The Bright Side of 3D Biology

Transcription

1 The Bright Side of 3D Biology Wolfgang Moritz, Ph.D. Head of Research and Applications at InSphero Discover Glo! Work Shop March 13th

2 Agenda 1. Introduction to InSphero 2. Do we need 3D culture? 3. Short overview of different 3D culture methods 4. InSphero s GravityPLUS system 5. Read outs for Imaging Biochemical assays 6. Examples Toxicity screening Metabolic investigations 2

3 Introduction InSphero InSphero in a Nutshell HQ in Switzerland, offices in the US and Germany Founded in 2009; 30+ employees (Switzerland/US) WINNER 2014 ISO 9001 certified since 2011 Strategic partners: Nature, April 2013 Strong customer base in pharmaceutical, chemical and cosemtics industry: 3

4 Offering a complete portfolio of 3D cell culture products and services Introduction InSphero GravityPLUS Platform 3D InSight Microtissues 3D InSight Growth Medium 3D InSight Services 3D InSight Assay Kits Cell 3 imager 4

5 InSphero s ready-to-use organotypic 3D InSight microtissue portfolio Introduction InSphero 5

6 Why 3D cell culture? Reasons to use 3D culture systems Tumor biology Recreation of biochemical and chemical gradients (oxgygen, nutrients, catabolites, juxtacrine factors, ph) Altered ECM compositions affects tumor signalling Stroma/Tumor interaction can better be simulated by co-culture systems in 3D, which is highly relevant for tumor survival/progression. Study of drug penetration (biologicals, ACD) or infiltrating immune cells in confrontational assays Drug Discovery/Safety Maintenance of epithelial phenotype in primary cultures Simulation of cell-cell interactions in reflecting organ biology more closely Recreate modulatory effects by non-parenchymal cells (e.g. endothelia cells, inflammatory cells, or stromals cells) For disease modelling. Certain pathologies dependent on a 3D configuration: inflammation, fibrosis, infection, cholestasis, cyst formation. Stem cell biology Epigenetic regulation by microenvironmental cues (physico-chemical gradients, cell-cell communication, ECM interaction) 6

7 Microtissue: an in vitro representative of a body s organ Why 3D cell culture? Organ OrganMicrotissue Human hepatic vasculature cast (adapted from Charlotte Debbaut; University of Gent) Microtissues represent: The minimal functional, non-perfused unit of an organ The in vivo correlate of a non-vascular portion of a tumor 7

8 Replication of solid tumor complexity Heterogenity cannot be reflected in 2D cultures Why 3D cell culture? Represents in vivo biology Simulates in vivo pharmacodynamics po 2 ph Metabolic activity Challenges classical in vitro assays Compound /substrate concentration Cell death periphery center periphery 8

9 Organs are represented by an complex network of different cell types. Example Liver Native Spheroid model Bile canaliculi network of human liver microtissues Different cell types Hepatocytes Kupffer cells Stellate cells Endothelial cells Liver progenitor cells Duct cells Leukocytes Intricate cellular arrangement is crucial for organotypic function (polarized alignement) Bile canaliculi network of native human liver Why 3D cell culture? Pictures kindly provided by J.Hengstler, IfADo, Germany 9

10 3D Culture Methods 3 basic - 3D concepts Hydrogel-based scaffolds Providing natural ECM for 3D arrangement Collagen, Matrigel, Alginate, PEG Physical scaffolds Providing an adhesive biomaterial to enforce 3D Polystyrene, ceramics, etc. Scaffold-free microtissue engineering No scaffold required Cells produce endogenous ECM 10

11 The hanging drop culture method How InSphero s GravityPLUS System works GravityPLUS System Benefits of system: Scaffold-free and highly functional Long lifetime Assay-ready Highly reproducible 11

12 norm ATP Microtissue read outs Read outs Imaging Biochemical Assays Histology Omics FACS Staurosporine [µm] 12

13 Label-free assaying of 3D models: Dainippon SCREEN Cell 3 imager Imaging 13

14 3D phenotypic efficacy study Plate scan with Cell 3 imager Imaging Day 0 Day 4 Gem/Doc Gem/Doc Day 7 ATP Size Gem/Doc Growth kinetics (size) Endpoint analysis, size and ATP (day 7) 14

15 Assay types for cell based experiments Biochemical Assays Medium Spt Non-lytic assays Reporter Assays Lytic assays Metabolism Carbohydrate, AA, Lipids, bile salts, xenobiotic metabolites (LC-MS) Reduction of tetrazolium salts or Resazurin (enzymatic, colometric, fluorometric) ATP, GSH/GSSG (ox. stress) (luminometric) Viability Cell death Proliferation Membrane integrity assays, e.g. LDH, DCP, ALP, αgst (enzymatic, colometric, fluorometric) Size (image analysis) Live/dead staining (Calcein-AM/Eth-HD), (microscopy) Stably or transient transfected cells expressing reporter genes, e.g. GFP, RFP, Luc (fluometric, luminometric) ATP, LDH, DCP, caspase (fluorometric, luminometric) Tissue/pathway specific function e.g. Albumin, Urea, Insulin, Glucagon, VEGF, Chemokines, Cytokines, Neurotransmitters (enzymatic, ELISA) CYP450 Stably or transient transfected cells expressing reporter genes driven by a tissue- or pathway specific promotor (fluometric, luminometric) camp, HDA, GPCR, phase I+II metabolic enzymes (luminometric) 15

16 Assay comparison Biochemical Assays STS HCT-116 microtissues (500 cells/drop) Dose-response with Staurosporin for 72 hrs Assay Endpoint IC50 CellTiter Glo Intracellular ATP (viability&metab olic activity) 9 nm GSH/GSSG Glo CytoTox One (intracellular LDH) CytoTox Glo Reduced Glutathion (Redox state) Intracellular LDH activity (viability) Extracellular DCP activity (necrosis) 72 nm 57 nm 121 nm 16

17 Promega s Biochemical assays evaluated with InSphero s microtissues Biochemical Assays Lytic Assays Cell Viability/Cytotoxicity CellTiter-Glo (2.0/3D) Apoptosis Caspase-3/7 Glo Redox State GSH/GSSG-Glo Metabolism P450-Glo Luciferin-IPA Non-Lytic Assays Cell Viability/Cytotoxicity CytoTox-ONE (fluo) NanoLuc RealTime Glo LDH Glo Metabolism Lactate Glo Glucose Glo Glucose Uptake Glo 17

18 New 3D tailored CellTiter Glo assay Biochemical Assays CellTiter-Glo 3D Assay o Ultra-Glo rluciferase o Add-mix-measure homogeneous assay format o Improved, highly efficient lytic reagent for ATP extraction Conventional Assay CellTiter Glo 3D ATP recovery: 27% ATP recovery: 93% 18

19 Latest and upcoming additions of Luciferase based assays Biochemical Assays RealTime-Glo MT Cell Viability Assay Viability/Metabolic assay Simple to handle Non-invasive Repeated or continous measurements possible Bioluminescent LDH release assay (in development) Cytotoxicity assay Highly sensitive Non-invasive Repeated measurements possible Backround affected by serum/pyruvate 19

20 Bioluminescent LDH release assay measured on human liver microtissues upon treatment with different liver toxic compounds Biochemical Assays Data kindly provided by Jolanta Vidugiriene, Promega 20

21 CellTiter Glo The «Screening Workhorse» Toxicity Screening 21

22 Toxicity Screening Establish Model for Long Term Toxicity Study Conventional strategy Human Liver Microtissues Chlorpromazin re-dosing Spt collection Spt Spt Chlorpromazin Anti-Psychotic drug, induces occasionally cholestatic and acute liver injury Day Viability/Metabolism Functionality Lytic Spt ATP (CellTiter Glo), requires multiple MTs Albumin (ELISA) Time consuming, expensive 22

23 Continuous monitoring of Toxicity Toxicity Screening Day 14 Vehicle Control Chlorpromazin 200 µm Shift of IC50 Assay IC50 [µm] Day 1 IC50 [µm] Day 4 IC50 [µm] Day 14 CellTiter Glo Shift of IC50 Albumin ELISA Cmax 0.94uM Disadvantage: CellTiter Glo is a lytic assay endpoint measurement Disadavantage: expensive and time consuming 23

24 Toxicity Screening Establish Model for Long Term Toxicity Study Human Liver Microtissues Chlorpromazin re-dosing Spt collection Spt Spt Spt Spt Chlorpromazin Anti-Psychotic drug, induces occasionally cholestatic and acute liver injury Day Viability non-lytic RealTime Glo (Promega) Necrosis Spt LDH Glo (Promega) Alternative strategy 24

25 RLU Toxicity Screening LDH stability in culture? LDH stability test (0.01units/ml) fresh 1 fresh 2 24h, 4 C 96h, 4 C 24h, 37 C in presence of hlimt 96h, 37 C in presence of hlimt 25

26 Toxicity Screening LDH measurement allows to follow toxic effect over time viable Assay IC50 [µm] CellTiter Glo 14.7 RealTime Glo ~ 23 Albumin ELISA 17.4 Cmax ~0.94 ongoing necrosis past necrosis 26

27 New release of luminescence based metabolite assays coming soon Metabolic Investigations 27

28 Combining viability assays with metabolite analysis all conveniently assessed by luminescent readings Metabolic Investigations 3) Drop in ATP content due to necrosis 1) Reduced ATP ontent due to metabolic switch? 2) Obviously no change in lactate production? 28

29 Thanks to Madison (WI): Dan Lazar Mike Valley Terry Riss Jolanta Vidugiriene Switzerland: Réka Nagy Andrea Lindemann Brigitta Saul Mareike Appelt Roland Schlatter Zoé Weydert Jennifer Bourland Simon Messner David Fluri Nadia Carco «HTS-DILI» «The Body on a Chip» 29