Factors to Consider for Designing and Optimizing Assays Applied to 3D Cultures

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1 Factors to Consider for Designing and Optimizing Assays Applied to 3D Cultures Terry Riss 2016.

2 Background and Justification for Using 3D Cell Culture Models Growing awareness of advantages of 3D cell cultures Physiological relevance Ability to model in vivo biology Many suppliers of materials to support 3D culture Hanging drop approach Hydrogel and matrix components Culture surfaces (plates) Chips utilizing microfluidics Link to webinar describing 3D model systems

3 Outline Factors to consider when characterizing assays applied to 3D structures Example assay modifications Live / dead assays Multiplexing with RNA extraction Apoptotic marker Luciferase reporter assay Appropriate controls Take home message

4 Major Take-Home Message There is an unmet need for validated assay methods to interrogate markers in 3D culture models

5 Factors to Consider Does Size of 3D Model Affect Assay Performance? Some things to think about Cell lysis Limitations for imaging Penetration of non-lytic probe reagents Mass of cells may overwhelm assay chemistry

6 What is Different about 3D Cell Culture? 2D 3D

7 Thickness for Reagent or Light Penetration 2D 3D Monolayer of cells Plastic surface ~5 µm deep ~ µm diameter spheres

8 Thickness for Reagent or Light Penetration 2D 3D Monolayer of cells Plastic surface ~5 µm deep

9 Need for Validated Assay Methods 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

10 Case Study: Hanging Drop Method & ATP Assay for Viability Hanging drop method (InSphero GravityPLUS ) Single microsphere per well Matrix free Size can be controlled Can easily see samples ATP Assay Most widely used assay for viable cells Fastest and most sensitive method Strong lytic capacity enabled by stabilized luciferase DNA binding dye to detect dead (lysed) cells

11 How the ATP Cell Viability Assay Works CellTiter-Glo Assay Reagent Lysis Solution ATPase Inhibitors Luciferin UltraGlo Luciferase Viable Cell ATP Dead Cell ADP Light Luciferin + Luciferase X No Reaction

12 How the ATP Cell Viability Assay Works Mix 2 min Wait 10 min Record RLU

13 How DNA Staining of Dead Cells Works (Nonpermeable Dye) CellTox TM Green Staining of dead cells results in a fluorescent signal that is stable. Viable Cell X Dead Cell Dye is excluded from live cells DNA dye only stains nucleus of dead cells or debris 13

14 CellTox Green Dye Staining of HCT-116 Spheroids Treated 48 hours with Panobinostat 0 (control) 38nM 10µM Andrew Niles 14

15 Combining Reagents to Test for Cell Lysis CellTox TM Green (DNA Staining) (ATP Detection) Mix Photograph Spheroids to Confirm Lysis

16 Critical Experiment that Prompted our Effort to Begin Validating Assays for 3D Experimental protocol combining DNA stain and ATP assay Spheroids grown to ~350 mm using hanging drop method DNA dye was added into the ATP assay reagent to detect cell lysis The standard mixing and equilibration steps were followed Spheroids were photographed using laser confocal microscopy Reagent 1 CellTiter-Glo Reagent ~350 mm spheroids Chad Zimprich, Mike Valley 16

17 How can you tell if all the ATP is extracted? What is an appropriate control?

18 Acid Extraction of ATP is the Gold Standard Treat sample with acid to extract ATP and inactivate ATPases Neutralize ph Measure using luciferase assay Compare value to standard curve made from ATP solutions processed identically

19 ATP (nm) Spheroid Size Affects Ability to Extract ATP HCT116 cells grown for 4 days seeded recovery +/- diam (um) 10, % 0.7% 614 5, % 3.3% 483 2, % 10.3% 424 1, % 14.0% % 10.6% 254 ~350 µm ~600 µm 2,000 1,800 1,600 1,400 1,200 1, CTG acid volume x 10^5 (um^3) Data suggest protocol needs modification to extract ATP

20 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 More physical disruption (shaking for 5 min) Longer incubation with lysis buffer (30min) CellTiter-Glo 3D Cell Viability Assay has been used with: hanging drop, inert scaffold, low binding plates, Matrigel and collagen embedded models

21 ATP Assay Improvements for 3D Cultures CellTiter-Glo CellTiter-Glo 3D Mix 2 min Wait 10 min Record RLU Mix 5 min Wait 25 min Record RLU

22 % Recovery of ATP from 6 Cell Lines Cultured with Matrigel: Comparison of ATP Assays

23 Can a permeable small molecule probe overcome limitations of lysing cells in large spheroids?

24 Some Assays Do Not Require Cell Lysis Small molecules can penetrate spheroids Tetrazolium (MTT) or resazurin redox substrates Pro-Substrate component of RealTime-Glo Cell Viability Assay DNA binding dyes (e.g. CellTox Green TM ) Protein-containing detection reagents Annexin V or antibody fragments

25 Why Not Just Use MTT or Resazurin Assays? MTT and resazurin assays have been used for 3D culture models (e.g. MTT used in MatTek's multilayered keratinocyte EpiDerm System) Limited information is available on reagent penetration 1 MTT and resazurin have been observed to be toxic to cells 2 Sensitivity of MTT assay limits signal to background ratio 1 Bonnier et al., Cell viability assessment using the Alamar blue assay: A comparison of 2D and 3D cell culture models. Toxicology in Vitro 29: , Assay Guidance Manual:

26 Toxicity of Balb 3T3 Cells Caused by Treatment with MTT for 4 Hours Time Zero 4 hours Images captured by Tracy Worzella using Incucyte instrument from Essen Biosciences Assay Guidance Manual:

27 New Chapter in AGM on 3D Spheroid Assays In vitro 3D Spheroids and Microtissues: ATP-based Cell Viability and Toxicity Assays Monika Kijanska and Jens Kelm

28 Signal-to-Background Comparison of Signal:Background Among Three Viability Assays Measuring Individual Spheroids 100,000 10,000 1, µm 340 µm MTT detection sensitivity is inadequate CellTiter-Glo 3D alamarblue MTT HCT116 cells were cultured in the InSphero GravityPLUS 3D Cell Culture system for 4 days to form 340 and 640 µm microtissues. Spheroids were processed to measure ATP (CellTiter-Glo 3D), resazurin reduction (alamarblue), or MTT reduction following standard procedures. 28

29 Real-Time Measurement of Cell Viability Using a Small Molecule Probe 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

30 Measure Changes in Viability Over Time Dose-response of Thapsigargin on A549 cells The luminescence signal was determined every hour for 72 h in a plate reader with gas control module (37 C/5%CO 2 )

31 Real Time Assay Signal Correlates with 3D Spheroid Size Why is signal linear with diameter?

32 Volume (Millions µm 3 ) Volume of Sphere is Not Linear with Diameter V = 4 3 πr Radius (µm)

33 Size Affects Physiology of Cells in 3D Structures Proliferating Quiescent Dead 100 µm 500 µm

34 Advantages of Assays with Permeable Probes Lysis of cells in spheroids is not necessary The real time approach provides kinetic information on viable cell number during the course of experiments Viable cells remain after exposure to reagent enabling more opportunities for multiplexing with other assays

35 Multiplexing Viability Assay with RNA Extraction from 3D Cultures 2016.

36 Multiplex RNA Extraction from Single Spheroids Following RealTime-Glo Cell Viability Assay Measure viability of spheroids on the fly using RealTime-Glo Extract RNA for expression analysis

37 Yield (ng) Multiplex RNA Extraction from Single Spheroids Following RealTime-Glo Cell Viability Assay Maxwell + RT-Glo Maxwell - RT-Glo ReliaPrep + RT-Glo ReliaPrep - RT-Glo Microtissue Diameter (µm) RealTime-Glo MT Cell Viability Assay to measure viability of different sizes of HEK293 cell spheroids followed by RNA extraction of the same samples using ReliaPrep RNA Tissue Miniprep System and Maxwell 16 LEV simplyrna Tissue Kit (with and without presence of RealTime-Glo Reagent)

38 RNA Integrity is Not Affected by Presence of RealTime-Glo Reagent RIN Values (RNA integrity) of RNA extracted from different size spheroids average ~9 (excellent) Cycle threshold of RNA isolated from 4 different cell types was not affected

39 Link to Article Describing RNA Extraction from 3D Cultures Total RNA Isolation from 3D Cell Cultures or Cells in Matrigel Matrix Using the ReliaPrep mirna Cell and Tissue Miniprep System

40 Measuring Apoptosis in 3D Cultures 2016.

41 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): ,

42 Homogeneous Real Time Apoptosis Assay Concept using Annexin Binding to PS Normal Cell - Viable Apoptotic Cell - Viable 2 Necrosis Non-Viable LgBiT Annexin V SmBiT Annexin V NanoBiT AnV:AnV Interaction CellTox Green Phosphatidyl Serine 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 Time following exposure to apoptosis inducer

43 Real Time Apoptosis and Secondary Necrosis Detection Apoptosis 2 Necrosis

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49 Time Course of Annexin V-fusion Protein Binding and DNA Staining with 3D Culture Model from a Researcher at Novartis Type II Raf Inhibitor (10µM)

50 For lytic apoptosis assays What if the marker you are trying to measure is not stable in high detergent concentrations?

51 Enzyme Markers May Lose Activity During Extraction with High Concentration of Detergent Example enzyme markers: Caspase to measure apoptosis LDH to detect cell death

52 Caspase-Glo 3/7 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)

53 Caspase Assay Protocol Optimization to Enhance Cell Lysis in Spheroids HCT116 cell spheroids grown to ~330mm using hanging drop method Add Caspase-Glo 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 5 min shake 30 min shake Increased shake time results in near complete lysis of cells in spheroids

54 General Recommendations for Improving Lysis of Large 3D Cell Structures Change detergent formulation when possible Incubate for longer time in lysis solution Increase physical disruption (shaking step)

55 Physical Disruption and Mixing Options Plate shaker The size of the well matters (i.e. 96 or 384 format) 1 Orbital shaker speed (~450 rpm) Electromagnetic device available (3600 cycles/min) 2 Trituration (pipetting up-and-down) can be used instead of shaking, but results in greater variability among replicates Ultrasonic treatment (potential for future) 1 Walling et al. Mixing in 384-Well Plates: Issues, Measurements, and Solutions. Assay Drug Dev Tech 5(2): ,

56 Seeing is believing or is it? Spheroids may appear intact using light microscopy even though cell membranes have been lysed

57 Low resolution visual appearance of 3D cultures may be misleading. Without a fluorescent marker, it may be difficult to tell if cells have been lysed

58 Protocol Used with Corning Spheroid Plates to Illustrate Spheroid Morphology After Treatment Add cells Photograph Incubate 4 days Photograph Add CellTiter-Glo 3D Reagent Shake 2 min Photograph

59 Morphology of Spheroids Before and After Adding CellTiter-Glo 3D Reagent

60 Luciferase Reporter Assays in 3D Culture 2016.

61 Can you use luciferase reporter assays with 3D culture models? What is an appropriate control to determine if you are extracting all the luciferase?

62 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 or strong detergent extraction as a control may destroy luciferase 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

63 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 ~ 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

64 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 ~ 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

65 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 ~ 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

66 Summary and Take Home Messages Most existing cell-based assays were originally designed for 2D monolayers or cells in suspension Size / mass of 3D structure may limit efficient lysis of cells or overwhelm assay chemistry Optimizing the detergent formulation, time of mixing with a plate shaker and the incubation time with lysis solution are recommended first steps for validating assays with lytic reagents Non-lytic assays using small molecule probes may be an option We recommend validating assays with each cell type and each 3D cell culture model system

67 2016. Questions Welcome