Automated measurement of cell motility and proliferation

Background

Time-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.

Results

We 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.

Conclusion

These 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.     

Early effects of chemical carcinogens as compared to induced cell proliferation. II. Automated image analysis

Static automated image analysis was applied to study early variations of chromatin structure in Feulgen-stained liver nuclei from rats injected i.p. with a single dose of dimethylnitrosamine (DMNA), a well known hepatocarcinogen. An increase of nuclear area and a correspondent decrease of average optical density (integrated optical density/area) was observed, as compared with controls, in nuclei from rats treated with 5.4 mg/kg of DMNA. These findings, which were comparable with those induced by partial hepatectomy, indicate the existence in DMNA-treated cells of a chromatin DNA relaxation similar to the G0-G1 transition previously described for human diploid fibroblasts stimulated to proliferate. Because similar results were independently obtained by flow microfluorimetry, it seems reasonable to hypothesize that chromatin decondensation could be a prerequisite for cancer induction.     

Automated quantitative analysis of epithelial cell scatter

Epithelial cell scatter is a well-known in vitro model for the study of epithelial-mesenchymal transition (EMT). Scatter recapitulates many of the events that occur during EMT, including the dissociation of multicellular structures and increased cell motility. Because it has been implicated in tumor invasion and metastasis, much effort has been made to identify the molecular signals that regulate EMT. To better understand the quantitative contributions of these signals, we have developed metrics that quantitatively describe multiple aspects of cell scatter. One metric (cluster size) quantifies the disruption of intercellular adhesions while a second metric (nearest-neighbor distance) quantifies cell dispersion. We demonstrate that these metrics delineate the effects of individual cues and detect synergies between them. Specifically, we find epidermal growth factor (EGF), cholera toxin (CT) and insulin to synergistically reduce cluster sizes and increase nearest-neighbor distances. To facilitate the rapid measurement of our metrics from live-cell images, we have also developed automated techniques to identify cell nuclei and cell clusters in fluorescence images. Taken together, these studies provide broadly applicable quantitative image analysis techniques and insight into the control of epithelial cell scatter, both of which will contribute to the understanding of EMT and metastasis.Key words: cell scatter, EMT, automated image analysis     

Quantitative analysis of T cell homeostatic proliferation

T cell homeostatic proliferation occurs on transfer of T cells into lymphopenic recipients; transferred cells undergo several rounds of division in the absence of specific antigen stimulation. For a quantitative analysis of this phenomenon, we applied a mathematical method to describe proliferating T cells to match peak distributions from actual CFSE dilution data. For in vitro stimulation of T cells with anti-CD3/anti-CD28, our simulation confirmed a high proportion of cells entering cell cycle with a low proportion undergoing apoptosis. When applied to homeostatic proliferation, it described striking differences in CD4 and CD8 T cell proliferation rates, and accurately predicted that successive divisions were accompanied by higher rates of apoptosis, limiting the accumulation of proliferating cells. Thus, the presence of multiple CFSE dilution peaks cannot be considered equivalent to lymphocyte expansion. Finally, genetic effects were identified that may help explain links between homeostatic proliferation and autoimmunity.     

The topographical regulation of embryonic stem cell differentiation

The potential use of pluripotent stem cells for tissue repair or replacement is now well recognized. While the ability of embryonic stem (ES) cells to differentiate into all cells of the body is undisputed, their use is currently restricted by our limited knowledge of the mechanisms controlling their differentiation. This review discusses recent work by ourselves and others investigating the intercellular signalling events that occur within aggregates of mouse ES cells. The work illustrates that the processes of ES cell differentiation, epithelialization and programmed cell death are dependent upon their location within the aggregates and coordinated by the extracellular matrix. Establishment of the mechanisms involved in these events is not only of use for the manipulation of ES cells themselves, but it also throws light on the ways in which differentiation is coordinated during embryogenesis.     

Automated cell isolation from photodegradable hydrogel based on fluorescence image analysis

We report an automated cell-isolation system based on fluorescence image analysis of cell aggregates cultured in a photodegradable hydrogel. The system incorporates cell culture in a humidified atmosphere with controlled CO₂ concentration and temperature, image acquisition and analysis, micropatterned light exposure, and cell collection by pipetting. Cell aggregates were cultured on hydrogels, and target cells were selected by phase contrast and fluorescence image analysis. After degradation of the hydrogel by exposure to micropatterned UV light, cell aggregates were transferred to a collection vessel by robotic pipetting. We assessed the system for hydrogel degradation, recovery of target cells, and contamination by off-target cells. We demonstrated two practical applications of our method: (i) in cell aggregates from MCF-7-RFP strains in which 18.8% of cells produced red fluorescent protein (RFP), we successfully obtained 14 proliferative fluorescence-positive cell aggregates from 31-wells, and all of the isolated strains produced a higher proportion of RFP production than the original populations; (ii) after fluorescent immunostaining of human epidermal growth factor receptor 2 (HER2) in cancer cells, we successfully isolated HER2-positive cells from a mixed population of HER2-positive and -negative cells, and gene sequence analysis confirmed that the isolated cells mainly contained the target cells.     

The extracellular topographical environment influences ovarian cancer cell behaviour

The importance of the biophysical cellular environment in cancer development has been increasingly recognised but so far has been only superficially studied. Here we investigated the effect of cell-like substrate topography on ovarian cancer cell behaviour and potential underlying signalling pathways. We observed changes in cell morphology in response to substrate topography, which implies modification of structure-function associations. Differences in focal adhesion signalling and Rho/ROCK activity suggested their involvement in the biomechanically-driven cellular responses. Cell-like topography was also shown to modulate the MAPK pathway and hence potentially regulate cell proliferation. The selective regulation of the cells by the mechanotransduction pathways that we noted has wide ranging implications for understanding cancer development. We established that the physical architecture of cell culture substrate is sufficient to influence cancer cell behaviour, independent of genetic composition or biochemical milieu.     

Immunocytochemical analysis of chromaffin cell proliferation in vitro.

The bromodeoxyuridine (BrdU) incorporation technique for immunocytochemical labeling of S-phase nuclei was optimized for the study of chromaffin cell proliferation. Sequential fixation in ethanol followed by paraformaldehyde, and the use of DNAse to render incorporated BrdU accessible to antibody, permitted permanent double staining for BrdU and tyrosine hydroxylase. The efficacy of the technique was demonstrated in microcultures of dissociated neonatal rat adrenal glands, in which chromaffin cells exhibited proliferative responses to nerve growth factor and fibroblast growth factor similar to those previously demonstrated by autoradiography. Growth factor responsiveness was observed in both serum-containing and serum-free medium.     

Image analysis of rat satellite cell proliferation in vitro

Myogenic cells were isolated from adult rat skeletal muscles and cultured in vitro. Cell proliferation was analyzed between days 1 and 14. The cell cycle phases were determined by examining Feulgen-stained cultures with a cell image processor. The nuclei were automatically analyzed by calculating 18 parameters relating to the texture and densitometry of chromatin and the shape of each nucleus. Cell cycle phases were characterized (Moustafa and Brugal, 1984). The recognition methods made it possible to analyse the nuclei of the myogenic cell populations which were either involved in each phase of the mitotic cycle, or left out of the cycle after fusion into myotubes.After 3 hr of culture 10% of the cell population was involved in the cell cycle. In the presence of foetal calf serum, this percentage increased until day 3 after plating. At that time, the DNA content of 28.2% of the cell population was higher than 3C, whereas it is 2C in G1 or G0 nuclei; image analysis showed that 42% of these cells were in S or G2 phase. From day 4, the proliferation rate gradually slowed down until day 8. After day 8, when numerous myotubes differentiated, the percentage of S and G2 phase cells had diminished to between 3 and 8%. The percentage of nuclei in G0 increased when the first myotubes differentiated around day 5. Myotube nuclei were largely in G0. When horse serum was added to the culture medium on day 4 to enhance myotube differentiation, significant cell proliferation was observed before cell fusion.These methods of analysis give the first daily pattern of myogenic cell proliferation and fusion in a cell population isolated from adult muscles.     

Automated migration analysis based on cell texture: method &; reliability

Background  

In this paper, we present and validate a way to measure automatically the extent of cell migration based on automated examination of a series of digital photographs. It was designed specifically to identify the impact of Second Hand Smoke (SHS) on endothelial cell migration but has broader applications. The analysis has two stages: (1) preprocessing of image texture, and (2) migration analysis.     

Automated monitoring of cell concentration and viability using an image analysis system

In order to automate measurements of cell concentration and viability in a suspended animal cell culture, we have developed anin situ microscopic image analysis system with an effective cell recognition algorithm. With a small amount of sample, this system can measure the cell density rapidly and aseptically. In addition, it can measure a cell size histogram including cell debris small particle distribution. These small particles have been found to be related to the viability of the mouse-mouse hybridoma STK1 cell line. By using cell debris small particle density as an indicator of cell viability, the developed system provides non-destructive viability monitoring without trypan blue staining.     

Computerized topographical analysis of functionally homogeneous neuronal sets.

A computer-assisted analysis of the spatial distribution of neurons having homogeneous characteristics is described in this paper. The camera lucida drawings of sections of a brain nucleus and the points representing the neurons labeled on the basis of a specific behavior of discharge rates were digitized on a personal computer Amiga 2000 or IBM compatible. Our software provided: a) the computerized, stereotaxically oriented reconstruction of the stored sections and of the plotted neurons; b) the identification within each section of the mass center (MC) of the units sharing a given behavior and of the area where the density of such neurons was maximal (MDA). The routine was tested on the spatial distribution of neuronal responses to serotonin in the lateral vestibular nucleus.     

Methods for topographical analysis of intra-nuclear BrdUrd-tagged fluorescence.

The observation of BrdUrd staining in the nuclei of cells from exponentially growing populations reveals different typical replicating patterns. We propose a methodological approach to order and characterize BrdUrd intranuclear distributions. First, visual ordering of the patterns is assessed using a spectral analysis coupled to a k-nearest neighbors clustering technique. Subsequently, nine topographical features are introduced to characterize the spatial distribution of BrdUrd-tagged fluorescence in the nuclei of proliferating cells. These topographical features are based on a structural approach. The localization of fluorescence spots is expressed in terms of the normalized distance from the nuclear border and its standard deviation. These topographical features are simple to calculate and easy to relate to visual experience.     

High-throughput analysis of single hematopoietic stem cell proliferation in microfluidic cell culture arrays

Heterogeneity in cell populations poses a major obstacle to understanding complex biological processes. Here we present a microfluidic platform containing thousands of nanoliter-scale chambers suitable for live-cell imaging studies of clonal cultures of nonadherent cells with precise control of the conditions, capabilities for in situ immunostaining and recovery of viable cells. We show that this platform mimics conventional cultures in reproducing the responses of various types of primitive mouse hematopoietic cells with retention of their functional properties, as demonstrated by subsequent in vitro and in vivo (transplantation) assays of recovered cells. The automated medium exchange of this system made it possible to define when Steel factor stimulation is first required by adult hematopoietic stem cells in vitro as the point of exit from quiescence. This technology will offer many new avenues to interrogate otherwise inaccessible mechanisms governing mammalian cell growth and fate decisions.     

Automated image analysis in autoradiography

Light microscope autoradiographs can be analysed with the aid of the Quantimet, a commercially available image scanning instrument. Standardization of the performance of the instrument was obtained by performing on-line data reduction and display with a small digital computer. The number of silver grains over individual cells can be rapidly and routinely determined, and excellent agreement can be achieved between visually determined and machine generated grain counts.     

Automated sequence reading and analysis

We report on a system developed by Bio-Rad Laboratories, Inc. which combines automated reading of DNA sequencing autoradiograms with comprehensive software for shotgun overlapping of the readings, analysis of the sequences derived and searching of databases. Reading is accomplished using a high speed optical scanner and pattern recognition software operating on a personal computer. Overlapping, analysis and database searching software each incorporate significant advances over prior systems.