Factors to Consider When Choosing Cell- Based Assays for Use with 3D Cultures

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1 Factors to Consider When Choosing Cell- Based Assays for Use with 3D Cultures Corning Webinar December 16, , Promega Corporation.

2 Outline Justification for using 3D cell culture Approaches to culture cells in 3D Factors to consider when validating assays applied to 3D cultures Limitations caused by size of the 3D structure Appropriate controls Example assays Promega Corporation 2

3 3D Cell Culture is Coming Your Way! Promega Corporation 3

4 3D Cell Culture is Coming Your Way! Promega Corporation 4

5 3D Cell Culture Models / Background & Justification Growing awareness of advantages of 3D cell cultures Physiological relevance / predictive value Ability to model in vivo biology Many suppliers of materials to support 3D culture Hydrogel and matrix components Culture surfaces (plates) Suppliers of validated assays for 3D cultures are lacking Promega Corporation 5

6 One of Many Examples to Determine Predictive Value of 3D Culture Models The observed differences in potency and efficacy of the cancer drugs in 3D models suggest that the biological implications of screening configurations should be taken into account to select superior cancer drug candidates in preclinical studies. Promega Corporation 6

7 Each Model has Advantages and Disadvantages Method Advantages Disadvantages Hydrogel Collagen Matrigel Agarose Alginate Physiological environment Easy to visualize Some can dissolve Animal-derived and poorly defined GF contamination Non-uniform structures Difficult to handle (4C) Hanging Drop Inert scaffold (Alvetex) Low binding plates Matrix free (or can add soluble matrix) Cells synthesize their own matrix Controlled uniform size Can form hypoxic center One sphere per well Compatible with automation / HTS 200 um thickness No hypoxia Easy to use and automate / HTS Easy to use and automate / HTS Form single spheroid per well Small size of sample Difficult to image large microtissues Cannot see cells during experiment Difficult to image Large flat surfaces form multiple nonuniform structures Difficult to image (high resolution) in round bottom plates Promega Corporation 7

8 Each Model has Advantages and Disadvantages Method Advantages Disadvantages Methyl cellulose Suspends cells throughout medium HTS compatible May be limited to only some cell types Viscosity Difficult to image Magnetic levitation Microfluidics chips Rapidly forms cell aggregates Can image aggregates with ipad Can make really cool toys Requires use of iron nanoparticles Requires magnetization device Nanoparticles may be cytotoxic and interfere with imaging Requires special engineering Very low throughput Promega Corporation 8

9 Unmet Need for Assay Methods to Interrogate 3D Cell Culture Models Most cell-based assays were designed for monolayer or suspension cultures. Promega Corporation 9

10 Unmet Need for Assay Methods to Interrogate 3D Cell 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?. Promega Corporation 10

11 Unmet Need for Assay Methods to Interrogate 3D Cell 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. Promega Corporation 11

12 First Approach Promega Chose was Hanging Drop Method & ATP Assay for Viability Hanging drop method Single spheroid per well Matrix free Size can be controlled Can easily estimate size with special plates ATP assay Most widely used assay for viable cells Fastest and most sensitive method Strong lytic capacity enabled by stabilized luciferase Promega Corporation 12

13 Why Not 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) Little information is available on penetration of reagents MTT and resazurin have been observed to be toxic to cells (Assay Guidance Manual: Sensitivity of MTT assay limits signal to background ratio Promega Corporation 13

14 Toxicity of Balb 3T3 Cells Caused by Treatment with Resazurin for 4 Hours Time Zero 4 hours Promega Corporation 14 Images captured by Tracy Worzella using Incucyte instrument from Essen Biosciences Assay Guidance Manual:

15 Signal-to-Background Comparison of Signal:Background Among Three Cell Viability Assays Measuring Spheroids 100,000 10,000 1, µm 340 µm CellTiter-Glo 3D alamarblue MTT HCT116 cells were cultured for 4 days to form 340 and 640 µm microtissues. Spheroids were processed to measure ATP (CellTiter-Glo 3D Assay), resazurin reduction (alamarblue), or MTT reduction following standard procedures. Promega Corporation 15

16 ATP Assay for Cell Viability (How it Works) ATP Assay Reagent Lysis Solution ATPase Inhibitors Luciferin UltraGlo Luciferase Viable Cell Dead Cell ATP ADP Light Luciferin + Luciferase X No Reaction Promega Corporation 16

17 ATP Assay for Cell Viability (How it Works) Viable Cell Dead Cell ATP ADP Promega Corporation 17

18 ATP Assay for Cell Viability (How it Works) ATP Assay Reagent Lysis Solution ATPase Inhibitors Luciferin UltraGlo Luciferase Viable Cell Dead Cell ATP ADP Promega Corporation 18

19 ATP Assay for Cell Viability (How it Works) ATP Assay Reagent Lysis Solution ATPase Inhibitors Luciferin UltraGlo Luciferase Viable Cell Dead Cell ATP ADP Promega Corporation 19

20 ATP Assay for Cell Viability (How it Works) ATP Assay Reagent Lysis Solution ATPase Inhibitors Luciferin UltraGlo Luciferase Viable Cell Dead Cell ATP ADP Light Luciferin + Luciferase X No Reaction Promega Corporation 20

21 DNA Dye Staining to Detect Dead Cells (How it Works) Non-permeable DNA dye Staining of dead cells results in a fluorescent signal that is stable. Viable Cell Dead Cell Promega Corporation 21

22 DNA Dye Staining to Detect Dead Cells (How it Works) Non-permeable DNA dye Staining of dead cells results in a fluorescent signal that is stable. Viable Cell X Dead Cell Dye is excluded from live cells Promega Corporation 22

23 DNA Dye Staining to Detect Dead Cells (How it Works) Non-permeable DNA dye Staining of dead cells results in a fluorescent signal that is stable. Viable Cell X Dead Cell Promega Corporation 23 Dye is excluded from live cells DNA dye only stains nucleus of dead cells or debris

24 Critical Experiment that Prompted Effort to Validate Assays Observation of Lytic Efficiency of ATP Assay Reagents Spheroids grown to ~350 mm Add ATP assay reagents + DNA dye to indicate lytic effectiveness Photograph using laser confocal microscopy Promega Corporation 24 Chad Zimprich, Mike Valley

25 Critical Experiment that Prompted Effort to Validate Assays Observation of Lytic Efficiency of ATP Assay Reagents Spheroids grown to ~350 mm Add ATP assay reagents + DNA dye to indicate lytic effectiveness Photograph using laser confocal microscopy Original Other ATP Assay Reagent CellTiter-Glo Reagent ~350 mm spheroids Promega Corporation 25 Chad Zimprich, Mike Valley

26 ATP (nm) % Recovery of ATP is Lower from Very Large Spheroids (Compared to Acid Extraction Control) 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 1,000 10,000 2,000 1,800 1,600 1,400 1,200 1, CTG acid volume x 10^5 (um^3) Data suggested protocol may need to be modified to capture higher % of ATP. Promega Corporation 26

27 CellTiter-Glo 3D Assay (New Formulation) ATP Assay Improvements for 3D Culture Systems Higher concentration of detergents to more effectively lyse cells in larger 3D structures Protocol modification to incorporate Physical disruption (shaking for 5 min) Longer incubation with lysis buffer (30 min) CTG 3D has been used with microtissues formed using: hanging drop inert scaffolds Hydrogels (Matrigel Matrix and collagen) Corning Spheroid Microplates (Ultra-low Attachment surface) Promega Corporation 27

28 ATP Recovery from Cells Cultured Four Days with Matrigel Matrix: Comparison of ATP Assays Promega Corporation 28

29 Samples in Corning Spheroid Plates Treated with CTG 3D and Shook 2 Min with Vibroturbulator Promega Corporation 29

30 Flow Diagram of Protocol used with Corning Spheroid Microplate Add cells Photograph Incubate 4 days Photograph Add CellTiter-Glo 3D Reagent Shake 2 min Photograph Promega Corporation 30 30

31 Images of 2D and 3D Cultures (Ultra-Low Attachment Plates) 3D cultures provide a more physiologically relevant model because they more accurately reflect the intricate microenvironment of tumors in vivo 1 Multicellular spheroids (MCS) are the most commonly used 3D tumor models HT-29 2D monolayer on TC-treated surface HT-29 3D multicellular spheroids on Ultra-Low Attachment surface 1 F. Pampaloni, et al., The third dimension bridges the gap between cell culture and live tissue. Nat. Rev. Mol. Cell Biol. 8(10), (2007) Corning Incorporated 31

32 Corning Spheroid Microplate Corning Ultra-Low-Attachment surface and unique round well-bottom design enable the formation and growth of a single, uniform spheroid per well with reproducible size Standard ANSI/SBS footprint dimensions for 96-well and 384-well formats Clear bottom to allow visualization and imaging Black sidewalls to reduce cross-talk and background noise in fluorescent- and luminescent-based assays 2014 Corning Incorporated 32

33 DU-145 Spheroids 72 hr 48 hr 24 hr Corning Spheroid Microplates Generate Uniform, Single MCS per Well in a 96-well Format (Corning Cat. No. 4520) 40 cells/well 200 cells/well 1,000 cells/well 5,000 cells/well 10,000 cells/well Scale bar = 1,000 μm 2014 Corning Incorporated 33

34 Key Claims Reproducible and uniform 3D MCS formation across all wells in plug and play microplate formats Single spheroid formed per well Centering of the spheroid in each well aids automated visualization Ease of media exchange and/or treatment with test compounds Generation, assaying, and processing of spheroids in the same well without the need for a transfer step Ultra-Low Attachment Surface is a hydrophilic, non-ionic coating designed to prevent cells from attaching Enables growth of distinct, unattached colonies, or spheroids 2014 Corning Incorporated 34

35 Simple Plug and Play Protocol 1. Plate cells 2. Generate and culture spheroids in plate 3. Assay spheroid in well 24 hr 48 hr 72 hr 4. Process/read plate to generate data 2014 Corning Incorporated 35

36 Cells Validated Using Corning Spheroid Microplate and Promega CellTiter-Glo 3D Cell Viability Assay Cell line MCF7 A549 IMR-32 Detroit 562 HCT 116 HT-29 BT-474 Tissue Human mammary gland, breast; Adenocarcinoma Human lung; Carcinoma Human brain; Neuroblastoma Human pharynx; SCC Human Colon; Colorectal carcinoma Human Colon; Colorectal adenocarcinoma Human mammary gland, breast/duct; Ductal carcinoma ATCC Cat. No. HTB-22 CCL-185 CCL-127 CCL-138 CCL-247 HTB-38 HTB-20 Multicellular formation Cluster Spheroid Spheroid Spheroid Spheroid Spheroid Spheroid HEK-293 Human embryonic kidney CRL-1573 Spheroid 5/9 m alpha3-18 DU 145 Hamster (CHO-K1 derived); M-CSF production Human prostate; Carcinoma CRL HTB-81 Cluster Spheroid 2014 Corning Incorporated 36

37 Assays with Corning Spheroid Microplates are Automation-friendly: Highly Reproducible Results with Minimal Variation Cells were dispensed into 384-well Spheroid microplates using a Multidrop Combi reagent dispenser (Thermo Scientific) After 72 hours in culture, spheroids were washed once with PBS using an AquaMax DW4 microplate washer (Molecular Devices) To quantify viable cells, a 1:1 mixture of CellTiter-Glo 3D reagent and media were added directly to wells Z values >0.5 and %CV <20% indicate an acceptable quality assay with low variability 2014 Corning Incorporated 37

38 Corning Spheroid Microplates and CellTiter-Glo 3D Cell Viability Assay for MCS Drug Screening MCS response after 72-hour treatment with a dose response of Doxorubicin (Representative data, n=8 wells per concentration). Cells were plated and cultured in spheroid microplates for 24 hours prior to treatment in a dose-dependent manner with Doxorubicin Doxorubicin was added directly into wells Toxicity was evaluated over a 72-hour period by adding CellTiter-Glo 3D reagent in a 1:1 ratio directly into wells and assessing cell viability 2014 Corning Incorporated 38

39 Summary Corning Spheroid Microplates are an optimal tool for generating, culturing, and assaying multicellular spheroids Uniform, single spheroid formation in every well aids in maintaining reproducibility and accuracy of assay The design of the spheroid microplates allows for incorporation of automation and high throughput screening applications Promega CellTiter-Glo 3D Cell Viability Assay is ideal for generating quantifiable data from multicellular spheroids Superior lytic ability and signal sensitivity allows for use over a broad range of spheroid types and sizes Homogenous one step reagent is optimal for automation and high throughput screening 2014 Corning Incorporated 39

40 Luminescence (RLU x 10-6 ) Fluorescence (RFU x 10-3 ) Multiplexing ATP Detection and DNA Staining using 3D Spheroids 3.5 CellTiter-Glo ATP 3D CellTox DNA Staining Green panobinostat (mm) HCT116 cells were cultured for 4 days to form ~350 mm microtissues. Samples were treated with CellTox Green and panobinostat for 48 hr. After recording fluorescence, an equal volume of CellTiter-Glo 3D Reagent was added, plate was shaken for 5 min, and the luminescence was recorded after a 30 min incubation. Promega Corporation 40

41 RealTime-Glo TM Cell Viability Assay Avoids Problems Associated with Lysis of Spheroids Live Cell Dead Cell NanoLuc protein sensor is present outside of the cells Pro-substrate enters the cell and is reduced by the cell to form a substrate for NanoLuc luciferase Substrate diffuses from the cell and is rapidly used by NanoLuc to produce light Promega Corporation 41

42 Measuring Changes in Cell Viability During Three Days of Culture Cells plated in media containing Real Time Cell Viability reagents Add drug Continuous luminescence readings Doxorubicin (nm) Analyze viability data Doxorubicin Time (h) IC50 (nm) 8 ND 16 ND Promega Corporation 42

43 Continuous Real-Time Monitoring of Drug Induced Changes in Cell Viability Cells plated in media containing Real Time Cell Viability reagents Continuous luminescence readings Cell Treatment Continuous luminescence readings Add digitonin Analyze viability data icell Cardiomyocytes (CDI) cultured in medium containing the Real Time Cell Viability reagent. After 2 days of cell growth, digitonin was added to a final concentration of 200 µg/ml. Promega Corporation 43

44 RealTime-Glo TM Assay Signal Correlates with Spheroid Size Promega Corporation 44

45 RealTime-Glo Assay of Single Spheroids (Cells Remain Viable Following Assay) Promega Corporation 45

46 Multiplex RNA Extraction from Single Spheroids Following Cell Viability Assay 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).. Promega Corporation 46

47 Validating Caspase Assays of 3D Spheroids a Different Approach Instability of the caspase enzyme limits ability to increase detergent concentration to lyse large spheroids Stability of luciferase in reagent is not an issue Caspase-Glo 3/7 Assay protocol was modified to incorporate: Physical disruption (shaking for 5 min) Longer incubation time with lysis buffer (30 min total) Promega Corporation 47

48 Caspase Assay Protocol Optimization to Facilitate Enhanced Spheroid Cell Lysis HCT116 cell spheroids grown to ~330 µm diameter Add Caspase-Glo 3/7 assay reagent + DNA dye to indicate lysis 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 improved spheroid cell lysis Promega Corporation 48

49 What is Appropriate Control for a Luciferase Reporter Assay of 3D Spheroids? How can you tell if the lysis buffer is extracting all the luciferase activity? Antibody detection is not reliable because it indicates quantity of antigen, not necessarily active luciferase. Correlation with ATP as a marker is an option. Promega Corporation 49 49

50 Measurement of Luciferase Reporter Activity from Different Size Microtissues Constitutive NanoLuc Reporter HCT116 cells expressing NanoLuc luciferase under a constitutive promoter (A) or a HIF-1 promoter (B) 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, and luminescence recorded after a total of 30 min incubation. Promega Corporation 50

51 Measurement of Luciferase Reporter Activity from Different Size Microtissues HIF-1 Hypoxia Marker HCT116 cells expressing NanoLuc luciferase under a constitutive promoter (A) or a HIF-1 promoter (B) 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, and luminescence recorded after a total of 30 min incubation. Promega Corporation 51

52 Measurement of Luciferase Reporter Activity from Different Size Microtissues HIF-1 Hypoxia Marker HCT116 cells expressing NanoLuc luciferase under a constitutive promoter (A) or a HIF-1 promoter (B) 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, and luminescence recorded after a total of 30 min incubation. Promega Corporation 52

53 Path Forward Improving Lysis of Large Spheroids General Approach Change detergent formulation when possible Longer incubation in lysis solution Incorporate mixing or physical disruption step Identify control to ensure marker is being measured Promega Corporation 53

54 Physical Disruption Options Plate shakers to mix 96 or 384 well plates (motion and speed matter; e.g. Union Scientific) Pipetting sample up-and-down can be used instead of shaking, but our results show greater variability among replicates Ultrasonic treatment (potential for future) Promega Corporation 54

55 Take Home Message There is a growing number of approaches to choose from for generating 3D spheroids Most in vitro cell-based assays were originally designed for 2D monolayers or cells in suspension Validation of assays with each cell type and 3D model is recommended Size / mass of 3D structure may limit efficient lysis of all cells or overwhelm assay chemistry Optimizing incubation time with lysis solution and mixing or physical disruption are recommended first steps for validating assays Promega Corporation 55

56 Take Home Message The ease of use of the Corning Spheroid Microplates in combination with Promega s cell health assays that have been demonstrated to work with spheroids provide an attractive option for performing 3D cell culture experiments. Promega Corporation 56 56

57 Questions Welcome Promega Corporation