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© FIMM - Institiute for Molecular Medicine Finlandwww.fimm.fi High Throughput Biomedicine Unit (HTB) at FIMM Technology Centre.

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Presentation on theme: "© FIMM - Institiute for Molecular Medicine Finlandwww.fimm.fi High Throughput Biomedicine Unit (HTB) at FIMM Technology Centre."— Presentation transcript:

1 © FIMM - Institiute for Molecular Medicine Finlandwww.fimm.fi High Throughput Biomedicine Unit (HTB) at FIMM Technology Centre

2 www.fimm.fi FIMM Technology Centre High-throughput Biomedicine Biomarker validation Metabolomics Genomics & Bioinformatics IT Imaging & clinical informatics State-of-the-art equipment ~ 50 Technology experts -PIs -Senior scientists -Post Docs -Masters -Bioinformaticians -Lab technicians National and international research core unit providing an extensive spectrum of biomedical research services. High-throughput Biomedicine

3 www.fimm.fi High density format 96 well plate Vol/well= 100ul 1536 well plate Vol/well= 4 ul Microarray format 384 well plate Vol/well= 25ul What is needed for a HTS?

4 www.fimm.fi High density format High throughput reader What is needed for a HTS?

5 www.fimm.fi What is needed for a HTS? High density format High throughput reader Data analysis

6 www.fimm.fi HTB Equipment 6 BeckmanCoulter integrated robotic system -used for splitting libraries on Labcyte ECHO source plates, by pipetting -running fully automated screens with cell or biochemistry based assays Motoman robotic arm Biomek FXp pipetting robot Multidrop Combi (x2) and Combi nl (x1) Cytomat 6001-plate incubator for cells Platelock-plate sealer V-spin-centrifuge Delidding stations and plate hotels on robot deck Paradigm plate reader Cytomat 24 plate hotel Labcyte Access robotic system -used for creating assay plates with chemicals and/or siRNAs -miniaturized cell-based and biochemical screening Labcyte robotic arm Labcyte 550 Omics2 Screening2 (2.5 nl droplet) Echo 525 (25 nl droplet) Labcyte LX bulk filler (4 source liquids) Xpeel-plate peeler Platelock-plate sealer V-spin-centrifuge Delidding stations and plate hotels on robot deck

7 www.fimm.fi Collaborations 7 550 525 550

8 www.fimm.fi 8 RNAi screeningChemical screeningMicroarray printing High Throughput Biomedicine Unit Sharing of chemicals with academic groups siRNA screens miRNA screens Combination screens of siRNA, miRNA, chemicals Drug Sensistivity and Resistance Testing (DSRT) Chemical Screens Biochemical Assays Reverse Phase Protein Lysate Array (RPPA) Serum printing Oligonucleotide printing Personalized medicine ex vivo testing of patient Cells with oncology drugs: Leukemia Ovarian cancer Glioblastoma Assay miniaturisation

9 www.fimm.fi 9 1. RNAi screening Screening is mainly performed on 384-well plates (96-well possible) Any adherent cell-line is applicable Screening volume/ sample 25 ul 1 plate: 320 samples + 64 controls Readout: high content microscopy, live cell microscopy or cell viability Analysis: image analysis, statistical analysis, pathway analysis siRNA library: Ambion Silencer Select full genome Custom made library: Qiagen or other vendors

10 www.fimm.fi Presentation name or name of the guestdd.mm.yyyy 10 Ambion Silencer® Select Human siRNA Library V4 Nuclear hormones (47) Ion channels (338) Proteases (494)GPCR (380) Kinases (710) Phosphatases (298) Druggable genome (9032) Extended Druggable genome (10 415) Human Genome Collection (21 585)

11 www.fimm.fi 8.9.2014 11 Incubation 72 h-7 days at 37 o CDispensing of detection reagent on cellsLuminescence / intensity readout Cell culture 384 well assay plates 1) Dispensing of transfection reagent on assay plates 2) Dispensing of cells on the plates siRNA Screen Workflow siRNA libraries on 384 well ECHO plates siRNA administration (nl) A B Fixing and staining of cellsMicroscopy readout

12 www.fimm.fi High Content Screening Image analysis Numbers cells Picture Microscope Microtiter plate

13 www.fimm.fi 13 2. Chemical Screening Drug sensitivity and resistance testing (DSRT) Custom screens (mammalian, yeast, prokaryotes) Biochemical screens Chemical library combined with siRNA KO Screening is mainly performed on 384-well plates (1536 well possible) Cell-lines, patient cells, suspension cells... Readout: can be chosen among several plate readers, microscopy

14 www.fimm.fi HTB chemical Libraries › HTB Unit has access to several chemical & genomic libraries:  306 FIMM and NIH National Cancer Institute oncology drug collection  446 NIH Clinical collection  2000+640 Microsource and ENZO, including FDA approved drug collection and natural products  1120 Tocris Tocriscreen mini  2500 NIH NCI (National Cancer Institute) collections  6000 Tripos Structures collection  15 000 ChemDiv Peptidomimetics  25 000 ChemDiv TP02 and TP03 collections  30 000 ChemBridge DivSet and CNS Set  30 000 Specs Consortium collection 14 10.10.2013

15 www.fimm.fi 8.9.2014 15 Cell culture 384 well assay plates Dispensing of cells on the plates -patient cells -cell lines Drug administration (nl) Incubation 72 h at 37 o C Dispensing of detection reagent on cells -cell viability mesurement Luminescence readout Drug sensitivity and resistance testing (DSRT) Oncology collection  306 drugs 5 conc.  10,000-fold conc. range 300 dose response curves Effective drugs Resistant drugs Microscopy readout under development

16 www.fimm.fi What is in the FIMM oncology collection? Rapamycin analogs mTOR/PI3K inhibitors PI3K inhibitors Bcl-2 inhibitors HSP90 inhibitors Survivin inhibitor HDACi:s Epigenetic modulators PARP inhibitors Hh inhibitors γ-secretase inhibitors Farnesyltransferase inhibitor p53 activators Metabolic inhibitors… 16 Currently 306 active substances: Conventional chemotherapeutics Immunosuppressants Tyrosine kinase-type inhibitors Abl, Src, EGFR, FGFR, VEGFR, JAK, IGF1R, PDGFR, Met, ALK Kit, Flt3…. S/T-type inhibitors Aurora, PLK1, MEK, TTK, PDK1, Akt, Wee1, PKCs, Cdks, Chk1… Scattered layout for controls: Benzethonium chloride for low control DMSO for high control Algorithms and scripts are being developed to QC and analyse the data 10.10.2013

17 www.fimm.fi 17 3. Microarray Printing Serum microarray RPPA 25 mm 75 mm Oligonucleotide microarray We hope to use the ECHO in the future to print arrays (Echo Array Maker software) Reverse Phase Protein Array (RPPA) Serum samples Oligonucleotide arrays Aushon 2470 contact printer

18 www.fimm.fi Cells on plates, Incubation 72h Cell lysis Heating of the plates at +95C Spotting with Aushon 2470 SyproRuby staining of the slides Scanning the slides for tot. protein Staining the slides with antibodies Scanning the slides with Li-Cor Odyssey Analysing the scans using Array-Pro Analyzer Transfer of the lysates on the arrayer compatible plates Reverse Phase Protein Array (under development)

19 www.fimm.fi 19 Preplating of chemicals on assay plates with ECHO Distributing of single aliquots to different research groups Partner in the DDCB network, a national infrastructure for drug discovery and chemical biology research in Finland Sharing of chemicals with academic groups 10.10.2013

20 www.fimm.fi 20 1. You have an idea for a screen 2. First planning meeting -To estimate the feasibility of the project, to give hints about assay development. 3. Assay development 4. Assay robustness and quality testing -The assay needs to fill quality measurements (e.g. Z’-value, signal- to-noise ratio) 5. Second planning meeting - To estimate wether screening can be started, planning the scedule 6. Screening -pilot screen -primary screen -(counter screen) 7. Data analysis -identification of hits, pathway analysis, statistical analysis 8. Validation -required for publications Overview of the screening process

21 www.fimm.fi 08/09/2014 21 Some statistics of last year: 392 DSRT sets (627,200 samples) 150 Custom designed chemical plates (48,000 samples) A large chemical screen with 100,000 compounds for an international pharma company 2 full genome siRNA screens with image analysis (64,755 siRNAs each) ~300 plates of custom designed siRNA/miRNA + compound screens (96,000 samples) 546 distributed compound aliquotes

22 www.fimm.fi 08/09/2014 22 Published papers Antila H. et al. 2014. Utilisation of in situ ELISA method for examining Trk receptor phosphorylation in cultured cells. J Neurosci Methods Stylinaou M. et al. 2013. Antifungal application of non-antifungal drugs. Antimicrob Agents Chemother. PemovskaT. et al. 2013.Individualized Systems Medicine (ISM) strategy to tailor treatments for patients with chemorefractory acute myeloid leukemia. Cancer Discovery Tang J. et al. 2013. Target Inhibition Networks: Predicting Selective Combinations of Druggable Targets to Block Cancer Survival Pathways. PLOS Computational Biology Nybond S. et al. 2013. Antimicrobial assay optimization and validation for HTS in 384-well format using a bioluminescent E. coli K-12 strain. EUFEPS Denisova O.V. et al. 2012. Obatolax, Saliphenylhalamide, and Gemcitabine Inhibit Influenza A Virus Infection. J.Biol. Chem. Koskela H.L.M. et al. 2012. Somatic STAT3 Mutations in Large Granular Lymphocytic Leukemia. NEJM

23 www.fimm.fi FIMM High Throughput Biomedicine Unit 23 Evgeny Kulesskiy (Graduate stud.) Karoliina Laamanen (MSc) Anna Lehto (BSc) Vilja Pietiäinen (PhD) Swapnil Potdar (MSc) Jani Saarela (PhD) Contact Details: Chemical screening, DSRT: jani.saarela@fimm.fi siRNA screening, imaging: carina.vonschantz-fant@fimm.fi Unit Head: Krister.wennerberg@fimm.fi Contact Details: Chemical screening, DSRT: jani.saarela@fimm.fi siRNA screening, imaging: carina.vonschantz-fant@fimm.fi Unit Head: Krister.wennerberg@fimm.fi Carina von Schantz-Fant (PhD) Laura Turunen (Lic.Sc.) Heidi Virtanen (PhD) Krister Wennerberg (PhD) Päivi Östling (PhD)


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