HeLa | EPFL Graph Search

Les cellules HeLa sont une lignée cellulaire cancéreuse utilisée en biologie cellulaire et en recherche médicale. Ces cellules proviennent d'un prélèvement de métastases effectué sans consentement sur une patiente Afro Américaine atteinte d'un cancer du col de l'utérus et décédée en 1951, Henrietta Lacks. Ses cellules sont d'un usage extrêmement courant dans les laboratoires de recherche de biologie. Une lignée « immortelle »
thumb|Image de culture de cellules HeLa Les cellules HeLa forment la première lignée cellulaire immortelle d'origine humaine jamais établie par George Gey (une lignée immortelle de cellules d'origine animale avait été créée 11 ans auparavant par Wilton Earle). Ces cellules présentent la particularité de se diviser indéfiniment. Elles n'ont pas de système de régulation, ce qui fait qu'elles ne peuvent que proliférer (dans des conditions de culture en laboratoire). Les chercheurs de l'université de Washington expliquent ce phénomène inhabituel par

Engineering Agrobacterium tumefaciens Adhesion to Target Cells

Zaïnebe Al-Mayyah, Alexandre Louis André Persat, Alix Kim Lan Pham, Xavier Jean-Yves Pierrat, Jeremy Pak Hei Wong

Agrobacterium tumefaciens is a plant pathogen commonly repurposed for genetic modification of crops. Despite its versatility, it remains inefficient at transferring DNA to many hosts, including to animal cells. Like many pathogens, physical contact between A. tumefaciens and host cells promotes infection efficacy. Thus, improving the strength and specificity of A. tumefaciens to target cells has the potential for enhancing DNA transfer for biotechnological and therapeutic purposes. Here, we demonstrate a methodology for engineering genetically encoded exogeneous adhesins at the surface of A. tumefaciens. We identified an autotransporter gene we named Aat that is predicted to show canonical beta-barrel and passenger domains. We engineered the beta-barrel scaffold and linker (Aat(beta)) to display synthetic adhesins susceptible to rewire A. tumefaciens to alternative host targets. As a proof of concept, we leveraged the versatility of a VHH domain to rewire A. tumefaciens adhesion to yeast and mammalian hosts displaying a GFP target receptor. Finally, to demonstrate how synthetic A. tumefaciens adhesion can improve transfer to host cells, we showed improved protein translocation into HeLa cells using a sensitive split luciferase reporter system. Engineering A. tumefaciens adhesion has therefore a strong potential in generating complex heterogeneous cellular assemblies and in improving DNA transfer efficiency against non-natural hosts.

AMER CHEMICAL SOC2022

Development of high content image-based cell viability assays

Julien Mena

Aim and applications: 1. Optimize fluorescence-based cellular assays for getting insights about mechanisms leading to cell death 2. Informative high content assays development with the aim to implement them in order to obtain preliminary toxicity information of compounds 3. Toxicity profiling of chemicals or biologicals used in screening, in particular hits behavior relative to the characterization of cell viability. Methods: Different fluorescent probes for cell viability were tested and optimized in HeLa cells by varying parameters of staining, controls and incubation times. Automated fluorescence microscopy was used to image cells in 96-well plates and automated image analysis provided tool for quantification of the response to the different assays. Image quality was estimated in each condition and ability to discriminate between positive and negative populations was statistically determined after image segmentation and features extraction. Results: Live/dead assay using calcein-AM and ethidium homodimer-1 showed significant difference in signal between living and dead cells but high variability was observed within the same population. Apoptosis probes FLICA and annexin V conjugate were tested on cells treated with staurosporine. FLICA staining gave low signal-to-background ratio and apoptotic population was not significantly discernible. Living, early apoptotic and necrotic/late apoptotic cells populations were segregated using annexin V assay. Autophagy detection using LC3B immunostaining was observable in cells treated with chloroquine. Developed image analysis method reliably segmented autophagosomes in autophagic cells but failed with dead cells. Assays were tested on a set of toxic compounds and results showed differences with information found on these substances. Conclusion: Fluorescent probes are a powerful tool to assess cell death mechanism in high content screening assay as they can report multiple biological activities at the same time. As information in this type of assay are gathered at a single cell level, high variability is expected and must be reduced as possible to successfully characterize the phenotypes encountered and reach screening requirements.

2010

Block copolymers for siRNA delivery

Marc Keller, Dominique André Rothenfluh

The discovery of the RNA interference (RNAi) pathway in plants in the early 90's and the subsequent demonstration of its presence in mammalian cells in 2001 were a major breakthrough in biological research. Indeed it was quickly realized that this highly specific mechanism of gene silencing could be harnessed to study genomics or interfere with disease causing genes. Because of the numerous potential applications, small interfering RNAs (siRNAs) which are able to induce the RNAi pathway became one of the most promising tools for molecular biology and started to be intensively studied. However the potential of siRNAs has not yet been fully exploited, mainly because of a lack of effective carrier capable to deliver them in all cell-types. In this project Poly(ethyleneglycol)-Poly(propylenesulfide)-Poly(ethyleneimine) (PEG-b-PPS-b-PEI), a block-co-polymer already developed by our research group for the delivery of DNA, has been used to deliver siRNA in different cell-types. Because the size of polyplexes, resulting from the complexation of polymer with siRNA, is supposed to be an important parameter for the transfection efficiency, a blend of PEG-b-PPS and PPS-b-PEI was also tested as it exhibits very small aggregates sizes. Block copolymers have been first synthesized and complexed with siRNA. The Physicochemical properties of the resulting polyplexes, such as the surface charge, the size, or the complexation efficiency were then assessed. Inhibition of Lamin A/C in HeLa and mesenchymal stem cells (MSCs) in culture was tried with Lipofectamine 2000 used as a positive control. Specific gene downregulation was determined by quantitative PCR (qPCR). PEG-b-PPS-b-PEI-siRNA and PEG-b-PPS/PPS-b-PEI-siRNA complexes exhibited a downregulation much weaker than that of Lipofectamine 2000 for both cell-types. Moreover the presence of tri-block or blend of di-blocks in the culture media induced Lamin A/C overexpression. Another siRNA model has been tested which targets the green fluorescent protein (GFP) in HeLa cells previously transfected with GFPcoding DNA. Finally the capacity of the polyplexes to protect siRNA against digestion by RNases and their stability in presence of heparin were also tested

2008