exosomes by TnTs, which facilitated intercellular transfer of these exosomes between connected cells. Mesothelioma cells connected via TnTs were also significantly enriched for lipid rafts at nearly a 2-fold higher number compared with cells not connected by TnTs. Our findings provide supportive evidence of exosomes as potential chemotactic stimuli for TnT formation, and also lipid raft formation as a potential biomarker for TnT-forming cells.

ACME: Automated Cell Morphology Extractor for Comprehensive Reconstruction of Cell Membranes

The quantification of cell shape, cell migration, and cell rearrangements is important for addressing classical questions in developmental biology such as patterning and tissue morphogenesis. Time-lapse microscopic imaging of transgenic embryos expressing fluorescent reporters is the method of choice for tracking morphogenetic changes and establishing cell lineages and fate maps in vivo. However, the manual steps involved in curating thousands of putative cell segmentations have been a major bottleneck in the application of these technologies especially for cell membranes. Segmentation of cell membranes while more difficult than nuclear segmentation is necessary for quantifying the relations between changes in cell morphology and morphogenesis. We present a novel and fully automated method to first reconstruct membrane signals and then segment out cells from 3D membrane images even in dense tissues. The approach has three stages: 1) detection of local membrane planes, 2) voting to fill structural gaps, and 3) region segmentation. We demonstrate the superior performance of the algorithms quantitatively on time-lapse confocal and two-photon images of zebrafish neuroectoderm and paraxial mesoderm by comparing its results with those derived from human inspection. We also compared with synthetic microscopic images generated by simulating the process of imaging with fluorescent reporters under varying conditions of noise. Both the over-segmentation and under-segmentation percentages of our method are around 5%. The volume overlap of individual cells, compared to expert manual segmentation, is consistently over 84%. By using our software (ACME) to study somite formation, we were able to segment touching cells with high accuracy and reliably quantify changes in morphogenetic parameters such as cell shape and size, and the arrangement of epithelial and mesenchymal cells. Our software has been developed and tested on Windows, Mac, and Linux platforms and is available publicly under an open source BSD license (https://github.com/krm15/ACME).

Abstract 2543: Thrombolytic Efficacy of Encapsulated Plasminogen

Introduction: Incidence of hemorrhage linked to treatment of ischemic stroke with tissue plasminogen activator (tPA) has led to interest in combination therapies such as plasminogen (Plg), with the goal of decreasing the administered volume of tPA. In-vitro studies using Plg have shown enhancement of lysis. However, due to rapid deactivation of Plg-derived plasmin in-vivo, local delivery of Plg via encapsulation by liposomes and release by ultrasound (US) may be necessary. This study examined the enhancement of tPA-induced lysis using encapsulated Plg (EPlg), in an in-vitro clot model. Methods: Blood was drawn from 26 subjects after local Institutional Review Board approval. Clots were made in 20-µL pipettes and placed in a water tank for microscopic visualization during treatment. Sample clots were exposed to tPA in human fresh-frozen plasma (hFFP) at a concentration of 3.15 µg/ml and EPlg at a concentration of 167 µg Plg/ml. Clots were exposed to no tPA (Control), tPA alone (+tPA), tPA and EPlg (+EPlg), tPA and 120-kHz US (+US), or tPA, EPlg, and 120-kHz US (+EPlg-US) for 30 minutes. The extent of thrombolysis was determined by assessing clot width as a function of time, using a time-lapse microscopic imaging technique; the fractional clot loss (FCL) at 30 minutes was used to determine thrombolytic efficacy. Results: Each treatment group had a minimum of 6 clots (range: 6-158) from 2 different donors (range: 2-24). FCL for +tPA was 38% (95% Confidence Interval: 36-40%); FCL for +EPlg was 39% (30-46%) (p=0.48 vs. +tPA). FCL for +EPlg-US was 49% (43-54%) (p<0.01 vs. +tPA). However, it did not exceed FCL for +US at at 70% (64-76%) (p<0.01 vs. +EPlg-US). Conclusions: Lysis with EPlg with tPA and US is more effective than tPA alone. This improvement of lytic efficacy using EPlg is dependent on concomitant use of US. Interaction between liposomes, Plg, and US is complex and merits further study.

Genome Size Is a Strong Predictor of Root Meristem Growth Rate

Variation in genome size (GS) has been linked to several facets of the plant phenotype. Recently it was shown that GS is significantly correlated with cell size and the duration of the cell cycle. Here we test the hypothesis that GS might also be a predictor of apical root meristem growth rate (RMGR). We studied eight species of eudicots with varying GS using time-lapse microscopic image analysis. A significant negative exponential relationship was observed between GS and RMGR. Our results show significantly decreased RMGR for large genome species. This relationship represents a significant consequence of GS expansion in plants and may partly explain why genome sizes tend to be small in eudicots. Interestingly, parasitic plants, which do not rely on root growth as much, often have large genomes.

ERK1/2 Activation Regulates the Wound Healing Process of Rabbit Corneal Endothelial Cells

Purpose: To evaluate the activation of ERK, a MAP kinase, in the regulation of proliferation and migration of rabbit corneal endothelial cells during the healing process after scrape wound injury. Methods: Cultured rabbit corneal endothelial cells were used in this study. Immunocytochemistry and Western blot analysis were performed to demonstrate the phosphorylation of ERK after scrape injury. The injured cell sheets were treated with different concentrations of ERK inhibitor, PD98059. The wound closure at 24, 48, and 72 hr following injury was evaluated. In order to evaluate the role of proliferation during wound healing, immunocytochemistry with Ki67 and 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt (MTS) assay were performed. The role of cell migration in wound healing was evaluated by quantification with the time lapse microscopic video imaging system. Results: Immunocytochemical and Western blot analysis revealed phosphorylation of ERK in the rabbit corneal endothelial cells after wounding. Inhibition of ERK phosphorylation by PD58059 resulted in a dose-dependent delay of wound closure. The inhibition of wound healing became significant from 48 to 72 hr in cells treated with 10 and 50 μ M of PD58059 (p < 0.05). ERK inhibition suppressed cell proliferation as indicated by Ki67 immunostaining and MTS assay. The time lapse microscopic video imaging analysis indicated delayed cell migration caused by PD98059. Conclusions: ERK activation regulates wound repair of rabbit corneal endothelial cells. Both cell proliferation and migration are involved in this process.

Automated measurement of cell motility and proliferation

BackgroundTime-lapse microscopic imaging provides a powerful approach for following changes in cell phenotype over time. Visible responses of whole cells can yield insight into functional changes that underlie physiological processes in health and disease. For example, features of cell motility accompany molecular changes that are central to the immune response, to carcinogenesis and metastasis, to wound healing and tissue regeneration, and to the myriad developmental processes that generate an organism. Previously reported image processing methods for motility analysis required custom viewing devices and manual interactions that may introduce bias, that slow throughput, and that constrain the scope of experiments in terms of the number of treatment variables, time period of observation, replication and statistical options. Here we describe a fully automated system in which images are acquired 24/7 from 384 well plates and are automatically processed to yield high-content motility and morphological data.ResultsWe have applied this technology to study the effects of different extracellular matrix compounds on human osteoblast-like cell lines to explore functional changes that may underlie processes involved in bone formation and maintenance. We show dose-response and kinetic data for induction of increased motility by laminin and collagen type I without significant effects on growth rate. Differential motility response was evident within 4 hours of plating cells; long-term responses differed depending upon cell type and surface coating. Average velocities were increased approximately 0.1 um/min by ten-fold increases in laminin coating concentration in some cases. Comparison with manual tracking demonstrated the accuracy of the automated method and highlighted the comparative imprecision of human tracking for analysis of cell motility data. Quality statistics are reported that associate with stage noise, interference by non-cell objects, and uncertainty in the outlining and positioning of cells by automated image analysis. Exponential growth, as monitored by total cell area, did not linearly correlate with absolute cell number, but proved valuable for selection of reliable tracking data and for disclosing between-experiment variations in cell growth.ConclusionThese results demonstrate the applicability of a system that uses fully automated image acquisition and analysis to study cell motility and growth. Cellular motility response is determined in an unbiased and comparatively high throughput manner. Abundant ancillary data provide opportunities for uniform filtering according to criteria that select for biological relevance and for providing insight into features of system performance. Data quality measures have been developed that can serve as a basis for the design and quality control of experiments that are facilitated by automation and the 384 well plate format. This system is applicable to large-scale studies such as drug screening and research into effects of complex combinations of factors and matrices on cell phenotype.