validating assays applied to 3d-cultures › media › worldwide › cls › documents › ... ·...

Promega CorporationPromega Corporation©2015 Promega Corporation.

[email protected]

Corning Panel Discussion / 3D-Culture Systems and Questions

AACR / April 3, 2017

Validating Assays Applied to 3D-Cultures

2Promega CorporationPromega Corporation©2015 Promega Corporation.

What is different about 3D cell culture?

2D 3D


Distance Required for Reagent Penetration

2D 3D

Plastic surface

Monolayer of cells

~5 µm deep ~50-500 µm diameter spheres


Unmet Need for Validated Assays to Interrogate Markers in 3D Culture Models

Most cell-based assays were designed for monolayer or

suspension cultures.

• Will reagents effectively lyse 3D structures?

• Will reagents penetrate to center of 3D spheroids?

• Will mass of cells block/quench signal before it reaches

detector?

Questions prompted collaborations with 3D culture system

providers to evaluate effectiveness of various assay

chemistries.


• Spheroids grown to ~350mm using hanging drop method

• Add ATP assay reagents + DNA dye to confirm lytic

effectiveness

• Photograph using laser confocal microscopy

~350 mm

spheroids

Reagent 1 Reagent 2

Chad Zimprich, Mike Valley

Observation of Lytic Efficiency of ATP Assay Reagents

Critical Experiment that Prompted our Effort to Begin Validating Assays for 3D


ATP Assay Improvements for 3D Cultures

1. Formulation changes: Higher concentration of

detergents to more effectively lyse cells in larger 3D

structures

2. Protocol modification to recommend

Physical disruption (shaking for 5 min)

Longer incubation with lysis buffer (30min)

• CellTiter-Glo® 3D Cell Viability Assay has been used

with: ultra low binding plates, hanging drop, Matrigel and

collagen embedded models, inert scaffolds…


Will Reagents Work in Hydrogels?

They should…Matrigel is “mostly” water


What if the Marker is Not Stable in Detergent?

Enzyme markers may lose activity during extraction with

high concentration of detergent

• Caspase – to measure apoptosis

• Luciferase – to detect genetic reporter activity


Caspase Assay of 3D Culture Models

• Stability of caspase enzyme limits ability to increase

detergent concentration in reagent formulation

• The reformulation strategy that was used for ATP assay

will not work

• To improve recovery of caspase activity, the assay

protocol was modified to incorporate

Physical disruption (shaking for 5 min)

Longer incubation with lysis buffer (30 min)


What is the Appropriate Control for a Luciferase Reporter Assay of 3D Spheroids?

• How can you tell if the lysis buffer is extracting all the

luciferase reporter activity?

• Acid extraction as a control may destroy activity.

• Antibody detection is not reliable because it indicates

quantity of antigen, not necessarily active luciferase.

• Correlation with a known marker such as ATP may be

an optional control.


Will Cell Permeable Probes Work in 3D Models?

• Can a permeable small molecule probe overcome

limitations of lysing cells in large spheroids?


Some Assays Do Not Require Cell Lysis

Small molecules can penetrate 3D models

• Tetrazolium (MTT) or Resazurin redox substrates

• Pro-substrate component of RealTime-Glo® MT Cell

Viability Assay

• DNA binding dyes (e.g. CellTox GreenTM)

• Annexin V or antibody fragments


CellTox Green Staining of HCT-116 Spheroids Treated 48 hours with Panobinostat

0 (control) 38nM 10µM

Andrew Niles

14Promega CorporationPromega Corporation©2013 Promega Corporation. Proprietary Information. Not for further distribution.

“Seeing is believing”…or is it?

• Spheroids may appear intact by light microscopy even

though cell membranes have been lysed

• Be aware of limits of ability to image using confocal

microscopy


Spheroids Before and After Detergent Lysis


Take Home Message

• Most in vitro cell-based assays were originally designed for 2D

monolayers or cells in suspension

• Size / mass of 3D structure may limit efficient lysis of all cells or

overwhelm assay chemistry

• Validation of assays with each cell type and each 3D culture model

is recommended

• Optimizing incubation time with lysis solution and mixing or

physical disruption are recommended first steps for validating

assays

• Non-lytic assay options are available using small molecule probes

Promega CorporationPromega Corporation©2015 Promega Corporation.

Questions Welcome


% Recovery of ATP from 6 Cell Lines Cultured with Matrigel: Comparison of ATP Assays


Caspase Assay Protocol Optimization to Facilitate Enhanced Microtissue Cell Lysis

• HCT116 cell spheroids grown to ~330mm using hanging drop method

• Add Caspase-3/7 assay reagent + DNA dye to indicate lytic effectiveness

• Shake with assay reagent for 5 or 30 min

• Image with confocal after a total of 30 min incubation with reagent

Increased shake time results in near complete spheroid cell lysis

5 min shake 30 min shake


Cell Permeable Probe used for a Real Time

Cell Viability Assay

• Luciferase and Pro-substrate are added as reagents to culture medium

• Pro-substrate enters the cell and is reduced to form a substrate for luciferase

• Substrate diffuses from the cell and is used by luciferase to produce light


Measure Changes in Viability Over Time

The luminescence signal was determined every hour for 72 h in a Tecan M200 plate

reader with gas control module (37C/5%CO2).

Dose-response of Thapsigargin on A549 cells


DNA Dye Staining to Detect Dead Cells (Overcomes some limitations of short half-life markers)

Dead CellViable Cell

Dye is excluded

from live cells

DNA dye only

stains nucleus

of “dead” cells

or debris

Non-permeable

DNA dye

(CellTox Green)

22

Staining of dead

cells results in a

fluorescent signal

that is stable.X


Detecting Appearance of Dead Cells in Real Time for Three Days

HepG2 cells treated with various doses of Terfenadine. CellTox™ Green Dye was

added at time of Terfenadine dosing. Fluorescence was measured every hour.

Increasing fluorescence indicates increase in number of dead cells.


Measurement of Luciferase Reporter

Activity from Different Size Microtissues

Constitutive NanoLuc® Reporter

HCT116 cells expressing NanoLuc® luciferase under a constitutive promoter were cultured for 4 days to form ~200-

700 mm microtissues. An equal volume of NanoGlo® Reagent or CellTiter-Glo® 3D Reagent was added to each well,

the plate shaken for 10 min, and luminescence recorded after a total of 30 min incubation.


Measurement of Luciferase Reporter Activity from Different Size Microtissues

NanoLuc reporter driven by HIF-1 promoter (Hypoxia marker)

HCT116 cells expressing NanoLuc® luciferase under a HIF-1 promoter were cultured for 4 days to form ~200-700

mm microtissues. An equal volume of NanoGlo® Reagent or CellTiter-Glo® 3D Reagent was added to each well, the

plate shaken for 10 min, and luminescence recorded after a total of 30 min incubation.


Will a large protein probe (such as Annexin V

or antibodies*) work as a detection reagent

with 3D culture models?

*Thurber & Wittrup. Cancer Res 68(9): 3334-3341, 2008.


NanoLuc

Homogeneous Real-Time Apoptosis Assay

Using Annexin-NanoBiT Binding to PS

Normal Cell - Viable Apoptotic Cell - Viable 2° Necrosis Non-Viable

Annexin V Annexin V

LgBiTSmBiT

PhosphatidylSerine

Time following exposure to apoptosis inducer

PS confined to inner leaflet

Cell membrane intact

Low luminescence

Low fluorescence

PS flipping to outer leaflet

Cell membrane intact

High luminescence

Low fluorescence

PS on outer leaflet and inside cell

Cell membrane compromised

High luminescence

High fluorescence

Necrosis

Detection

Reagent


Real Time Apoptosis and Secondary

Necrosis Detection

28


Exposure (hr)

Annexin Binding Membrane Integrity

Intrinsic Inducer with Tumor Spheroids

Morphology

and CellTox™

Green Staining

HCT-116

colorectal

cancer

“tumors”


Type II Raf Inhibitor Annexin-NB

Type II Raf Inhibitor CellTox Green

Preliminary evidence from a researcher at Novartis suggesting

Annexin V-fusion proteins work with 3D culture model


Type II Raf Inhibitor (10µM)

Time course of Annexin V-fusion protein binding and DNA Staining

with 3D culture model from a researcher at Novartis


How can you determine the viability of cells on

chips or in large 3D printed tissues using

non-invasive techniques?


Application Notes Describing Additional 3D Assays

3D Applications Notes

DNA isolation from 3D microtissues using the Maxwell RSC System -

http://inside.promega.com/Sales/SciApps/AppNoteLibrary/DNA%20Isolation%20from%203D%20microtissue%20using%20the%

20Maxwell%C2%AE%20RSC.pdf

Total RNA isolation from 3D cell cultures or cell in Matrigel with the Maxwell RSC miRNA Tissue kit -

http://inside.promega.com/Sales/SciApps/AppNoteLibrary/Maxwell%20miRNA%203D.pdf

Nano-Glo Dual luciferase reporter assay on 3D microtissues -

http://inside.promega.com/Sales/SciApps/AppNoteLibrary/Nano%E2%80%90Glo%C2%AE%20Dual%E2%80%90Luciferase%C2%

AE%20Reporter%20Assay%20on%203D%20microtissues.pdf

P450-Glo CYP3A4 Assay on 3D microtissue -

http://inside.promega.com/Sales/SciApps/AppNoteLibrary/P450%E2%80%90Glo%E2%84%A2%20CYP3A4%20Assay%20on%203

D%20microtissues.pdf

Isolation of RNA from 3D microtissues after RealTime-Glo MT cell Viability assay (RT-glo Maxwell)-

http://inside.promega.com/Sales/SciApps/AppNoteLibrary/RNA%20Isolation%20from%203D%20microtissues%20after%20RealT

ime%E2%80%90Glo%E2%84%A2%20MT%20Cell%20with%20Maxwell%C2%AE16%20LEV.pdf

Isolation of RNA from 3D microtissues after RealTime-Glo MT cell Viability assay (RT-Glo + ReliaPrep)-

http://inside.promega.com/Sales/SciApps/AppNoteLibrary/RNA%20Isolation%20from%203D%20microtissues%20after%20RealT

ime%E2%80%90Glo%E2%84%A2%20MT%20Cell%20with%20ReliaPrep.pdf

http://inside.promega.com/Sales/SciApps/AppNoteLibrary/DNA Isolation from 3D microtissue using the Maxwell%C2%AE RSC.pdf

http://inside.promega.com/Sales/SciApps/AppNoteLibrary/Maxwell miRNA 3D.pdf

http://inside.promega.com/Sales/SciApps/AppNoteLibrary/Nano%E2%80%90Glo%C2%AE Dual%E2%80%90Luciferase%C2%AE Reporter Assay on 3D microtissues.pdf

http://inside.promega.com/Sales/SciApps/AppNoteLibrary/P450%E2%80%90Glo%E2%84%A2 CYP3A4 Assay on 3D microtissues.pdf

http://inside.promega.com/Sales/SciApps/AppNoteLibrary/RNA Isolation from 3D microtissues after RealTime%E2%80%90Glo%E2%84%A2 MT Cell with Maxwell%C2%AE16 LEV.pdf

http://inside.promega.com/Sales/SciApps/AppNoteLibrary/RNA Isolation from 3D microtissues after RealTime%E2%80%90Glo%E2%84%A2 MT Cell with ReliaPrep.pdf

validating assays applied to 3d-cultures › media › worldwide › cls › documents › ... ·...

Documents