1,721,046 research outputs found
AnaSP: A software suite for automatic image analysis of multicellular spheroids
No full textToday, more and more biological laboratories use 3D cell cultures and tissues grown in vitro as a 3D model of in vivo tumours and metastases. In the last decades, it has been extensively established that multicellular spheroids represent an efficient model to validate effects of drugs and treatments for human care applications. However, a lack of methods for quantitative analysis limits the usage of spheroids as models for routine experiments. Several methods have been proposed in literature to perform high throughput experiments employing spheroids by automatically computing different morphological parameters, such as diameter, volume and sphericity. Nevertheless, these systems are typically grounded on expensive automated technologies, that make the suggested solutions affordable only for a limited subset of laboratories, frequently performing high content screening analysis. In this work we propose AnaSP, an open source software suitable for automatically estimating several morphological parameters of spheroids, by simply analyzing brightfield images acquired with a standard widefield microscope, also not endowed with a motorized stage. The experiments performed proved sensitivity and precision of the segmentation method proposed, and excellent reliability of AnaSP to compute several morphological parameters of spheroids imaged in different conditions. AnaSP is distributed as an open source software tool. Its modular architecture and graphical user interface make it attractive also for researchers who do not work in areas of computer vision and suitable for both high content screenings and occasional spheroid-based experiments
CellTracker (not only) for dummies
No full textMOTIVATION: Time-lapse experiments play a key role in studying the dynamic behavior of cells. Single-cell tracking is one of the fundamental tools for such analyses. The vast majority of the recently introduced cell tracking methods are limited to fluorescently labeled cells. An equally important limitation is that most software cannot be effectively used by biologists without reasonable expertise in image processing. Here we present CellTracker, a user-friendly open-source software tool for tracking cells imaged with various imaging modalities, including fluorescent, phase contrast and differential interference contrast (DIC) techniques. AVAILABILITY AND IMPLEMENTATION: CellTracker is written in MATLAB (The MathWorks, Inc., USA). It works with Windows, Macintosh and UNIX-based systems. Source code and graphical user interface (GUI) are freely available at: http://celltracker.website/. CONTACT: [email protected] information: Supplementary data are available at Bioinformatics online
MicroMos: an open source software tool to obtain high-resolution panoramic images of 2D cell cultures
No full textOBJECTIVE:
In order to perform significant analyses, the microscope’s user is typically interested in acquiring high-detailed images representing an entire histological sample or well containing cells. Due to the finite size of the camera’s field of view, the microscopist has to find the proper trade-off between higher magnification factor and extension of the observed area.
Here we present MicroMos version 3.0, an open-source tool for building mosaics by stitching together more partially overlapping images. The method proposed is based on visual information only and the mosaics are built by incrementally stitching couples of images. The radiance of the original sample is preserved by compensating the vignetting effect of each stitched image, and the mosaics obtained can be used for quantitative analyses.
By exploiting MicroMos we studied confluence and proliferation of mesenchymal stromal cells (MSC) adherent on OSPROLIFE (Eurocoating, Cirè-Pergine, Italy), a commercial biomaterial in the form of granules. In order to acquire statistically significant data we analysed mosaics built using MicroMos, implementing customized algorithms to segment the MSC and automatically estimate of the percentage of the area of the granules covered by cells.
MATERIALS AND METHODS:
To validate the proposed mosaicing method we performed several experiments under different working conditions. In particular, in order to assess the quality of the mosaics obtained using different warping models and tonal adjustments we used six different sets of images of histological samples and cell cultures. We aligned the images according to different registration models and computed several metrics to estimate which registration method performs as the best.
Then, to study confluence and proliferation of MSC adherent on OSPROLIFE granules, we prepared several samples each containing 50 mg of OSPROLIFE granules and a different number of MSC. Cell confluence was evaluated by adding 4 μM Calcein AM before acquiring sequence of partially overlapping images.
RESULTS:
In our experiments we tested different registration approaches, confirming quite unexpectedly that the translational model does not always act as the best, although the motion of the microscope’ sample holder is apparently translational. Indeed, the sample holder could be slightly inclined, hence yielding non-negligibly affine, or even projective, transformations between subsequent images.
CONCLUSION:
MicroMos version 3.0 is freely distributed as an open source tool, endowed with a graphical user interface, at the website: https://sourceforge.net/p/micromos/. Its usability makes building mosaics of microscope images at subpixel accuracy easier. Furthermore, optional parameters for building mosaics according to different strategies make MicroMos an easy and reliable tool to compare different registration approaches, warping models and tonal corrections. Finally, by analysing mosaics of granules at different time intervals we also obtained quantitative data regarding cell proliferation, confirming that MSC adhere onto the OSPROLIFE granules and proliferate over time
AnaSP: a software suite to automatically analyse spheroid used in high throughput experiments.
No full textCancer multicellular spheroids: Volume assessment from a single 2D projection
No full textVolume is one of the most important features for the characterization of a tumour on a macroscopic scale. It is often used to assess the effectiveness of care treatments, thus mak-ing its correct evaluation a crucial issue for patient care. Similarly, volume is a key feature ona microscopic scale. Multicellular cancer spheroids are 3D tumour models widely employedin pre-clinical studies to test the effects of drugs and radiotherapy treatments. Very fewmethods have been proposed to estimate the tumour volume arising from a 2D projectionof multicellular spheroids, and even fewer have been designed to provide a 3D reconstruc-tion of the tumour shape. In this work, we propose Reconstruction and Visualization from aSingle Projection (ReViSP), an automatic method conceived to reconstruct the 3D surface andestimate the volume of single cancer multicellular spheroids, or even of spheroid cultures.As the input parameter ReViSP requires only one 2D projection, which could be a widefieldmicroscope image. We assessed the effectiveness of our method by comparing it with otherapproaches. To this purpose, we used a new strategy that allowed us to achieve accuratevolume measurements based on the analysis of home-made 3D objects, built by mimickingthe spheroid morphology. The results confirmed the effectiveness of our method for both3D reconstruction and volume assessment. ReViSP software is distributed as an open source tool
Comparison of methods to generate multicellular spheroids with characteristics compliant with 3D high-content screening experiments
No full textOBJECTIVE:
High-content screening (HCS) is a powerful approach to discover new drugs and carry out basic biological research. HCS is widely performed in pharmaceutical and biotechnology industries, but always by using 2D cell cultures. In the literature, there are several papers proving that many cell lines behave differently when cultured as monolayer rather than when cultured in 3D. Accordingly, in vitro-built 3D multicellular spheroids are receiving increased interest with evidence showing that the biological response is closer to that of in vivo cells. Today, more and more biological laboratories are using spheroids to test drugs and radiotherapy treatments. However, a well-established and general purpose validated protocol to generate spheroids with characteristics compliant with 3D HCS experiments does not exist. Here we compared several generation methods with the aim of investigating the possibility to generate in a short time hundreds of homogeneous 3D spheroids, suitable for HCS experiments.
MATERIALS AND METHODS:
We analysed dimension, shape, and homogeneity of Mesenchymal Stromal Cell (MSC) and A549 lung cancer cell spheroids generated by using: (a) a NASA rotatory bioreactor (Synthecon, Houston, USA); (b) several Perfecta3D (3DBiomatrix, Ann Arbor, USA) and GravityPLUS (InSphero, Schlieren, Switzerland) hanging drop plates; (c) magnetic levitation method with magnetic nanoparticle; (d) pellet culture method. To compute the morphological features we used AnaSP (http://sourceforge.net/p/anasp/) and ReVISP (http://sourceforge.net/p/revisp/).
RESULTS:
Spheroids generated by using magnetic levitation and hanging drop plates are quite small (diameter < 500 μm) and irregular (sphericity index < 0.90). Instead, spheroids generated with the NASA bioreactor are irregular, but characterized by a large dimension. In addition, after waiting few days, the so-called spheroidization time, they become much more spherical. Spheroids generated with the pellet culture method have a large dimension and a spherical shape. As regards the number of spheroids generated, magnetic levitation and NASA bioreactor provide an easy solution to generate a number of spheroids compliant with HCS experiments in a very short time. The pellet culture method depends on the number of chambers in the centrifuge, and more rounds are typically required to generate a sufficient number of spheroids. A high number of hanging drop plates has to be used to generate a sufficient amount of spheroids. To generate the spheroids the NASA bioreactor required two weeks, magnetic levitation and hanging drop plates one week, pellet culture method one day.
CONCLUSION:
An absolute best solution does not exist to generate spheroids for HCS experiments. Both NASA bioreactor and pellet culture method seem to be a good choice if a number of large spherical spheroids is required. However, due to the short time needed to generate the spheroids, and the absence of a spheroidization time, the pellet culture method seems to be the best solution at one’s disposal
Shape-based viability of 3D multicellular spheroids
No full textThree-dimensional (3D) multicellular aggregates, typically known as “spheroids”, are used as in vitro model for several applications, ranging from drug testing in case of cancer spheroids, to regenerative medicine applications for Mesenchymal Stromal Cells (MSC) ones. In particular, MSC spheroids have been demonstrated to improve several MSC features, such as multilineage potential, secretion of proangiogenic and chemotactic factors, and resistance against hypoxic condition. Accordingly they represent an extremely promising tool for many clinical applications.
Many approaches have been proposed to generate spheroids and each approach has its own disadvantages. Whichever generation approach is used, a certain degree of shape and volume heterogeneity is always appreciable in the spheroids obtained. Different volumes determine a different viability of the spheroids. In particular, large spheroids (starting from about 500 μm in diameter) are characterized by an external proliferating zone, an internal quiescent zone (caused by limited distribution of oxygen, nutrients and metabolites), and a necrotic core. On the contrary, small spheroids are typically composed by proliferating cells only. It is intuitive understanding that a volume-based preselection is necessary to obtain statistical significant data when the spheroids are used as an in vitro model for drug testing. Less intuitive is understanding that spheroids of same volume, but different shape, are characterized by a different general viability.
In this work we proved that a shape-based preselection of the spheroids used as in vitro model, and not only a volume-based preselection, is necessary to obtain statistical significant data from the experiments performed.
First of all we generated spheroids of MSC and cells of different cancer lineages by using the well-known pellet culture method simply based on a centrifuge used to collapse in a spherical aggregate single cells contained in a tube. Then, we automatically computed several morphological parameters, including the volume of the spheroids obtained, by using the open-source software AnaSP (freely available at http://sourceforge.net/p/anasp/). We selected two groups of 15 spheroids of similar volume, but different sphericity index, and we compared their metabolic activity as an index of viability.
The data obtained by using the luminescence CellTiter-Glo®3D Cell Viability assay (Promega) showed after one week of culture a significantly reduced viability of the group of spherical spheroids with respect to the irregular-shaped group (p <0.05). This was probably due to a reduced distance between each cell and the culture medium interface in the non-spherical subset, leading to a wider zone of active cell proliferation.
In conclusion, the present work highlighted the importance of monitoring the morphological parameters of 3D spheroids, and the necessity of preselecting the spheroids both for volume and shape in order to obtain statistical significant data when the spheroids are used as in vitro model for drug and treatment testing
Over time homogeneity and stability of mesenchymal stromal cells 3D spheroids built using base-level laboratory equipment
No full textMesenchymal Stromal Cells (MSC) are widely used for tissue engineering applications because of their capacity to promote tissue regeneration while modulating immune responses. Three-dimensional (3D) MSC aggregates, also known as multicellular spheroids, better preserve phenotype and innate properties with respect to cells cultured in monolayer. They are the preferred format for many cell therapy and tissue engineering applications, and for bioprinting uses.
Many approaches have been proposed in literature to generate spheroids composed of cancer cells, and most of them can be applied when constructing MSC spheroids. Efficiency, cost, and size of the produced spheroids are typically considered to compare different generation approaches. The pellet culture method provides a cost-effective and extremely rapid method to generate 3D MSC spheroids. Only a benchtop centrifuge and sterile polypropylene conical tubes, typically available in every biological laboratory, are required. However, although the pellet culture method is widely used to generate MSC spheroids, few studies have been performed to analyse the morpho-biological homogeneity and stability during time in culture of the obtained spheroids.
In this study we monitored for two months changes in size and morphology of a set of spheroids generated by the pellet culture method. Each spheroid was imaged with brightfield microscopy twice a week. AnaSP (open-source software freely available at: http://sourceforge.net/p/anasp) was used to compute volume, sphericity and jagging (i.e. indentation degree of the spheroid's border) of each spheroid over time.
The volume of the MSC spheroids showed a decreasing trend. However, just fifteen days from spheroid generation, all the values remained constant and similar, demonstrating good volume stability and homogeneity after an initial settling time. Sphericity of the MSC spheroids kept always high values, thus indicating that the shape is generally close to a perfect sphere. Jagging degree values were always very low, proving that MSC spheroids are characterized by a compact border. We also performed confocal and histological analyses to better investigate their stability. Hematoxylin&Eosin stained cryosections were analysed at different time points to evaluate the internal cellular architecture. Then, the cell viability and distribution on the spheroid surface was evaluated with Live&Dead staining and confocal imaging. No significant changes in internal distribution and spheroid compactness were observed up to 30 days from the spheroids generation.
In conclusion, we can assume that the MSC spheroids generated are homogenous and stable for a long time interval. Accordingly, the proposed enhanced pellet culture method can be used as a reference system for laboratories interested in easily obtaining, without need for specialized equipment, stable homogeneous populations of MSC spheroids to be used in long term studies and applications
Heterogeneous self-tracked health and fitness data integration and sharing according to a linked open data approach
Full text linkThe huge volume of data gathered from wearable fitness devices and wellness appliances, if effectively analysed and integrated, can be exploited to improve clinical decision making and to stimulate promising applications, as they can provide good measures of everyday patient behaviour and lifestyle. However, several obstacles currently limit the true exploitation of these opportunities. In particular, the healthcare landscape is characterised by a pervasive presence of data silos which prevent users and healthcare professionals from obtaining an overall view of the knowledge, mainly due to the lack of device interoperability and data representation format heterogeneity. This work focuses on current, important needs in self-tracked health data modelling, and summarises challenges and opportunities that will characterise the community in the upcoming years. The paper describes a virtually integrated approach using standard Web Semantic technologies and Linked Open Data to cope with heterogeneous health data integration. The proposed approach is verified using data collected from several IoT fitness vendors to form a standard context-aware resource graph, and linking other health ontologies and open projects. We developed a web portal for integrating, sharing and analysing through a customisable dashboard heterogeneous IoT health and fitness data. In this way, we are able to map information onto an integrated domain model by providing support for logical reasoning
Colour deconvolution: stain unmixing in histological imaging
Full text linkMOTIVATION: Microscopy images of stained cells and tissues play a central role in most biomedical experiments and routine histopathology. Storing colour histological images digitally opens the possibility to process numerically colour distribution and intensity to extract quantitative data. Among those numerical procedures is colour deconvolution, which enables decomposing an RGB image into channels representing the optical absorbance and transmittance of the dyes when their RGB representation is known. Consequently, a range of new applications become possible for morphological and histochemical segmentation, automated marker localisation and image enhancement.AVAILABILITY AND IMPLEMENTATION: Colour deconvolution is presented here in two open-source forms: a MATLAB program/function and an ImageJ plugin written in Java. Both versions run in Windows, Macintosh, and UNIX-based systems under the respective platforms. Source code and further documentation are available at: https://blog.bham.ac.uk/intellimic/g-landini-software/colour-deconvolution-2/.SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online
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