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

2

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

3


Major Utilities of Cells in Biomedical Research

Basic Biology Predictive Testing

Production Therapeutics per se

4


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

5

CELL TYPE

ORGANISATION

ADVANCED CELL SYSTEMS

DYNAMIC


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

6


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

7


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

8

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


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


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

10


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


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


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


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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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)


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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


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


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)


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


Control MOI3 MOI6

+ Polybrene8µg ml-1

MOI3 + Poly MOI6 + Poly

IncuCyte ZOOM


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Bulking up & selection of stable NR-SKOV-3 cell population

IncuCyte ZOOM


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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


Confluence

Mean 18%

Range 15-22

SD 2%

NR-SKOV3

(2K per well)

NR-SKOV3+

PBMCs

(8K per well)

IncuCyte ZOOM


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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


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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


PRODUCTIVITY


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

✔ Follow up e-mail:

White Paper Download

Copy of Slides Download (pdf)

Webcast recording

✔ Publications

✔ Customer reviews

27

http://www.selectscience.net/products/incucyte-zoom-continuous-live-cell-imaging-and-analysis-system/?prodID=116928

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