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Development of in vitro sponge cell culture - Wageningen UR

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<strong>Development</strong> <strong>of</strong><strong>in</strong> <strong>vitro</strong> <strong>sponge</strong> <strong>cell</strong> <strong>culture</strong>IPOP symposium 29 th <strong>of</strong> JanuaryKlaske J. Schippers 1Dirk E. Martens 1Shirley A. Pomponi 2René H. Wijffels 11. Bioprocess Eng<strong>in</strong>eer<strong>in</strong>g, Wagen<strong>in</strong>gen University2. Harbor Branch Oceanographic Institution, USA


Why <strong>sponge</strong>s?Sponges are a source for bioactivecompounds with pharmaceutical <strong>in</strong>terestantibiotic, antifoul<strong>in</strong>g, antiviral,anti<strong>in</strong>flammatory, cytotoxic, antitumordiscodermalideantitumor2 <strong>of</strong> 16avarolanti HIVhalichondr<strong>in</strong> Banticancer


3 <strong>of</strong> 16Why <strong>sponge</strong>s?


What are <strong>sponge</strong>s?AnimalSessile, benthic organismInvertebrateFilter feeder4 <strong>of</strong> 16


How to produce?primmorphswelldef<strong>in</strong>ed & controlledex situproduction <strong>of</strong> bioactivecompounds<strong>cell</strong> <strong>culture</strong>wild harvestchemical synthesismari<strong>culture</strong>5 <strong>of</strong> 16


How to make a <strong>sponge</strong> <strong>cell</strong> <strong>culture</strong>?Ca 2+ /Mg 2+ free seawater,<strong>cell</strong> stra<strong>in</strong>er 70 µm, centrifugationSupernatantconta<strong>in</strong>s bacteriaDysidea avaraPellet conta<strong>in</strong>s<strong>sponge</strong> <strong>cell</strong>sCryopreserveReady forexperiments10 5 10 6 <strong>cell</strong>s/mland measureviabilityDissolve <strong>in</strong><strong>sponge</strong> <strong>cell</strong><strong>culture</strong> medium6 <strong>of</strong> 16


In <strong>vitro</strong> <strong>sponge</strong> <strong>cell</strong> <strong>culture</strong>major bottleneckscontam<strong>in</strong>ation bysymbiontslack <strong>of</strong> proliferation7 <strong>of</strong> 16


Contam<strong>in</strong>ation by symbionts What to do?antibioticsscreen<strong>in</strong>g <strong>of</strong> eukaryotic populationcollect <strong>cell</strong>s from larvae/embryo’s8 <strong>of</strong> 16


Screen<strong>in</strong>g: 18S rDNAPCR DGGEAx<strong>in</strong>ella corrugatamtime seriesmtime seriesmHaliclona oculata9 <strong>of</strong> 16


Lack <strong>of</strong> proliferation What to do?viability assayapoptosisprimary <strong>cell</strong> <strong>culture</strong>simmortaliz<strong>in</strong>g10 <strong>of</strong> 16


Viability assayFluorescence microscopeFDA green alivePI red dead20 umDysidea avara11 <strong>of</strong> 16


Immortaliz<strong>in</strong>gImmortaliz<strong>in</strong>g genes:hTERTSV40EBVtransfection should be optimizedexpression<strong>of</strong> gene20 umGFP <strong>in</strong> Haliclona oculata12 <strong>of</strong> 16


Transfection optimization Is DNA taken up by <strong>cell</strong> ?50 umlip<strong>of</strong>ection withfluorescentlabeled DNA Is gene be<strong>in</strong>g expressed? promotor metabolic activeRNApolpromotorgene13 <strong>of</strong> 16


Future Pro<strong>of</strong> apoptosis and proliferation <strong>in</strong> <strong>cell</strong> <strong>culture</strong>s Develop proliferat<strong>in</strong>g primary <strong>cell</strong> <strong>culture</strong> Develop transfection method <strong>in</strong> <strong>sponge</strong> <strong>cell</strong>s Immortalize <strong>sponge</strong> <strong>cell</strong>s14 <strong>of</strong> 16


Summary Create immortalized <strong>sponge</strong> <strong>cell</strong> <strong>culture</strong> Produce bioactive compound Cultivation <strong>in</strong> bioreactor?15 <strong>of</strong> 16


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