Presentation is loading. Please wait.

Presentation is loading. Please wait.

High Throughput Biomedicine Unit (HTB) at FIMM Technology Centre

Similar presentations


Presentation on theme: "High Throughput Biomedicine Unit (HTB) at FIMM Technology Centre"— Presentation transcript:

1 High Throughput Biomedicine Unit (HTB) at FIMM Technology Centre

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

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

4 High throughput reader
What is needed for a HTS? High throughput reader High density format

5 High throughput reader
What is needed for a HTS? High throughput reader High density format Data analysis

6 HTB Equipment 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 Collaborations 550 525 550

8 High Throughput Biomedicine Unit Personalized medicine
RNAi screening Chemical screening Microarray printing 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 Sharing of chemicals with academic groups Personalized medicine ex vivo testing of patient Cells with oncology drugs: Leukemia Ovarian cancer Glioblastoma Assay miniaturisation

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 Reporter assays, cell death assays, soon beta-arrestin complementation assays, aplha screens

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) dd.mm.yyyy Presentation name or name of the guest

11 siRNA administration (nl)
siRNA Screen Workflow Cell culture 384 well assay plates siRNA administration (nl) 1) Dispensing of transfection reagent on assay plates 2) Dispensing of cells on the plates siRNA libraries on 384 well ECHO plates Luminescence / intensity readout Dispensing of detection reagent on cells Incubation 72 h-7 days at 37oC Source plate 384 wells, working volumes: Standard vol plate µl Low dead vol plate µl In acoustic droplet ejection volumes are transferred from one well to another in 2,5 nl droplets. A Microscopy readout Fixing and staining of cells B

12 High Content Screening
cells Picture Microscope Microtiter plate Image analysis Numbers

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 Reporter assays, cell death assays, soon beta-arrestin complementation assays, aplha screens

14 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 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 ChemDiv Peptidomimetics ChemDiv TP02 and TP03 collections ChemBridge DivSet and CNS Set Specs Consortium collection

15 Drug sensitivity and resistance testing (DSRT)
Cell culture 384 well assay plates Drug administration (nl) Dispensing of cells on the plates -patient cells -cell lines Oncology collection 306 drugs 5 conc. 10,000-fold conc. range Incubation 72 h at 37oC Dispensing of detection reagent on cells -cell viability mesurement Luminescence readout Source plate 384 wells, working volumes: Standard vol plate µl Low dead vol plate µl In acoustic droplet ejection volumes are transferred from one well to another in 2,5 nl droplets. 300 dose response curves Microscopy readout under development Resistant drugs Effective drugs

16 What is in the FIMM oncology collection?
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… Rapamycin analogs Epigenetic modulators mTOR/PI3K inhibitors PARP inhibitors PI3K inhibitors Hh inhibitors γ-secretase inhibitors Bcl-2 inhibitors Farnesyltransferase inhibitor HSP90 inhibitors p53 activators Survivin inhibitor Metabolic inhibitors… HDACi:s 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 16

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

18 Reverse Phase Protein Array (under development)
Cells on plates, Incubation 72h Heating of the plates at +95C Transfer of the lysates on the arrayer compatible plates Spotting with Aushon 2470 Cell lysis Technically, minuscule amounts of a) cellular lysates, from intact cells or laser capture microdissected cells, b)body fluids such as serum, CSF, urine, vitreous, saliva, etc., are immobilized on individual spots on a microarray that is then incubated with a single specific antibody to detect expression of the target protein across many samples SyproRuby staining of the slides Analysing the scans using Array-Pro Analyzer Scanning the slides with Li-Cor Odyssey Staining the slides with antibodies Scanning the slides for tot. protein

19 Sharing of chemicals with academic groups
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

20 Overview of the screening process
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

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 06/04/2017

22 Published papers Antila H. et al Utilisation of in situ ELISA method for examining Trk receptor phosphorylation in cultured cells. J Neurosci Methods Stylinaou M. et al Antifungal application of non-antifungal drugs. Antimicrob Agents Chemother. PemovskaT. et al Individualized Systems Medicine (ISM) strategy to tailor treatments for patients with chemorefractory acute myeloid leukemia. Cancer Discovery Tang J. et al Target Inhibition Networks: Predicting Selective Combinations of Druggable Targets to Block Cancer Survival Pathways. PLOS Computational Biology Nybond S. et al 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 Obatolax, Saliphenylhalamide, and Gemcitabine Inhibit Influenza A Virus Infection. J.Biol. Chem. Koskela H.L.M. et al Somatic STAT3 Mutations in Large Granular Lymphocytic Leukemia. NEJM 06/04/2017

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


Download ppt "High Throughput Biomedicine Unit (HTB) at FIMM Technology Centre"

Similar presentations


Ads by Google