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Quantitative microscopy using 3D
multicellular spheroids:
generation, imaging, and analysis
Seoul, South Korea
27th August 2016 – 03rd September 2016
Eng. Filippo Piccinini, PhD
University of Bologna, Italy
Filippo Piccinini - details
First Name, Surname Filippo Piccinini
Place of birth Forlimpopoli, FC, Italy
Date of birth April 20, 1985
Bachelor degree Biomedical Engineer, University of Bologna, September 2004 - July 2007, score: 110/110 cum LAUDE
Master degree Biomedical Engineer, University of Bologna September 2007 - October 2009, score: 110/110 cum LAUDE
PhD degree European Doctorate in Information Technology
Email f.piccinini@unibo.it
Italian mobile +39 3495000398
Website www.filippopiccinini.it
Current position
Post Doc Research Fellow, Advanced Research Centre on Electronic Systems (ARCES)
Computer Vision Group (CVG) University of Bologna, Italy
Supervisor Prof. Alessandro Bevilacqua
Where people typically think Italian researchers work Where my mother thinks I typically work
University of Bologna - Where I work Where I work - University of Bologna
2
Where I work - University of Bologna
UNIVERSITY OF
BOLOGNA
Computer Vision Group – Research interests
IMAGE PROCESSING AND ANALYSIS
Outdoor imaging Aerospace imaging Biomedical imaging
Filippo Piccinini, memberships
Italian Mesenchymal Stem Cell Group (GISM), www.gismonline.it, Founder Member
Italian Society of Biochemistry and Molecular Biology (SIB), www.biochimica.it
Italian National Bioengineering Group (GNB), www.bioing.it
European Association for Cancer Research (EACR), www.eacr.org
European Light Microscopy Initiative (ELMI), http://elmi.embl.org/home/
Network of European Bioimage Analysts (NEUBIAS), http://eubias.org/neubias/
Filippo Piccinini, main foreign collaborators
Prof. Peter Horvath, Biological Research Centre (BRC), Hungarian Academy of Sciences, Szeged, Hungary.
Prof. Kevin Smith, KTH Royal Institute of Technology, School of Computer Science and Communication, Stockholm, Sweden.
Dr. Gábor Csúcs, Light Microscopy and Screening Center, Swiss Federal Institute of Technology, ETH Zurich, Switzerland.
Prof. Valérie Vilgrain, Department of Radiology, University Beaujon Hospital, Paris-Clichy, France.
Dr. Vilja Pietiainen, Institute for Molecular Medicine Finland, University of Helsinki, Helsinki, Finland.
Filippo Piccinini, developed software tools
CellTracker, for tracking cells cultured in vitro http://celltracker.website/
Advanced Cell Classifier, for classifying cells in high-content screening images http://www.cellclassifier.org/
MicroMos, for building a panorama, starting from a set of overlapping images
http://www.filippopiccinini.it/Mosaicing/index.html
AnaSP, software suite to segment brightfield images of multicellular spheroids http://sourceforge.net/projects/anasp/
ReViSP, for volume estimation and 3D rendering of multicellular spheroids http://sourceforge.net/projects/revisp/
CIDRE, for correcting the illumination field of microscopy images http://www.nature.com/nmeth/journal/v12/n5/full/nmeth.3323.html
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Filippo Piccinini, publications
14) F. Piccinini, A. Tesei, A. Bevilacqua, Single-image based methods used for non-invasive volume estimation of cancer
spheroids: a practical assessing approach based on entry-level equipment. Computer Methods and Programs in Biomedicine,
advanced online publication, July 2016
13) C. Bellotti, S. Duchi, A. Bevilacqua, E. Lucarelli, F. Piccinini, Long term morphological characterization of Mesenchymal
Stromal Cells 3D spheroids built with a rapid method based on entry-level equipment. Cytotechnology, advanced online
publication, March 2016
12) M. Zanoni, F. Piccinini, C. Arienti, A. Zamagni, S. Santi, R. Polico, A. Bevilacqua, A. Tesei, 3D tumor spheroid models for in
vitro therapeutic screening: a systematic approach to enhance the biological relevance of data obtained. Scientific Reports, 6:
19103, January 2016
11) F. Piccinini, A. Kiss, P. Horvath, CellTracker (not only) for dummies. Bioinformatics, 32(6): 955–957, March 2016
10) K. Smith, Y. Li, F. Piccinini, G. Csucs, C. Balazs, A. Bevilacqua, P. Horvath, CIDRE: an illumination-correction method for
optical microscopy. Nature Methods, 12(5): 404–406, May 2015
STATISTICS
Peer reviewed scientific articles: 22
- Journal publications: 14
- Conference proceedings: 8 First author publications: 10
Last author publications: 2
Total impact: 56.4050 IF Average impact: 4.3388 IF
Total number of citations: 170
H-index: 8
KEYWORDS
3D cell cultures
Light –sheet microscopy
Open-source software tools Mesenchymal stromal cells
Cell tracking
Cell classification
Filippo Piccinini, publications
09) F. Piccinini, AnaSP: a software suite for automatic image analysis of multicellular spheroids. Computer Methods and
Programs in Biomedicine, 119(1): 43–52, April 2015
08) F. Piccinini, A. Tesei, C. Arienti, A. Bevilacqua, Cancer multicellular spheroids: Volume assessment from a single 2D
projection. Computer Methods and Programs in Biomedicine, 118(2):95–106, February 2015
07) F. Piccinini, A. Tesei, G. Paganelli, W. Zoli, A. Bevilacqua, Improving reliability of live/dead cell counting through automated
image mosaicing. Computer Methods and Programs in Biomedicine, 117(3):448-463, December 2014
06) F. Piccinini, M. Pierini, E. Lucarelli, A. Bevilacqua, Semi-quantitative monitoring of confluence of adherent mesenchymal
stromal cells on calcium-phosphate granules by using widefield microscopy images. Journal of Materials Science: Materials in
Medicine, 25(10):2395-2410, October 2014
05) F. Piccinini, E. Lucarelli, A. Gherardi and A. Bevilacqua, Automated image mosaics by non-automated light microscopes: the
MicroMos software tool. Journal of Microscopy, 252(3):226-250, December 2013
04) Z. Bulj, S. Duchi, A. Bevilacqua, A. Gherardi, B. Dozza, F. Piccinini, G. A. Mariani, E. Lucarelli, S. Giannini, D. Donati and S.
Marmiroli, Protein kinase B/AKT isoform 2 drives migration of human mesenchymal stem cells. International Journal of
Oncology, 42(1):118-126, January 2013
03) F. Piccinini, A. Tesei, W. Zoli and A. Bevilacqua, Extending the Universal Quality Index to assess N-image fusion in light
microscopy. International Journal of Bioelectromagnetism, 14(4):217-222, December 2012
02) F. Piccinini, A. Tesei, W. Zoli and A. Bevilacqua, Extended depth of focus in optical microscopy: assessment of existing
methods and a new proposal. Microscopy Research and Technique, 15(11):1582-1592, November 2012
01) F. Piccinini, E. Lucarelli, A. Gherardi and A. Bevilacqua, Multi-image based method to correct vignetting effect in light
microscopy images. Journal of Microscopy, 248(1):6-22, October 2012
Outline
Spheroid generation
Pellet culture method
ReViSP and AnaSP
CellTracker
Advanced Cell Classifier
New project
Outline
Spheroid generation
Pellet culture method
ReViSP and AnaSP
CellTracker
Advanced Cell Classifier
New project
SPHEROID
“3D multicellular aggregate built in
vitro and used as a model for testing
drugs and radiotherapy treatments”
“In 1970 Sutherland proposed multicellular aggregates of "spherical" shape, called
spheroids, as a reliable 3D tumour model grown in vitro. These multicellular
aggregates morphologically resemble nodules seen in animal and human
carcinomas, and this is the reason behind the name spheroids.”
Cell culture: 3D VS 2D
3D CELL CULTURE 2D CELL CULTURE
Physiologic cell-cell contact Cell-cell contact only on the cell edges, and cells mostly in contact with plastic
Cells interact with extracellular matrix Cells contact extracellular matrix mostly on one surface
Diffusion gradient of drugs, gases, nutrients, and waste
No gradients present
Co-culture of multiple cells mimics microenvironment
Co-cultures unable to establish a microenvironment
MORE SIMILAR TO
IN VIVO TUMOURS
4
Spheroid placed in low-attachment multi-well plates
to test drug dosages or radiotherapy treatments
Plate preparation
Phantoms for linear accelerator
Round-bottom 96-well
low attachment plates
Oncology drug collection
Institute for Molecular Medicine Finland (FIMM) drug collection,
composed by 393 approved
and emerging investigational
oncology drugs.
PROBLEM
A cell-culture model used for testing drugs and radiotherapy treatments, in
replicates (for instance using several wells of a multi-well plate), must be
homogeneous and stable over time. Otherwise the data obtained are not reliable
and are dependent by the original status of the cells
HOW TO GENERATE HOMOGENEOUS AND
STABLE 3D SPHEROIDS?
DAY07, spherical shape DAY07, irregular shape
M. Zanoni, F. Piccinini, C. Arienti, A. Zamagni, S. Santi, R. Polico, A. Bevilacqua, A. Tesei, 3D tumor spheroid models for in vitro
therapeutic screening: a systematic approach to enhance the biological relevance of data obtained. Scientific Reports, 6: 19103,
January 2016
Courtesy by 3DBiomatrix Courtesy by Synthecon Inc.
Common systems to build spheroids
Antigravity bioreactor Hanging drop plates
Courtesy by Hamilton
Magnetic levitator Pellet culture method
Comparison of systems to build spheroids
What do you want?
Parameters:
Number of homogeneous spheroids needed (High-content screening?)
Size of the necrotic core (Presence of a magnetic bead?)
Shape of the spheroids (Spherical? Irregular?)
Diameter of the spheroids (100µm or 1 mm?)
Parameters cell-line
dependent
Comparison of systems to build spheroids
What do you want?
Parameters:
Number of homogeneous spheroids needed (High-content screening?)
Size of the necrotic core (Presence of a magnetic bead?)
Shape of the spheroids (Spherical? Irregular?)
Diameter of the spheroids (100µm or 1 mm?)
Parameters
cell-line
dependent
Cell used:
A549 lung
cancer
TIME
REQUIRED
[days]
NO. CELL
REQUIRED
(×106)
EQUIVALENT DIAMETER
[µm]
(range, mean±SD, CV, n)
AMOUNT OF SPHERICAL
SPHEROIDS
(SI ≥ 0.90)
AMOUNT OF
LARGE SPHEROIDS
(diameter > 500 μm)
NASA
BIOREACTOR 15 40 500–1100, 897±98, 11.0, 192 HIGH HIGH
HANGING DROP
PLATES 7 0.5 200–500, 359±95, 26.5, 38 LOW LOW
PELLET CULTURE
METHOD 1 20 800–900, 880±21, 2.4, 20 HIGH HIGH
MAGNETIC
LEVITATION 7 0.5 200–500, 347±87, 25.1, 28 LOW LOW
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Correlation between viability and shape
We hypothesized that the 3D shape reflects a different general viability of
the spheroids. To better investigate
this correlation, we selected 30
spheroids of similar volumes (0.112 ±
0.013 mm3) but belonging to the spherical (n = 15; sphericity ≥ 0.90) or
non-spherical subtypes (n = 15;
sphericity < 0.90) to analyze how
different shapes influence the
metabolic state of spheroids. The data obtained from the luminescence
metabolic assay performed after one
week of culture showed a significantly
reduced viability of spherical spheroids
with respect to the irregular-shaped group (P = 0.045). 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
200 µm = maximum distance from border of the proliferating cells
Spherical shape
Ellipsoidal shape
Systems to build spheroids
What do you want?
High number of spheroids of same volume, large dimension, and spherical shape.
Cell used:
A549 lung
cancer
TIME
REQUIRED
[days]
NO. CELL
REQUIRED
(×106)
EQUIVALENT DIAMETER
[µm]
(range, mean±SD, CV, n)
AMOUNT OF SPHERICAL
SPHEROIDS
(SI ≥ 0.90)
AMOUNT OF
LARGE SPHEROIDS
(diameter > 500 μm)
NASA
BIOREACTOR 15 40 500–1100, 897±98, 11.0, 192 HIGH HIGH
HANGING DROP
PLATES 7 0.5 200–500, 359±95, 26.5, 38 LOW LOW
PELLET CULTURE
METHOD 1 20 800–900, 880±21, 2.4, 20 HIGH HIGH
MAGNETIC
LEVITATION 7 0.5 200–500, 347±87, 25.1, 28 LOW LOW
Systems to build spheroids
What do you want?
High number of spheroids of same volume, large dimension, and spherical shape.
Cell used:
A549 lung
cancer
TIME
REQUIRED
[days]
NO. CELL
REQUIRED
(×106)
EQUIVALENT DIAMETER
[µm]
(range, mean±SD, CV, n)
AMOUNT OF SPHERICAL
SPHEROIDS
(SI ≥ 0.90)
AMOUNT OF
LARGE SPHEROIDS
(diameter > 500 μm)
NASA
BIOREACTOR 15 40 500–1100, 897±98, 11.0, 192 HIGH HIGH
HANGING DROP
PLATES 7 0.5 200–500, 359±95, 26.5, 38 LOW LOW
PELLET CULTURE
METHOD 1 20 800–900, 880±21, 2.4, 20 HIGH HIGH
MAGNETIC
LEVITATION 7 0.5 200–500, 347±87, 25.1, 28 LOW LOW
Systems to build spheroids
What do you want?
High number of spheroids of same volume, large dimension, and spherical shape.
Cell used:
A549 lung
cancer
TIME
REQUIRED
[days]
NO. CELL
REQUIRED
(×106)
EQUIVALENT DIAMETER
[µm]
(range, mean±SD, CV, n)
AMOUNT OF SPHERICAL
SPHEROIDS
(SI ≥ 0.90)
AMOUNT OF
LARGE SPHEROIDS
(diameter > 500 μm)
NASA
BIOREACTOR 15 40 500–1100, 897±98, 11.0, 192 HIGH HIGH
HANGING DROP
PLATES 7 0.5 200–500, 359±95, 26.5, 38 LOW LOW
PELLET CULTURE
METHOD 1 20 800–900, 880±21, 2.4, 20 HIGH HIGH
MAGNETIC
LEVITATION 7 0.5 200–500, 347±87, 25.1, 28 LOW LOW
Conclusion – Morphological pre-selection
A morphological pre-selection of the spheroids, based on volume and sphericity is needed to obtain reliable data when using spheroids as in vitro models
F. Piccinini, AnaSP: a software suite for automatic image analysis of multicellular
spheroids. Computer Methods and Programs in Biomedicine, 119(1):2015.
Outline
Spheroid generation
Pellet culture method
ReViSP and AnaSP
CellTracker
Advanced Cell Classifier
New project
6
C. Bellotti, S. Duchi, A. Bevilacqua, E. Lucarelli, F. Piccinini, Long term morphological characterization of Mesenchymal Stromal
Cells 3D spheroids built with a rapid method based on entry-level equipment. Cytotechnology, advanced online publication,
March 2016 Pellet culture method
Cost-effective and extremely rapid method to generate MSC spheroids proposed by Johnstone et al. (In vitro chondrogenesis of bone marrow-derived mesenchymal
progenitor cells. Exp Cell Res, 1998).
It only requires a benchtop centrifuge and sterile polypropylene conical tubes
The generation efficiency is particularly high (one spheroid per tube) and the size of the spheroids can be tuned by controlling the number of cells seeded in the tubes.
Spheroid homogeneity
Cost of the system
Production efficiency
Tunable spheroid size
Pellet culture method
SPHEROIDS PRODUCTION
2.5×105 cells suspended in 0.5 ml DMEM-HG supplemented with 10% FBS and placed in a 1.5 mL polypropylene conical tube with a screw cap.
Aliquots were spun in a benchtop centrifuge at 500 g for 5 min.
Tubes incubated in humidified atmosphere at 37 °C with 5% CO2, with loosened caps to ensure adequate gas exchange.
After 72 h the pellets become compact spherical aggregates.
0.5 mm
Brightfield image acquisition
Plate preparation
Image acquisition
Original Corrected (*)
(*) K. Smith, Y. Li, F. Piccinini, et al., CIDRE: an illumination-correction
method for optical microscopy. Nature Methods, 12(5):2015
Illuminationcorrection
K. Smith, Y. Li, F. Piccinini, G. Csucs, C. Balazs, A. Bevilacqua, P. Horvath, CIDRE: an illumination-correction method for optical
microscopy. Nature Methods, 12(5): 404–406, May 2015
AnaSP: software suite to analyse several features
AnaSP (ANAlysis of SPheroid) SPHEROID SEGMENTATION
MORPHOLOGICAL FEATURES COMPUTATION
Software freely available at: http://sourceforge.net/p/anasp
F. Piccinini, AnaSP: a software suite for automatic image analysis of multicellular
spheroids. Computer Methods and Programs in Biomedicine, 119(1):2015.
7
Volume, Sphericity, and Jagging degree
25 MSC SPHEROIDS ANALYSED FOR A TWO-MONTH PERIOD
The intervals within the bounds represent µ ± 2σ
Volume, Sphericity, and Jagging degree
25 MSC SPHEROIDS ANALYSED FOR A TWO-MONTH PERIOD
The intervals within the bounds represent µ ± 2σ
External and internal architecture analysis
Cryostat Leica CM 1900
HISTOLOGICAL ANALYSIS CONFOCAL ANALYSIS
Nikon Eclipse Ti microscope equipped with an A1R confocal laser
External and internal architecture analysis
LIGHT SHEET MICROSCOPE
Zeiss Light Sheet v2.1
PERFECTA3D and PROMEGA assays
External and internal architecture analysis
HISTOLOGICAL ANALYSIS (H&E staining)
CONFOCAL ANALYSIS (Live&Dead assay)
Conclusion
The pellet culture method can be used efficiently to obtain homogenous
MSC spheroids with a high sphericity and a “smooth” surface
The MSC spheroids produced are morpho-biologically stable for at least
fifteen days after two weeks from generation
The two main outcomes of this work are showing that:
This is the first long-term analysis providing morphological and
biological data of MSC spheroids maintained in culture for two months
The pellet culture method can be used as a reference for laboratories
interested in easily obtaining stable homogeneous populations of MSC
spheroids without having to use specialized equipment
8
Outline
Spheroid generation
Pellet culture method
ReViSP and AnaSP
CellTracker
Advanced Cell Classifier
New project
F. Piccinini, A. Tesei, C. Arienti, A. Bevilacqua, Cancer multicellular spheroids: Volume assessment from a single 2D projection.
Computer Methods and Programs in Biomedicine, 118(2):95–106, February 2015
F. Piccinini, AnaSP: a software suite for automatic image analysis of multicellular spheroids. Computer Methods and Programs
in Biomedicine, 119(1): 43–52, April 2015
“Spheroid shape and volume are
relevant features for the data
reliability when spheroids are
used as an in vitro model”
State-of-the-art approaches for volume estimation
MODEL ASSUMPTIONS
Local spherical symmetry
Force of gravity
FORMULAS TYPICALLY USED
State-of-the-art approaches for volume estimation
MODEL ASSUMPTIONS
Local spherical symmetry
Force of gravity
FORMULAS TYPICALLY USED
SPHERE method
ELLIPSOID method
9
Methods from other application fields
SIERACKI method
M.E. Sieracki, C.L. Viles, K.L. Webb, “Algorithm to estimate cell
biovolume using image analyzed microscopy”, Cytometry Part A
10(5):551-557, 1989
ZEDER method
M. Zeder, E. Kohler, L. Zeder, J. Pernthaler, “A novel algorithm for
the determination of bacterial cell volumes that is unbiased by cell
morphology”, Microscopy and Microanalysis 17(5):799-809, 2011
Our approach
3D RECONSTRUCTION METHOD
Spheroid
segmentation
3D map
creation
Protuberance
segmentation
Surface
visualization
Surface
quantization
Image
preprocessing
Our approach
3D RECONSTRUCTION METHOD
DEPTH-OF-FOCUS RECONSTRUCTION
Spheroid
segmentation
3D map
creation
Protuberance
segmentation
Surface
visualization
Surface
quantization
Image
preprocessing
Input Output Input
Output
VIGNETTING CORRECTION
Our approach
3D RECONSTRUCTION METHOD
SPHEROID SEGMENTATION
Spheroid
segmentation
3D map
creation
Protuberance
segmentation
Surface
visualization
Surface
quantization
Image
preprocessing
Our approach
3D RECONSTRUCTION METHOD
PROTUBERANCE SEGMENTATION
Spheroid
segmentation
3D map
creation
Protuberance
segmentation
Surface
visualization
Surface
quantization
Image
preprocessing
Our approach
3D RECONSTRUCTION METHOD
3D MAP CREATION
Spheroid
segmentation
3D map
creation
Protuberance
segmentation
Surface
visualization
Surface
quantization
Image
preprocessing
10
Our approach
3D RECONSTRUCTION METHOD
INTERCONNECTING THE 3D PARTS
Single connection Parts interconnected with cylinders
Spheroid
segmentation
3D map
creation
Protuberance
segmentation
Surface
visualization
Surface
quantization
Image
preprocessing
Our approach
3D RECONSTRUCTION METHOD
SURFACE QUANTIZATION
Spheroid
segmentation
3D map
creation
Protuberance
segmentation
Surface
visualization
Surface
quantization
Image
preprocessing
Analysis of a culture of spheroids
Mosaicing technique to acquire a high-detailed image of an entire well
F. Piccinini, A. Bevilacqua, E. Lucarelli, Automated image mosaics by non-automated light microscopes: the MicroMos software tool. Journal of Microscopy, 252(3):226–250, 2013.
Analysis of a culture of spheroids
Mosaicing technique to acquire a high-detailed image of an entire well
F. Piccinini, A. Bevilacqua, E. Lucarelli, Automated image mosaics by non-automated light microscopes: the MicroMos software tool. Journal of Microscopy, 252(3):226–250, 2013.
F. Piccinini, E. Lucarelli, A. Gherardi and A. Bevilacqua, Automated image mosaics by non-automated light microscopes: the
MicroMos software tool. Journal of Microscopy, 252(3):226-250, December 2013
Analysis of a culture of spheroids
3D reconstruction of all the spheroids
Volume of the single spheroids
INPUT
OUTPUT
11
ReViSP software tool
Reconstruction and Visualization
from a Single Projection (ReViSP)
Microscope image GUI of ReViSP 3D mesh of the spheroid
ReViSP does not require any prior information except the binary mask of the spheroid to be reconstructed
ReViSP software tool
Reconstruction and Visualization
from a Single Projection (ReViSP)
Microscope image GUI of ReViSP 3D mesh of the spheroid
ReViSP does not require any prior information except the binary mask of the spheroid to be reconstructed
ReViSP live demo
Software freely available at: http://sourceforge.net/p/revisp
The ground truth volume of a spheroid
cannot be easily computed
problem
Ground truth volume
C: CONVERSION COEFFICIENT (voxels/mm3)
B: DENSITY
A: VOLUME BY OBJECT WEIGHTING
Sample object
Precision balance Graduated cylinder
Camera calibration grid
Synthetic objects (Group1)
SPHERICAL
OVOID
EIGHT
IRREGULAR
12
Synthetic objects (Group2 with protuberances)
ONE PROTUBERANCE TWO PROTUBERANCES THREE PROTUBERANCES
Volume assessment: Experimental results
Absolute percentage error, objects Group1
SPHERE ELLIPSOID SIERACKI ZEDER ReViSP
Spherical 0.69 1.99 1.09 0.74 1.26
Ovoid 23.97 8.15 2.45 2.01 2.08
Eight 21.91 33.12 4.34 4.96 1.65
Irregular 9.00 16.10 3.79 7.34 3.55
Average E% 13.89 14.84 2.92 3.76 2.14
Absolute percentage error, objects Group2
SPHERE ELLIPSOID SIERACKI ZEDER ReViSP
One protuberance 17.89 23.03 5.28 7.16 1.02
Two protuberances 11.79 6.45 13.77 9.35 5.89
Three protuberances 19.23 162.96 37.88 10.66 6.55
Average E% 16.30 64.15 18.98 9.06 4.49
Conclusion
Proved that common methods used to estimate volume of
spheroids are characterized by high errors.
Provided an approach conceived for 3D reconstruction and
visualization of a culture of spheroids.
Proposed a perspective gold standard method to estimate
the volume of a spheroid.
The main outcomes of ReViSP are:
Software freely available at: http://sourceforge.net/p/revisp
AnaSP: software suite to analyse several features
Rembrandt, 1629 Rembrandt, 1634 Rembrandt, 1640 Rembrandt, 1660
DAY08 DAY15 DAY22 DAY35 DAY01
15th July 1606
DAY08
AnaSP: software suite to analyse several features
AnaSP GUI
SPHEROID SEGMENTATION
MORPHOLOGICAL FEATURES COMPUTATION
Software freely available at: http://sourceforge.net/p/anasp
AnaSP: software suite to analyse several features
13
Outline
Spheroid generation
Pellet culture method
ReViSP and AnaSP
CellTracker
Advanced Cell Classifier
New project
F. Piccinini, A. Kiss, P. Horvath, CellTracker (not only) for dummies. Bioinformatics, 32(6): 955–957, March 2016
F. Piccinini, A. Kiss, P. Horvath, CellTracker (not only) for dummies. Bioinformatics, 32(6): 955–957, March 2016
CellTracker , freely available open-source software tool
http://celltracker.website/ ~1500 visitors a month!!!!!!!!!!!
CellTracker software ~200 download a month!!!!!!!!!!!!!!!
CellTracker GUI
CellTracker statistics
Outline
Spheroid generation
Pellet culture method
ReViSP and AnaSP
CellTracker
Advanced Cell Classifier
New project
Advanced Cell Classifier
Advanced Cell Classifier,
an open source software for automatically classifying
cells in high-content screening images
14
Advanced Cell Classifier
GRAPHICAL USER INTERFACE
Advanced Cell Classifier
CELL PHENOTYPES
Outline
Spheroid generation
Pellet culture method
ReViSP and AnaSP
CellTracker
Advanced Cell Classifier
New project
High-content screening in 3D
Generation of homogeneous and stable cancer spheroids to be used
as 3D tumour models.
Developments of automated image-processing methods and
machine learning approaches enabling cell phenotypic identification.
Validation of an approach to isolate single cells inside a 3D spheroid
by using a laser micro-dissector.
“[…] Recent improvements of computational capacity and automated
microscopy paved a new era in HCS, the 3D one […]”
SPECIFIC GOALS
OVERALL OBJECTIVE
develop computational and assay automation methods to perform 3D HCS
High-content screening in 3D
OVERVIEW
15
High-content screening in 3D
SPHEROID GENERATION
Anna Tesei, Oncology Research Hospital, Meldola, Italy
Laszlo Puskas, AVIDIN company, Szeged, Hungary
HIGH-CONTENT SCREENING
Leo Prince, OcellO company, Leiden, Netherlands
Main activities and collaborators
LIGHT-SHEET MICROSCOPY ANALYSES
Spartaco Santi, Digital Microscopy Center, Bologna, Italy
DRUG TESTING
Vilja Pietiainen, Institute for Molecular Medicine Finland, Helsinki, Finland
SOFTWARE DEVELOPMENT
Peter Horvath, Biological Research Center, Szeged, Hungary
Alessandro Bevilacqua, University of Bologna, Italy
SINGLE-CELL ISOLATION
Peter Horvath, Biological Research Center, Szeged, Hungary
High-content screening in 3D
Final dream?
Establishing a facility to perform 3D high-content screening analyses,
including spheroid generation, image acquisition, and cell classification
WHY AM I HERE IN SEOUL?
To see if there are opportunities for me,
new collaborations,
and to meet other researchers working in the field!
Advanced Research Center on Electronic Systems (ARCES), Computer Vision Group
(CVG), University of Bologna, Italy
Prof. Alessandro Bevilacqua
Dr. Alessandro Gherardi
Silvia Malavasi
Serena Baiocco
Biological Image Analysis and Machine Learning Group (BIOMAG), Biological
Research Center, Szeged, Hungary
Prof. Peter Horvath
Tamas Balassa
Abel Szkalisity
Csaba Molnar
Krisztian Koos
Arpad Balin
Biosciences Laboratory, Istituto Scientifico Romagnolo per lo Studio e la Cura dei
Tumori (IRST) IRCCS, Meldola, Italy.
Dr. Anna Tesei
Dr. Chiara Arienti
Michele Zanoni
Alice Zamagni
Orthopaedic Pathology and Osteoarticular Tissue Regeneration / Digital Microscopy
Center, Rizzoli Orthopaedic Institute, Italy
Dr. Enrico Lucarelli
Dr. Spartaco Santi
Dr. Barbara Dozza
Dr. Serena Duchi
THANK YOU
Dr. Chiara Bellotti
Dr. Elisa Martella
Other colleagues
Prof. Tullio Salmon Cinotti, Bologna, Italy
Prof. Kevin Smith, Stockholm, Sweden
Prof. Laszlo Puskas, Szeged, Hungary
Dr. Gabor Csucs, ETH Zurich, Switzerland
Dr. Vilja Pietiainen, Helsinki, Finland
Dr. Lassi Paavolainen, Helsinki, Finland
Acknowledgments THANK YOU
Eng. Filippo Piccinini PhD, www.filippopiccinini.it
Email: f.piccinini@unibo.it
Mobile: +39 3495000398
Skype: filippo.piccinini85
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