joseph b. martin conference center, boston, ma cover of assay … · 2019-05-02 · high content...

High Content 2017September 13th-15th4th Annual Conference

San Diego Conference Center, San Diego, CA

Assay Development

Considerations for HCSSource: NIH/NCATS

Assay Guidance Guidelines

Joe TraskSenior Application Scientist

PerkinElmer, Inc.

email: [email protected]

High Content 2018September 18th-20th

5th Annual Conference

Joseph B. Martin Conference Center, Boston, MA

http://www.ncats.nih.gov/

September 18, 2018

OJ TraskAssay Development Educational Course

5h Annual Conference, Boston, MA

WHY ASSAY DEVELOPMENT ???

http://www.nature.com/news/1-500-scientists-lift-the-lid-on-reproducibility-1.19970

SEE VIDEO !!!

1,500 scientists lift the lid on reproducibility,

Nature Survey May 2016

http://www.sbi2.org/

September 18, 2018



WHAT IS “HIGH CONTENT” IMAGING?

High Content screening is an automated cell biology method drawing on optics, chemistry, biology and image analysis to permit rapid, highly parallel biological research and drug discovery. (Source: Wiki)

“High Content” in the context of image

analysis provides a few to hundreds of

measureable “features” per cell or object

being interrogated without multiplexing.

Multiplexing adds to the overall value of

the data but it is not high content on its

own.

HC Features – these are measureable

statistical pieces of information of

structures within the cell. A simple

structure are spots or edges; complex

structures are considered objects


September 18, 2018



HIGH CONTENT _________?

HCS – High Content Screening

HCA – High Content Analysis

HCI – High Content Imaging

IC – Image Cytometry

Computer-assisted automated microscopy

Computer-assisted image analysis


THE IMAGE IS DATA…THE GOOD, BAD, & UGLY

Cytotoxic Fluorescent

Media Anisomycin

Size

Shape

Intensity

Texture

Dynamics

Distribution

Multiparametric

Multivariate

Rarely

Homogeneous

September 18, 2018



IMAGE PROCESSING OVERVIEW

Figure adopted by Defineins, IncSource: Adopted from Definiens


September 18, 2018



ASKING THE RIGHT QUESTIONS…

• What is an HCS assay going to provide me that other assays do not?

• Will it provide more relevant information than other assays?

• Does the data outputs provide not only a result but also locate the target’s activity in the cell (location of a phosphorylated protein)

• Can phenotypic or target-based measurements including morphology be quantified?

Question that should be asked:

Same label, different distributionLocation,

Location,

Location.

Modified from presentation at the RNAi Global Initiative Teleconference - HCA Assay Development, 17-Sep-2009, OJ Trask

pH3-AF647, captured on Operetta CLS, 63xW


September 18, 2018



HCS EXPERIMENTAL WORKFLOW

Seed Treat StainGrowCells

Cell models

Compounds

Multi-well

plates

AntibodiesFluorescent

Dyes

Wetware

siRNAs

CRISPR

Chemistry

AnalyzeImage

HCS

Hardware

Liquid Handling

Robotics

Data analysis

Knowledge

High Content ProfilerTM

Software

StatisticsData

Management

MachineLearning

Slide courtesy of PerkinElmer


September 18, 2018



HCI ASSAY DEVELOPMENTBASIC PRINCIPLES & THINGS YOU

NEED TO KNOW…

https://www.ncbi.nlm.nih.gov/books/NBK100913/



September 18, 2018




September 18, 2018



PROBING CELL HEALTH & STRESS INDICATORS

Presented at SLAS, 2014, The Hamner Institute, J. Trask


September 18, 2018




NEED TO KNOW…




14 Cellular Imaging & Analysis Webinar Series2017-May-24

Advanced & Complex Cell Models

Demand for better in vitro models to recapitulate the in vivo environment

Modelcells, tissue, organisms

Alternative Animal Studies Develop

Biology

Cancer Biology

Toxicology

Neurobiology

Drug Discovery / Pre-clinical

Immunology

Metabolic Diseases

September 18, 2018



CELL MODEL CHOICEIS IT REPRESENTATIVE OF BIOLOGY OR PHENOTYPE?

Example in Toxicology: Human in vitro Liver Cell Models

Model Source

Hep G2 Hepatocellular carcinoma, epithelial, 15yo caucasian male; Wistar

Institute, 1980.

HuH 7 Differentiated hepatocyte derived cellular carcinoma cell line

originally taken from a liver tumor in a 57yo Japanese male in 1982

THLE-2 Derived from primary normal adult liver, left lobe, by infection with

SV40 large T antigen. NIH, 1993.

HepaRG™ terminally differentiated hepatic cells derived from a human hepatic progenitor

cell line that retains many characteristics of primary human hepatocytes.

Stem cells: (1) ES and (2) iPS derived

Primary, cryopreserved

Primary, freshly isolated – Hu difficult to get

Coculture systems, e.g., hepatocytes with stellate, kupffer, and stroma cells

Tissue

Co

mp

lex

ity


September 18, 2018



Types

• Immortalized Cell Lines

• Stem Cells

• Primary Cells

• Explant Tissue

• Organisms

Characteristics

• What is the source

• What background information is available

• Genotype / Phenotype

• Engineered

• Mixed coculture

• Adherent or suspension

• 2D, 3D, 4D (t)?

Are the cells conducive to imaging?


September 18, 2018



CHOICE OF CELL LINE

Resources• Literature references

(http://www.Pubmed.com)

• Existing protocols

(http://www.currentprotocols.com/WileyCDA)

• American Type Culture Collection (ATCC) (http://www.atcc.org)

• Stem Cell Consortium at NIH CRM https://commonfund.nih.gov/stemcells/lines

• Scientific Societies & Journals

• Other commercial sources


September 18, 2018



HELA CELL LINEIS IT EVERYWHERE?

• 1951, Henrietta Lacks (HeLa), a patient who died of her cancer.

• The cells from Lacks' cancerous cervical tumor were taken without her knowledge or consent by researcher George Gey.

• 1952, HeLa, the first human cells grown in a lab that were naturally “immortal”

• 1954, Jonas Salk developed a vaccine for polio using these cells

• 1955, HeLa cells were the first human cells successfully cloned

• 1967, Stanley Gartler & 1975, Walter Nelson-Rees were the first to publish on the contamination of various cell lines by HeLa

• Scientists have grown an estimated 20 tons of her cells

• Approximately 11,000 patents involving HeLa cells

• HeLa contaminated Cell Lines

• Why does ATCC continue to distribute HeLa Contaminated Cell Lines? ATCC continues to distribute these cell lines, even though they have been shown to be contaminated with HeLa, because researchers need them for purposes beyond use as models for specific disease/original source tissue. For example, ATCC® CCL-17 ™, KB cells are used as a model for folate receptors; ATCC® CCL-23 ™, HEp-2 cells are used as a host to grow Chlamydia and RSV. Source: https://www.atcc.org/Global/FAQs/3/6/HeLa%20contaminated%20Cell%20Lines-1207.aspx


September 18, 2018



CELL LINECELL DENSITY EFFECTS ON BIOLOGY & WINDOW

Lower Cell Seeding Density Higher Cell Seeding Density

Neurite Outgrowth

Receptor Internalization

Migration / Tracking

WoundHealing


September 18, 2018



ENGINEERED CELL LINE

Making your own constructs and cell lines:

Requires molecular biology expertise

transient transfections, stable transfections, or retroviral

Requires flow cytometry or other techniques to generate clonal populations

Typically easy to culture, may need to maintain under selection

Usually maintain viability and phenotype over several passages

Need to prove the biology works

MK2-EGFP RetrovirusConstruct

Untreated

Anisomycin / TNF-a

REF: Trask OJ , et al., Assay development and case history of a 32K-biased library high-content MK2-EGFP translocation screen to identify p38 mitogen-

activated protein kinase inhibitors on the ArrayScan 3.1 imaging platform. Methods Enzymol. 2006;414:419-39.


September 18, 2018



CHOICE OF CELL LINE

HCS Assay with Non-adherent Cells

Jurkat cells plated on fibronectin coated 96-well platesStained with Hoechst (left) and Phalloidin (right)

U937 cells plated on

96 well LiveCell

Array microplate

20um diameter picowells

(left) Hoechst stained (right)


September 18, 2018



CHOICE OF CELL LINE…THE RIGHT STUFF

Cell growth conditions • Each cell type / cell model has specific medium optimized for growth

• Serum type is usually specified with recommended medium type

• Some cells may require Horse serum or other sera in addition to FBS

• Serum is almost always required for growth conditions unless the medium has been optimized for serum free conditions

• Typical serum range is 0-20%

• Additional growth components may be required

• Conditioned medium may be needed for primary cells

• Cell treatment options• Medium used during the treatment may differ from growth media

• Serum type and concentration may vary in assay conditions with low or serum free conditions used for compound testing (to remove effects of serum)

• Incubation times for treatment of stimulants and or compounds are dependent on the response desired and biological relevance


September 18, 2018



CELL CULTURE HIGH PASSAGE LEADS TO SIGNAL LOSS

Limit extended passage of cells

Maintain the quality & reproducibility of your HCS assay


September 18, 2018



CELL CULTURE PASSAGE OF CELLS AFFECTS SIGNAL

ER

K-1

/2 C

yto

Nu

cD

iff

ƞg/ml PMA

Fo

ld W

ind

ow

p13 p28 p40

Work done by Debby Nickischer, 2002-04, Sphinx Laboratories, Eli Lilly


September 18, 2018



CELL CULTURE PASSAGE OF CELLS AFFECTS SIGNAL

Cell Passage Number Comparison. Different cell splitting passages of HeLa cells seeded at 5,000 cells/well overnight

and treated with dose response of TNF-α for ~35 minutes, then fixed and stained to measure NF-κB translocation. Plates

were analyzed on HCS imager to determine NF-κB translocation using CytoNuc Difference calculation; data was

normalized to control and plotted in GraphPad Prism using non-linear regression 3-parameter fit.


September 18, 2018



CELL CULTURE IMPACT OF SPLITTING CELLS ON DIFFERENT DAYS

Source: Presented at 2001 IBC High Content Imaging Workshop, Bal Harbor, FL, J. Trask


September 18, 2018



CELL SEEDING DENSITYCELL DENSITY AFFECTS ASSAY WINDOW

AND IMAGE ANALYSIS ALGORITHM PERFORMANCE

REF: https://www.ncbi.nlm.nih.gov/books/NBK100914/


September 18, 2018




NEED TO KNOW…




September 18, 2018




September 18, 2018



BEWARE: NOT ALL MICROPLATES ARE EQUAL

Polymer Based Glass

→ANSI/SBS/SLAS Standard

→ Not all wells have same surface area

or volume capacities

Biggest differences:

• Bottom thickness

• Bottom thickness variability

• Skirt height


September 18, 2018



BEWARE: REVIEW IMAGE WELLS FOR FOCUS

Unevenness Across the Well Image Quality: The Affects on Algorithm

Reference: Paul A. Johnston and Oscar J. Trask (2017 – in press) Methods in Molecular Biology Series: High

Content Screening A Powerful Approach to Systems Cell Biology and Drug Discover. ISBN 978-1-4939-7355-2

Objects Ident =35 Objects Ident =20

Match objective lens with the microplate thickness!384w microplate


September 18, 2018



MICROPLATE COATINGAFFECTS BIOLOGICAL OUTCOME




September 18, 2018





September 18, 2018



Source:

BD website



September 18, 2018



3D microplate formats-Most are composed of Matrigel, Hydrogel, Agrose,

ECM or other substrate

-Take hours to days to form spheroids

-Not all cells form spheroids

Types

• Suspension or “hanging drop”

• Ultra low attachment

• Magnetic Beads

• Biomaterials/films

• Others?


Which Model: ULA Microplates & Spheroid Phenotypes


Imaging

Cell Models: InSphero GravityPLUSTM

HCT116

Opera 20xW, z-stack maximum intensity

projection. Hoechst, GFP, RFP

NIH3T3

Imaging

20x

100 µm

Cell SeedingMicrotissue

Maturation

Microtissue

Transfer

Microtissue

Assay


September 18, 2018




NEED TO KNOW…




September 18, 2018



Verify your probe choice is detectable with instrument

• Resolution required & wavelength

SBI2 Educational Course, Steve Haney


September 18, 2018



Source: 2016 SBI2 Educational Course, Steve Haney


September 18, 2018



TYPICAL HCS PLATE PREPARATION -FIXATION

• Test with each biology and cell model

• Type of fix

• Age / stability

• Amount (%)

• Pre-warm to 37oC

FixationFix 30 min, Perm 15 min

0

50

100

150

200

250

300

350

1 2

avg

cyto

nu

c d

iff .9

1

1.8

2

3.7

4

% fix

Formaldehyde

Untreated Treated


September 18, 2018



TYPICAL HCS PLATE PREPARATION –FIXATION PERMEABILIZATION

• Permeabilization

• Triton X-100 or other detergent

• Permeabilizing fixed cell membranes

• allows antibodies and dyes access


September 18, 2018



REAGENT EVALUATION& DETERMINATION

Antibody Titration Experiment

Graph of commercially available antibodies evaluated

X-axis represents 1oAb concentration with 10μg/ml 2oAb;

Y-axis represents average fluorescent intensity of NF-κB difference between the cytoplasm and nucleus

subtracted from the 2oAb control. Values listed below x-axis are the raw data (CytoNuc Diff).

Considerations

• Establish background /non-

specific binding of 2oAb

• Maintain 2oAb Conc during 1o

Ab titration

• If multiplexing, test cross-talk

of fluorescent spectra & non-

specific binding of other

antibodies or probes.

• Fixation & permeablization

methods are not standardized

___proteins may require further

optimization.


September 18, 2018



REAGENT STABILITY LOT-TO-LOT & FREEZE-THAW CYCLES

Stability of cytokines following multiple freeze-thaw

cycles. Following the reconstitution of the cytokine per manufacture

suggestion, cytokine reagents were store at –80oC, and then allowed

to thaw at room temperature before use. Samples were then re-frozen

at –80oC multiple times. Translocation of NF-kB was performed on

HeLa cells following treatment with IL-1α (left) or TNF-α (right) as

previously described and data was normalized to control and plotted

in GraphPad Prism using non-linear regression 3-parameter fit.



September 18, 2018



PROBE STABILITY POST FIXATION

Stability of NF-kB-p65-AF488 complex post staining and fixation. Using several plates, HeLa cells seeded

at 5,000 cells/well overnight and treated with 25ηg/ml of TNF-α for ~35 minutes, then fixed and stained to

measure NF-κB translocation at the maximum signal. At different time points (days), plates were analyzed

on HCS imager to determine NF-κB translocation fluorescent intensity measurements using CytoNuc

Difference calculation; raw data was used and plotted for comparison.



September 18, 2018




NEED TO KNOW…




September 18, 2018



The liquid plumr effect – at high concentrations of DMSO• Can cause inconsistent effect on cells in areas of well• Can kill cells

Ways to solve• Pre-dilution in media• Mixing during addition

Cells don’t receive compound

Cells receive too much compound

High Concentration DMSO Low Concentration DMSO

Compound Distribution among cells more even


September 18, 2018



DMSO TOLERANCE



September 18, 2018



COMPOUND SELECTION IMPLEMENTATION OF CONTROLS

• Known inhibitors – Biology Control


September 18, 2018



COMPOUND SELECTION CHOICE OF STIMULI

Stimulant Concentration CurvesErk Cyto-Nuc Activation

0.000000010.00000010.0000010.00001 0.0001 0.001 0.01 0.1 1 10 100 1000

50

100

150

200

250

300

350

400

450

500

550 OSM

PMA

TGF

EGF

TNF

[Log] ng/ml

Mean

Cyto

Nu

cD

iff

R²

OSM

0.9731

PMA

0.9197

TGF0.9717

EGF

0.9016

TNF0.7092

EC50

OSM

0.1852

PMA

1.204

TGF0.003484

EGF

0.4793

TNF0.03396



September 18, 2018



COMPOUND SELECTION CHOICE OF STIMULI

Activation of NF-κB-p65 with Different Stimuli. HeLa cells seeded at 5,000 cells/well overnight and treated with

stimuli for 30 minutes, then fixed and labeled with NF-κB-p65-AF488 and Hoechst33342 to measure NF-κB

translocation. Plates were analyzed on HCS imager to determine NF-κB translocation using CytoNuc Difference

calculation; data expressed as raw unit values (y-axis) from algorithm using non-linear regression 3-parameter fit

was done in GraphPad Prism; standard deviation error bars (n=3) was removed for visualization.



September 18, 2018




NEED TO KNOW…




September 18, 2018



When using secondary treatment / stimuli – things to ask?

• Pre-treat with compound

• Simultaneous addition of all test articles

• Are treatment times amenable for screening

Long Incubations

• Bolus treatment

• Remove ALL and add ALL compounds/medium

• Remove ½ volume and add ½ volume compounds/medium

Frames per second

HeLa-MK2-EGFP cells, 1 frame/ 2min for 64 min

TNFɑ added frame 1 (time 0), Compressed run time 25 secs


September 18, 2018



KINETICS… DETERMINING A ROBUST STIMULI RESPONSE

pE

RK

-1/2



September 18, 2018



KINETICS… DETERMINING A ROBUST STIMULI RESPONSE

NF-κB Translocation time course kinetics. HeLa cells seeded at 5,000 cells/well overnight and treated

with 25ηg/ml of IL-1α over time, at 5 or 10 time minute intervals, cells were fixed and then stained to

measure NF-κB translocation. Plates were analyzed on HCS imager to determine NF-κB

translocation using CytoNuc Difference calculation; data was normalized and plotted in GraphPad

Prism using non-linear regression one-site binding to calculate the ½ time response, 24 minutes.



September 18, 2018




NEED TO KNOW…




September 18, 2018



HCS STATISTICAL MEASUREMENTSHOW MANY FIELDS PER WELL TO

DETERMINE STATSISTICAL SIGNIFCANCE?

REF: Trask O. Joseph Jr., Moore Amanda, and LeCluyse Edward L., A Micropatterned Hepatocyte

Coculture Model for Assessment of Liver Toxicity Using High-Content Imaging Analysis, ASSAY and

Drug Development Technologies 2014 12 1 , 16 -27

*

**

***


September 18, 2018



HCS STATISTICAL MEASUREMENTS3D ASSAY OPTIMIZATION Z’-FACTOR

-0.22

0.210.17

0.53

0.67

60µm

1

-

92%

2

60

85%

3

30

77%

5

15

62%

13

5

0

Ref: Curiosity of PerkinElmer and InSphero

Number of Planes

Distance, µm

Decrease in Acquisition


September 18, 2018



• Review of information for final conditions

• Cell model; microplate type & coating

• Cell Seeding density & medium conditions

• Optimal stimuli and/or reference compound & dose

• Optimal time of stimulus and/or treatment

• Optimal staining conditions; live, fixed, stability

• Validation - 3 day or 3 independent experiential IC/EC50

• Determine repeatability and reproducibility of the assay

• Well to well variance measurements by:

• Full plate of untreated/DMSO/Stimuli and compound

• ½ plate of each by columns/rows or checkerboard

• Reference library of compounds @ defined dose


September 18, 2018



ST

IMU

(%)

-20

020

4060

8010

012

0

1 12 24 36 48 60 72 84 96mTOP=83.083 mSTD=-247.4 mBKGD=2.0905 m Cont50=165.0 tHits=19 mTopCV=12.0% mBotCV=7.9%

Book:Page= A04267:022 Screen: BCATENINTRANSLOC RAPID80Plates: 1 to 20 Overlay plots

A B C D E F G H

ST

IMU

(%)

-20

020

4060

8010

012

0

ST

IMU

(%)

-20

020

4060

8010

012

0

1 12 24 36 48 60 72 84 961 12 24 36 48 60 72 84 96mTOP=83.083 mSTD=-247.4 mBKGD=2.0905 m Cont50=165.0 tHits=19 mTopCV=12.0% mBotCV=7.9%


A B C D E F G H

Hit Cytotoxic Compound Fluorescent Compound

Ref: Borchert K, et al., High-content screening assay for activators of the Wnt/Fzd

pathway in primary human cells. Assay Drug Dev Technol. 2005 Apr;3(2):133-41.

IMAGE ARTIFACTSCYTOTOXICITY & FLUORESCENT


September 18, 2018



ST

IMU

(%)

-20

020

4060

8010

012

0

1 12 24 36 48 60 72 84 96mTOP=83.083 mSTD=-247.4 mBKGD=2.0905 m Cont50=165.0 tHits=19 mTopCV=12.0% mBotCV=7.9%


A B C D E F G H

ST

IMU

(%)

-20

020

4060

8010

012

0

ST

IMU

(%)

-20

020

4060

8010

012

0

1 12 24 36 48 60 72 84 961 12 24 36 48 60 72 84 96mTOP=83.083 mSTD=-247.4 mBKGD=2.0905 m Cont50=165.0 tHits=19 mTopCV=12.0% mBotCV=7.9%


A B C D E F G H

Hit Cytotoxic Compound Fluorescent Compound

Review Images!!!

Ref: Borchert K, et al., High-content screening assay for activators of the Wnt/Fzd

pathway in primary human cells. Assay Drug Dev Technol. 2005 Apr;3(2):133-41.

IMAGE ARTIFACTSCYTOTOXICITY & FLUORESCENT


Assay Development for

High Content Screening August 07, 2017

OJ TraskNIH/NCATS Assay Guidance Workshop for High Throughput Screening and Lead Discovery

William F. Bolger Center, Potomac, Maryland

1. Assay Development Guidelines for Image-Based High Content Screening, High

Content Analysis and High Content Imaging. William Buchser, Mark Collins, Tina

Garyantes, Rajarshi Guha, Steven Haney, Vance Lemmon, Zhuyin Li, and O. Joseph Trask.

2. Advanced Assay Development Guidelines for Image-Based High Content

Screening and Analysis. Mark-Anthony Bray, Anne Carpenter, and Imaging Platform, Broad

Institute of MIT and Harvard.

3. Nuclear Factor Kappa B (NF-κB) Translocation Assay Development and

Validation for High Content Screening. O. Joseph Trask Jr.

4. High Content Screening with Primary Neurons. Hassan Al-Ali, Murray Blackmore, John L

Bixby, and Vance P. Lemmon.

5. New chapters under consideration

• Fluorescent artifacts – Q4 (authors: Steve Haney, Paul Johnston, Steve Titus, Joe Trask)

• 3D imaging

• Machine learning and analytics

• Toxicology

• Others…

NIH/NCATS ASSAY GUIDANCE MANUAL HCI CHAPTERS


NIH / NCATS Assay Guidance Manual

Assay Development for High Content Screening & Best Practices for 3D HCS

September 18, 2018

OJ Trask

RESOURCES


www.sbi2.org [email protected]

High Content Screening (2018). A Powerful Approach to

Systems Cell Biology and Phenotypic Drug Discovery

Editors: Johnston, Paul A., Trask, Oscar J. (Eds.)

ISBN 978-1-4939-7357-6

• “3D High-Content Screening of Organoids for Drug Discovery”, Dan LaBarbera, Ph.D.,

Univ of Colorado

• “HCS for predictive toxicology and in vitro pathology using simple 3D microtissues”,

Kim Boekelheide, M.D., Ph.D., Brown University

• “Face-to-face with cancer: The power of high-content imaging and analytics to illuminate

cell population dynamics”, Shannon Mumenthaler, Ph.D., Univ of Southern CA.


joseph b. martin conference center, boston, ma cover of assay … · 2019-05-02 · high content...

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