2016 INCUCYTE™ LIVE -CELL ANALYSIS WEBCASTS · PDF fileResolving the Changing Needs of...
Transcript of 2016 INCUCYTE™ LIVE -CELL ANALYSIS WEBCASTS · PDF fileResolving the Changing Needs of...
Resolving the Changing Needs of Cellular Research With Continuous Live-cell Analysis
Del Trezise, PhD.
Smart Cell Monitoring2016 INCUCYTE™ LIVE-CELL ANALYSIS WEBCASTS
Webinar Contents
✔ Fundamental Challenges in Biomedical Research
✔ Changing Landscape of Cell Systems
✔ Continuous Live-Cell Analysis
✔ Current Approaches
✔ Workflow Examples
✔ Wrap Up & Perspective
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WEBINAR: Smart Cell Monitoring & Analysis
Fundamental Challenges in Biomedical ResearchBridging between model systems and true physiology
How relevant are the models that we use? Do they reliably inform us? Are they predictive and ‘translational’?
Recognising added complexity
Genetics, Epi-genetics, Environment Patients are unique and change over time
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WEBINAR: Smart Cell Monitoring & Analysis
Major Utilities of Cells in Biomedical Research
Basic Biology Predictive Testing
Production Therapeutics per se
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WEBINAR: Smart Cell Monitoring & Analysis
Cellular Models:A Changing Landscape
Mono Cultures (2D)
Co-cultures (2D)
3D Spheroids
Microtissues &Organ on a chip
Immortalised cell lines
Primary cells
Stem cell derived cells
Patientspecific cells
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CELL TYPE
ORGANISATION
ADVANCED CELL SYSTEMS
DYNAMIC
WEBINAR: Smart Cell Monitoring & Analysis
Features of Advanced Cell SystemsPrimary Cells, Stem Cells, Patient-Specific Cells, Co-cultures etc.
Typically harder to source & maintain
Often limited supply More expensive to obtain & work with More challenging workflows
Cell properties change over time
Sometimes of limited lifespan Differentiation/De-differentiation Cell/Cell communication
PRECIOUS
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WEBINAR: Smart Cell Monitoring & Analysis
Comparison of Cell Model Systems: High level Attributes
Model System Relative Cost Ease of use Availability Relevance
Host cell Linese.g. CHO, HEK
Tumour Cell Linese.g. MDA-MB231, Jurkat
Primary Cellse.g. human blood cells
Co-/multi culturese.g. neurones & astrocytes
iPSC-Derived Cellse.g. cardiomyocytes
Microtissues/Organoidse.g. hepatic structures
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WEBINAR: Smart Cell Monitoring & Analysis
Key requirements for working with advanced cell models
✔ Analytical methods that are relevant and information rich
✔ Methods that can track parameters over time
✔ Methods that are cell sparing
✔ Methods that are user-friendly
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CONTINUOUS MONITORING & ANALYSIS is ideally suited to meet these needs
Commensurate with advanced cell system
Understand temporal change
e.g. Streamlined workflows, Miniaturization
Focus on the biology
WEBINAR: Smart Cell Monitoring & Analysis
Continuous Monitoring & Analysis
• More accurate baseline• Trends & outliers recognition• Early detection & decision making
Snapshot Continuous
vs.
• Non-perturbing (lifestyle & ‘white-coat’ syndrome)
• Simpler & cheaper
Benefits
A
B
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e.g. Heart Rate, Blood Pressure
WEBINAR: Smart Cell Monitoring & Analysis
Continuous Live-Cell Analysis: What is it?
Continuous (or semi-continuous) cellular measurement
✔ Relevant analyses in real-time
✔ From living non-perturbed cells
✔ Non-exhaustive
✔ Long term as required
✔ Applicable across workflows
e.g. Cell function, morphology
Environmental control is key
Cells are not depleted
Hours, days, weeks
Culture, manipulation, assay
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WEBINAR: Smart Cell Monitoring & Analysis
Workflow applications of continuous live-cell analysis
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Continuous Analysis
Thawing
Harvesting
Separating
Sub-culturing
Bulk/Scale Up
Re-stocking
Cell Plating & QC
Assay Development & Optimisation
Phenotypic Assays & Screens
Mechanism of Action Studies
Gene Transfection• DNA, RNA• CRISPR/Cas• siRNA, miRNAs
Labels & Reporters
Reprogramming & Differentiation
Clonal dilution
CultureAssay
Manipulate
WEBINAR: Smart Cell Monitoring & Analysis
Continuous Live-Cell AnalysisComparison vs End Point Workflow
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Endpoint
Single time point
Continuous
e.g. Cell Health, Morphology, Growth
e.g. Transfection, Plating, Pre-treatments
Kinetic assay
Culture Manipulate Assay
WEBINAR: Smart Cell Monitoring & Analysis
Continuous Live-Cell Analysis: Electrical✔ Impedance or extracellular field potential (MEA)
✔ In situ (incubator) via specialist electrode plates
✔ Signals are non-invasive, label-free, long-term
✔ Applications - Neuronal, Cardio-myocyte, Other
✔ Surrogate for a number of cellular phenotypes e.g. proliferation, adhesion
✔ Impedance signals can be difficult to interpret. High cell numbers usually required. Consumables are costly.
Zeng et al., 2016 doi:10.1038/srep19449
Kamioka, H et al: Neurosci. Lett. 1996, 206, 109–112.
A549 cells
Mouse spinal cord
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A
B
WEBINAR: Smart Cell Monitoring & Analysis
Continuous Live-Cell Analysis: Imaging✔ Time-Lapse Images of Cells
✔ In situ (incubator) via regular tissue culture consumables (e.g. flasks, plates)
✔ Applications – Cell Health, Cell QC, Phenotypic Assays
✔ On-the fly image analysis = data
✔ Low number of cells required
✔ Label-free (Phase, Brightfield) or Fluorescence. Morphological or live-cell reporter readouts
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WEBINAR: Smart Cell Monitoring & Analysis
Continuous Live-Cell Analysis: Imaging Solution Types
Type Environmental Control Flexibility in Optics Capacity, Throughput Automated Analysis
Compact Microscopee.g. Juli, CytoSmart
Live Cell Imagere.g. IncuCyte ZOOM
Microscope Workstatione.g. Nikon Biostation
High Content Imagere.g. MD ImageXpress
(In Incubator)
(In Incubator)
(Own Housing)
(On Stage)
(1 plate/flask)
(6 plates/flasks)
(Automation)
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NB Capacity/ Throughput & Integrated Turn-key Analysis scoring refers to Continuous Analysis (not End-point) Applications
0 50 100 150 2000
20
40
60
80
Time (h)
Neu
rite
leng
th (m
m/m
m2 )
Example 1: Differentiation of human cortical NSCs into neurones &development of long-term stable neural network
Plate cells
Differentiation stimulus
NeuriteNetwork
Masked Images for quantification
IncuCyte ZOOM HD Phase Images 20x
15K cells well-1 on laminin-coated 96-well plate, 24 wells
Neurite Outgrowth (Day 0 - Day 8)
WEBINAR: Smart Cell Monitoring & Analysis
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IncuCyte ZOOM HD Phase 20x Network stabilisation day 5-20
Proliferation day 5-200 100 200 300 400 500
0
40
80
120
Time (h)
Neur
ite le
ngth
(mm
/mm
2 )
0 100 200 300 400 5000
40
80
120
Time (h)
Con
fluen
ce (%
)
NEURITE LENGTH CONFLUENCE
WEBINAR: Smart Cell Monitoring & Analysis
Example 1: Differentiation of human cortical NSCs into neurones &development of long-term stable neural network
✔ Neural network (5-20 days) maintained in subset of wells only (17/24)
✔ Drop off observed in 7/24 wells – coincident with increase in confluence
✔ Explained by proliferation of non-terminally differentiated precursors
PRODUCTIVITY
WEBINAR: Smart Cell Monitoring & Analysis
Value of Continuous Live-Cell AnalysisExample 1: Neural Stem Cell Differentiation & Network Formation
Real time observation and measurement
Verification of cell health & morphology throughout duration of study
Living non-perturbed cells - no fix/stain/wash artefacts Full time-course of neurite outgrowth & stabilisation Identification of precursor proliferation complication
Reduced ‘hands-on’ time & lab cost
Automated real time image gathering & analysis 24 wells x 78 time-points = 1880 data points <0.5 million total cells = acceptable cost No labelling antibodies Cells still viable for follow on studies e.g. ICC
BIOLOGICAL INSIGHT
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Tumour cell apoptosis (green)
Tumour cell count (red)
WEBINAR: Smart Cell Monitoring & Analysis
Example 2: Immune-cell/tumour cell co-culture killing assays for immuno-oncology
Target cells SKOV-3 Ovarian Cancer stably transfected with NucLight Red
Effector Cells Human Peripheral Blood Mononuclear cells (PBMCs)
+
Caspase 3/7 Substrate
IL-2/CD3
Transfection of SKOV-3 Target Cells with NucLight Red
WEBINAR: Smart Cell Monitoring & Analysis
Control MOI3 MOI6
+ Polybrene8µg ml-1
MOI3 + Poly MOI6 + Poly
IncuCyte ZOOM
Culture Manipulate Assay
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WEBINAR: Smart Cell Monitoring & Analysis
Bulking up & selection of stable NR-SKOV-3 cell population
IncuCyte ZOOM
Culture Manipulate Assay
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1 2 3 4 5 6 7 8 9 10 11 12
A 17 21 17 22 21 20 21 21 16 19 18 18
B 16 17 19 19 17 18 15 19 17 19 15 19
C 17 22 21 19 19 19 14 19 17 19 17 17
D 16 17 19 20 19 17 16 16 17 20 16 15
E 19 18 17 18 18 20 15 15 17 18 18 17
F 21 19 19 17 19 17 15 17 18 20 17 18
G 16 18 20 20 20 16 16 19 16 17 18 17
H 17 19 18 19 16 17 16 16 18 15 17 15
96-well Plate QC ± PBMCs
WEBINAR: Smart Cell Monitoring & Analysis
Confluence
Mean 18%
Range 15-22
SD 2%
NR-SKOV3
(2K per well)
NR-SKOV3+
PBMCs
(8K per well)
IncuCyte ZOOM
Culture Manipulate Assay
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WEBINAR: Smart Cell Monitoring & Analysis
Co-culture immune-cell killing assay
Live SKOV-3 Cell Count
Hours
SKOV3 + PBMCs + IL-2/CD3
SKOV3 SKOV3 + PBMCs
Apoptotic SKOV-3 Cell Count
SKOV3 + PBMCs + IL-2/CD3
SKOV3 SKOV3 + PBMCs
Culture Manipulate Assay
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WEBINAR: Smart Cell Monitoring & Analysis
Immune-cell killing (ADCC) assay: 4 x parallel plates
Plate 1 0.66 Plate 2 0.63
Plate 3 0.83 Plate 4 0.73
1
3
2
4
Plate QC: Z’
IC50 9.1 ng ml-1
-4 -3 -2 -1 0 140000
60000
80000
100000
log [Herceptinl] µg ml-1
AU
C (1
-144
h)
Total Study: 3.1 x106 PBMCs
IncuCyte ZOOM
Culture Manipulate Assay
PRODUCTIVITY
WEBINAR: Smart Cell Monitoring & Analysis
Value of Continuous Live-Cell AnalysisExample 2: Immune Cell /Tumour Cell co-culture & killing assay
Real time observation and measurement
Full time-course of direct readout (target cell death) Multiplex readouts & cell morphology build confidence Movie verifies interaction between target & effector cells
Reduced ‘hands-on’ time & lab cost
Streamlined reagent & assay build & validation Automated real time image gathering & analysis - minimal
time required by experimenter 4 x 96w x 2 metrics x 64 time-points = 49,152 data Cells still viable for follow on studies e.g. Flow, ELISAs
BIOLOGICAL INSIGHT
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Wrap up & Perspective
✔ Cellular research landscape is changing – greater complexity, ‘precious’ & ‘dynamic’ cells
✔ Developments in continuous live-cell analysis methods are well aligned to the new requirements
✔ Continuous live-cell imaging methods provides both valuable biological insight and enhanced
productivity, at scale and across cellular workflows
✔ Continuous live-cell imaging and analysis is emerging as a powerful tool for the cell biologist,
particularly for those working with advanced cell systems
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Further Information & Next steps
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