AnaSP: A software suite for automatic image analysis of multicellular spheroids

Piccinini, Filippo

doi:10.1016/j.cmpb.2015.02.006

Cited by 59 publications

(54 citation statements)

References 50 publications

(56 reference statements)

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“…First of all, the acquired images were flat-field corrected for inhomogeneous illumination intensity (Smith et al 2015). Then, all the images were segmented and analysed by using AnaSP, the open-source software described in Piccinini (2015), freely available at: http://sourceforge. net/p/anasp.…”

Section: Msc Isolation and Culturementioning

confidence: 99%

Long term morphological characterization of mesenchymal stromal cells 3D spheroids built with a rapid method based on entry-level equipment

et al. 2016

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Three-dimensional (3D) spheroids of mesenchymal stromal cells (MSC) have been demonstrated to improve a wide range of MSC features, such as multilineage potential, secretion of therapeutic factors, and resistance against hypoxic condition. Accordingly, they represent a promising tool in regenerative medicine for several biological and clinical applications. Many approaches have been proposed to generate MSC spheroids. They usually require specific generation systems, such as rotatory bioreactors or low-attachment plates, and each approach has its own disadvantages. Furthermore, an over-time analysis of morphological homogeneity and architectural stability of the spheroids generated is rarely provided. In this work we adapted the ''pellet culture'' method to obtain homogenous spheroids of MSC and maintain them in vitro for long term studies. We analysed their outer and inner structure over a 2-month period to provide morphological and architectural information regarding the spheroids generated. Quantitative and qualitative data were obtained using brightfield and confocal microscope imaging coupled to a computational analysis to estimate volume, sphericity, and jagging degree. In addition, histological evaluation was performed to more thoroughly assess the cellular composition and the internal architecture of the 3D spheroids. The results provided show that MSC spheroids generated with the proposed approach are homogeneous and stable, from both morphological and architectural points of view, for a period of at least 15 days, approximately between day 15 and day 30 after their generation. Accordingly, the approach proposed serves as a rapid, cost-effective, and efficient method to generate and maintain MSC spheroids using common entry-level laboratory equipment only.Keywords Mesenchymal stromal/stem cells Á 3D multicellular spheroids Á Pellet culture method Á Morphological analysis Á Microscopy Á Computational analysis Chiara Bellotti and Serena Duchi contributed equally to this work.

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Section: Msc Isolation and Culturementioning

confidence: 99%

Long term morphological characterization of mesenchymal stromal cells 3D spheroids built with a rapid method based on entry-level equipment

et al. 2016

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“…Software for spheroid image analysis has largely focused on static images generated by high throughput screening [4][5][6][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27]. Existing software programs for analyzing spheroid imaging are described in Table 1.…”

Section: Introductionmentioning

confidence: 99%

TASI: A software tool for spatial-temporal quantification of tumor spheroid dynamics

Konen

Brat

Marcus

et al. 2017

Preprint

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Spheroid cultures derived from explanted cancer specimens are an increasingly utilized resource for studying complex biological processes like tumor cell invasion and metastasis, representing an important bridge between the simplicity and practicality of 2D monolayer cultures and the complexity and realism of in vivo animal models. Temporal imaging of spheroids can capture the dynamics of cell behaviors and microenvironments, and when combined with quantitative image analysis methods, enables deep interrogation of biological mechanisms. This paper presents a comprehensive open-source software framework for Temporal Analysis of Spheroid Imaging (TASI) that allows investigators to objectively characterize spheroid growth and invasion dynamics. TASI performs spatiotemporal segmentation of spheroid cultures, extraction of features describing spheroid morpho-phenotypes, mathematical modeling of spheroid dynamics, and statistical comparisons of experimental conditions. We demonstrate the utility of this tool in an analysis of non-small cell lung cancer spheroids that exhibit variability in metastatic and proliferative behaviors. INTRODUCTION3D spheroid models of cancer have been widely used to investigate mechanisms of invasion and metastasis [1][2][3][4] and the impact of drugs on metastatic potential [5][6][7]. Spheroid cultures help to bridge the gap between simplistic 2D in vitro cultures and complex in vivo mouse models, and have been used to study complex biological processes that are strongly coupled to tissue microenvironments. Cell-cell and cell-matrix interactions in spheroid cultures are more similar to animal models and human disease than 2D in vitro models, yet spheroids can be grown rapidly, are relatively inexpensive and are easier to image than in vivo models. The relative ease in imaging spheroid models makes them especially amenable to investigating temporal processes where dynamic behaviors and interactions can be captured. Metastatic and invasive processes are fundamentally dynamic, and temporal imaging of spheroids can provide important insights into how cancer cells divide [8], invade, and metastasize [1,5,9,10]. For example, measuring growth kinetics of tumor spheroids has been used for anti-cancer drug screening [5,7]. Co-culturing of multiple cell types in 3D spheroids has also been used to investigate cell-cell interactions in microenvironments [11,12].Software for spheroid image analysis has largely focused on static images generated by high throughput screening [4][5][6][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27]. Existing software programs for analyzing spheroid imaging are described in Table 1. Software for measuring spheroid dynamics has received relatively less attention [4,13,[23][24][25][26][27][28][29]. An interactive system for segmenting and measuring spheroid volume and dimensions was developed in [26]. Of the software packages available for spheroid analysis, none provides end-to-end statistical analysis and visualization of imaging measurements. The prim...

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“…ReViMS provides several automatic and manual tools for segmenting z - stacks of fluorescence images (3,12). To analyze spheroids, an automatic method was employed based on a fixed threshold determined by averaging the values obtained according to the Otsu and Triangle histogram segmentation methods (Supplementary Material Part 2).…”

mentioning

confidence: 99%

“…Today, the use of cancer spheroids as 3-D in vitro models is an established methodology. Several morphological and biological features of cells in culture can be automatically determined using publicly available software (3). However, the volume of the tumor spheroids before and after treatmentone of their most important characteristicsis challenging to estimate (4).…”

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confidence: 99%

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ReViMS: Software Tool for Estimating the Volumes of 3-D Multicellular Spheroids Imaged Using a Light Sheet Fluorescence Microscope

et al. 2017

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Cancer 3-D spheroids are widely used to test drugs and radiotherapy treatments. These 3-D cell clusters range from tens to hundreds of micrometers in size, with shapes that typically differ from a perfect sphere. Change in spheroid volume is one of the most important parameters for evaluating treatment efficacy, and using light sheet fluorescence microscopes (LSFM), optical sections of samples in that size range can be obtained. However, there remains a lack of validated methods for quantifying the volumes of 3-D multicellular aggregates. Here, we present Reconstruction and Visualization from Multiple Sections (ReViMS), an open-source, user-friendly software for automatically segmenting z-stacks of fluorescence images and estimating the volumes of 3-D multicellular spheroids. To assess the precision and accuracy of the volume estimates obtained with ReViMS, we used several cancer spheroids imaged with LSFM. Both the precision and accuracy were >95%, demonstrating the effectiveness of ReViMS.

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AnaSP: A software suite for automatic image analysis of multicellular spheroids

Cited by 59 publications

References 50 publications

Long term morphological characterization of mesenchymal stromal cells 3D spheroids built with a rapid method based on entry-level equipment

Long term morphological characterization of mesenchymal stromal cells 3D spheroids built with a rapid method based on entry-level equipment

TASI: A software tool for spatial-temporal quantification of tumor spheroid dynamics

ReViMS: Software Tool for Estimating the Volumes of 3-D Multicellular Spheroids Imaged Using a Light Sheet Fluorescence Microscope

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