A new tool for screening : Cellular imaging (HCA/HCS)
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1 Biotechnology company specialized in cellular imagery which provides HCA/HCS services A new tool for screening : Cellular imaging (HCA/HCS) Applications : Efficacy & Toxicoloxy assays Julian BURSZTYKA, PhD Nathalie MAUBON, PhD
2 Plan Company Context Applications Services offering 2
3 Plan Company Context Applications Services offering 3
4 HCS Pharma Offices a Laboratories in Rennes (Biopôle) dedicated to : - Cell culture - Automation : multiprobe (Perkin), ALH 3000 (Caliper) - Imaging: BD Pathway, Operetta Caliper ALH , 96 & 384-channel & gripper Multiprobe IIex Perkin 4-channel independent CIR * Authorisation BD Pathway 855 CIR = Crédit Impôt Recherche = Research Tax Credit 4
5 Strenghthened links with the University of Rennes Platform Access -MRIc : Photonic Microscopy, Transmission Electron Microscopy (TEM)... Confocal Leica SP8 SP5 Confocal olympus Video DMRIB - H2P2 : histopathologie & cytologie Laser Microdissection Automation of markings Scan histologie 5
6 Partnerships with research units of the University of Rennes UMR 6290 CNRS Université de Rennes 1 Specialists of interactions UV/skin, melanoma study 6
7 Plan Company Context Applications Services offering 7
8 Cellular Imaging: Increase in information vs. classical in vitro assays Classical in vitro assays (HTS) Plate reader +/- cell culture Cellular Imaging (HCS) Imager + cell culture Resuts A1 354 A A3 214 A4 143 Count WL1 WL2 WL3 size A A A A One value per well (absorbance, fluorescence, luminescence, etc.) Few information on biological content No measure on individual cell Multiples values per well, means on populations, statistics on individual cells Information on biological content : number of cells, sizes, shapes Identification cell by cell, measures of subpopulations and identification of rare cell events
9 Cell Imaging Benefits Highly visual Multiparametric Nucleï Co-culture neurons / macrophages Mitochondria Screening / Phenotypic analysis ROS Enriched information on biological content Primary Keratinocytes 9
10 Cellomic place in preclinical studies Cellomic (Phenotypic screening) Genomic Transcriptomic Proteomic Study of genes Study of ARN Study of proteins * * Study of cells * Studies of proteins by proteomic allow to identify a lot of proteins but do not give information on their localisation or activity. Phenotyping screening allow up to 3 markers but the Imaging Technology can bring the informtions on localisation and activity. Other advantages of cellomic: - In genomic studies, genes are not always the same level of expression - In transcriptomic studies, ARN activity is not always understood 10
11 Quantification by image analysis Image Segmentation: detection of regions of interest Analysis : intensity, surfaces, textures, colocalisation, translocation, Quantification 11
12 Plan Company Context Applications Services offering 12
13 HCS applications Dermatology/Pharmacology : Phenotypic screening & content screening : potentially in the same experiment (Up to 4 simultaneous markings) Phenotypic Screening : Cell cycle analysis, apoptosis, morphology, texture, measurement of ROS, mitochondrial viability Content screening : induction, phosphorylation, translocation of a targeted protein, colocalization of two targets 13
14 HCS applications Toxicology/Safety : Neogenesis Neurites aspect Double strand breaks, micronucleï UV Protection, moisturizing, redness Cardiotoxicity assays Co-cultures, 3D Primary cells, cell lines Steatosis, phospholipidosis 14
15 HCS applications Practical examples : UV protection (Measurement of ROS after UV-irradiation) Moisturizing (Measurement of AQP3 in the membrane of keratinocytes) Anti cellulite (inhibition of the differentiation of 3T3 cells in adipocytes) Customized experiments in close partnership 15
16 Genotoxicity : g-h2ax (Double-stranded DNA break marker) on human primary keratinocytes Etoposide CTRL DMSO Hoechst P-Histone H3 g-h2ax Merge CTRL Etoposide 16
17 Genotoxicity : g-h2ax (Double-stranded DNA break marker) on human primary keratinocytes Etoposide CTRL DMSO Quantification : Upstream screening and early removal of toxic assets for R&D of active ingredients 17
18 Genotoxicity : Comet Assay Development of a high throughput assay: 18
19 General toxicity : Steatosis & phospolipidosis 48h of treatment on HepG2 cell lines CTRL cells Rifampicin Cyclosporin A Sertraline Amiodarone Hoechst (blue), steatosis (green), phospholipidosis (red) Steatosis Phospholipidosis Cell Count 19
20 Measure of Aquaporine 3 (moisturizing parameter) on primary human keratinocytes Primary human keratinocytes undergoing differentiation Differentiated primary human keratinocytes In blue: Nucleï (hoechst), In yellow: aquaporin 3 (ICC-IF) Benchmark inducer = glyceryl glucoside - + Quantification
21 Antioxidant protection on primary human keratinocytes NAC - + Menadione CTRL DMSO NAC = N-acetylcystein Menadione Menadione+ NAC Ctrl + 0,5% DMSO ROS (Inducing Factor) 1,99 1,42 1 NAC protecting factor = 57,7% 100 µm Menadione alone or with 50 µm N-acetylcystein, n = 6 ROS = Reactive Oxygen Species In development: UV-Induced cell protection 21
22 Antiinflammatory effect on primary human keratinocytes In development: Inhibition of nuclear translocation of NF-kB, Inhibition of Nitric Oxid production, IL-8 Primary human keratinocytes Blue : Hoechst stained nucleï Green : NF-kB/P65 (ICC-IF) 22
23 Plan Company Context Applications Services offering 23
24 Typical assay Always Includes at least one cell count (toxicity) Includes the quantification of one or more endpoints of interest. = Multi-parametric assays - Free choice of number of repliquas and concentrations - Test of actives in mixes (Only one requirement = solubility) 24
25 Services offering Qualified models, routine studies Custom-models developement CIR authorisation 25
26 Thanks for your attention HCS Pharma: Expert assessment Efficiency Close partnership Transparency Acquisition & images analysis Experimentation Results & statistics Reporting and follow up Study design 26
27 CONTACTS Nathalie MAUBON PhD in biology CEO, CSO Julian BURSZTYKA PhD in toxicology COO HCS Pharma 6 Rue Pierre Joseph Colin RENNES Phone : Mail : cosmeto@hcs-pharma.com 27
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