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Abstract
To find anti-cancer drugs, different cellular modifications can be looked at: cell death effect on proliferating ...
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High Content Screening of automated wound healing and cytotoxicity assays in 2D and 3D culture

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To find anti-cancer drugs, different cellular modifications can be looked at: cell death effect on proliferating cells either in 2D culture or on spheroids (3D culture), anti-mitotic, anti-migration/invasion, anti-angiogenesis effects… Different assays can be performed to follow these different effects. High content screening is a multi-parametric technology allowing searching for poly-effects drugs.
In this poster, several well known compounds were tested in 3 different assays: cytotoxicity in 2D culture, cytotoxicity in spheroids (3D culture) and scratch assay (also named wound healing). These 3 models were automated in 96-well plates.

Publié dans : Santé & Médecine
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High Content Screening of automated wound healing and cytotoxicity assays in 2D and 3D culture

  1. 1. Abstract To find anti-cancer drugs, different cellular modifications can be looked at: cell death effect on proliferating cells either in 2D culture or on spheroids (3D culture), anti-mitotic, anti-migration/invasion, anti-angiogenesis effects… Different assays can be performed to follow these different effects. High content screening is a multi-parametric technology allowing searching for poly-effects drugs. In this poster, several well known compounds were tested in 3 different assays: cytotoxicity in 2D culture, cytotoxicity in spheroids (3D culture) and scratch assay (also named wound healing). These 3 models were automated in 96-well plates. Methods Results Conclusions & Perspectives  Automation on cytotoxicity assay on 3D culture in 96-well microplate and scratch assay (would healing) was successful.  EC50 results on cytotoxicity assay in 3D culture show a shift towards higher concentrations compared to 2D culture. This is due to the time required for the compound to reach the center of the spheroid. Depending on this time, the shift may be higher or lower. These 3D results are more relevant compared to the in vivo situation. Nevertheless, to be the most relevant as possible to the in vivo tumor, the spheroid needs to reach a consistent size. However, a necrotic core appears from the 3rd day in this condition (seeding: 2000 cells/well).  Perspective: the relevance of this 3D model would be increased by the addition of angiogenesis step with co-culture of HUVEC.  Scratch assay is another assay which proliferation/migration is followed and depending of the profile of the results, cell cycle arrest inducers (as colchicine) can be distinguished from cell death inducers. The two actions of Vinblastine (cell cylce arrest between 0.01 and 0.1 µM and cell death inducer at higher concentration) can be better observed in this assay than in the cytotoxicity assay. Cell culture: Hela (HT29, HepG2 or Caco-2: data not shown) were routinely maintained in DMEM (v/v) supplemented with 10% FBS. Depending of the cell line, cells were seeded between 2 500 and 10 000 cells/well in 96-well plates for 2D culture (Corning® cell-bind or Greiner® plates) or at 2000 cells/well in 96-well Corning® Spheroid microplates for 3D culture. Then, cells were incubated at 37 °C in 5 % CO2 for plating. The medium was changed every 3 days if needed. Cytotoxicity assay on 2D and 3D culture: After 48h of plating, cytotoxicity assay was performed by replaced medium in each well by fresh medium with or without different anti-cancer drugs at different concentrations added by Hoechst (1/1000) and propidium iodide (1/1000). Wound healing assay: When cells are at confluence, a scratch was performed in each well on a robotic platform with a 96-well head (Caliper ALH3000, Perkin Elmer). Medium was changed and replaced as previously. Then cells were incubated at 37 °C in 5 % CO2 for several days. Live cells imaging: Each day, pictures were taken on operetta (Perkin Elmer). Analysis and visualization were performed on Columbus (Perkin Elmer). Cytotoxicity assay on 2D & 3D culture Colchicine Automated wound healing on 96-well plates To get this poster, please flash the QR- code You can use the I-NIGMA application from your store Cmax - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Cmin CTRL Cisplatine Colchicine Cyclophosphamide Etoposide Paclitaxel Staurosporine Vinblastine Compound X T0 T48h Cmax - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Cmin CTRL Blue: Hoecsht, Yellow: Propidium iodide %Surfacescratched/T0 [Colchicine] (µM) CTRL T0 CTRL T48h %Surfacescratched/T0 [vinblastine] (µM) Blue: Hoecsht, Yellow: Propidium iodide Heatmap of T48h plate CTRL cells Vinblastine 1 µM EC50=106 nM EC50=149 nM 2D 3D 100 µM 30 µM 10 µM 3 µM 1 µM 300 nM 100 nM 30 nM 10 nM 3 nM CTRL 2D 3D Vinblastine 2D 2D 3D CTRL cells Etoposide 1 µM 100 µM 30 µM 10 µM 3 µM 1 µM 300 nM 100 nM 30 nM 10 nM 3 nM CTRL Etoposide 30nM> EC50 >100nM EC50> 100 nM High Content Screening of automated wound healing and cytotoxicity assays in 2D and 3D culture MAUBON N.1*, ROUDAUT M.1, NDOYE A.2 & BURSZTYKA J.1 1 HCS Pharma, 6 rue Pierre Joseph Colin, 35000 Rennes 2 Perkin Elmer , 16 av Québec, Bât Lys, 91140 Villebon sur Yvette 1*nathalie.maubon@hcs-pharma.com Vinblastine Cmax - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Cmin CTRL 2 days 3 days 4 days 5 days 2 days 3 days 4 days 5 days Cmax - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Cmin CTRL etoposide Time and dose effect on 3D culture

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