Diese Präsentation wurde erfolgreich gemeldet.
Die SlideShare-Präsentation wird heruntergeladen.
Wir verwenden Ihre LinkedIn Profilangaben und Informationen zu Ihren Aktivitäten, um Anzeigen zu personalisieren und Ihnen relevantere Inhalte anzuzeigen. Sie können Ihre Anzeigeneinstellungen jederzeit ändern.
HCS Pharma
Biocentre Fleming Bat A
250 rue Salvador Allende
59120 LOOS
BIOMIMESYS® is a registered trademark
of HCS Pharma...
HCS Pharma
Biocentre Fleming Bat A
250 rue Salvador Allende
59120 LOOS
BIOMIMESYS® is a registered trademark
of HCS Pharma...
HCS Pharma
Biocentre Fleming Bat A
250 rue Salvador Allende
59120 LOOS
BIOMIMESYS® is a registered trademark
of HCS Pharma...
HCS Pharma
Biocentre Fleming Bat A
250 rue Salvador Allende
59120 LOOS
BIOMIMESYS® is a registered trademark
of HCS Pharma...
Nächste SlideShare
Wird geladen in …5
×

0

Teilen

Jetzt herunterladen Herunterladen

Herunterladen, um offline zu lesen

Simplifying 3d cell culture generation for high content screening with BIOMIMESYS® and the viaflo 96-384

Jetzt herunterladen Herunterladen

Herunterladen, um offline zu lesen

Growing interest in phenotypic screening, together with evidence that the drug response of cells grown in three-dimensional (3D) structures more closely resembles in vivo activity, has made high throughput, 3D fluorescence imaging an attractive screening option for drug discovery. However, creating 3D spheroids compatible with high content screening can be a difficult and expensive process.

BIOMIMESYS® is a range of patented hyaluronic acid scaffolds for 3D cell culture. They are made of RGDS- and galactosaminegrafted hyaluronic acid, using an adipic acid dihydrazide crosslinker and extracellular matrix proteins (eg. type I, IV or VI collagen) to accurately mimic the in vivo extracellular environment. BIOMIMESYS® is suitable for automated testing thanks to the uniform thickness of the scaffold – around 650 μm – with an average porosity of 100 to 200 μm

This application note describes a straightforward workflow for 3D cell seeding and spheroid formation using HCS Pharma’s BIOMIMESYS® plates in combination with INTEGRA’s VIAFLO 96/384 pipetting system. This process ensures rapid, controllable and reproducible 3D cell cultures, providing researchers with a highly efficient method to produce physiologically-relevant cellular models in a high throughput format

Ähnliche Bücher

Kostenlos mit einer 30-tägigen Testversion von Scribd

Alle anzeigen

Ähnliche Hörbücher

Kostenlos mit einer 30-tägigen Testversion von Scribd

Alle anzeigen
  • Als Erste(r) kommentieren

  • Gehören Sie zu den Ersten, denen das gefällt!

Simplifying 3d cell culture generation for high content screening with BIOMIMESYS® and the viaflo 96-384

  1. 1. HCS Pharma Biocentre Fleming Bat A 250 rue Salvador Allende 59120 LOOS BIOMIMESYS® is a registered trademark of HCS Pharma +33 769 999 137 www.biomimesys.com hello@biomimesys.com A groundbreaking 3D cell culture technology Application Note 3D CELL CULTURE GENERATION FOR HIGH CONTENT SCREENING WITH BIOMIMESYS® AND THE VIAFLO 96/384 INTRODUCTION Growing interest in phenotypic screening, together with evidence that the drug response of cells grown in three-dimensional (3D) structures more closely resembles in vivo activity, has made high throughput, 3D fluorescence imaging an attractive screening option for drug discovery. However, creating 3D spheroids compatible with high content screening can be a difficult and expensive process. BIOMIMESYS® is a range of patented hyaluronic acid hydroscaffolds™ for 3D cell culture. They are made of RGDS- and galactosamine- grafted hyaluronic acid, using an adipic acid dihydrazide crosslinker and extracellular matrix proteins (eg. type I, IV or VI collagen) to accurately mimic the in vivo extracellular environment. BIOMIMESYS® is suitable for automated testing thanks to the uniform thickness of the hydroscaffold™ – around 300 μm with an average porosity of 100 to 200 μm (Figure 1) This application note describes a straightforward workflow for 3D cell seeding and spheroid formation using HCS Pharma’s BIOMIMESYS® plates in combination with INTEGRA’s VIAFLO 96/384 pipetting system. This process ensures rapid, controllable and reproducible 3D cell cultures, providing researchers with a highly efficient method to produce physiologically- relevant cellular models in a high throughput format. Figure 1 : Schematic of BIOMIMESYS® synthesis (upper panel) and SEM image of the resulting hydroscaffold™ (lower panel). MATERIALS AND METHODS Cell seeding onto BIOMIMESYS® plate with VIAFLO 96/384 The VIAFLO 96/384 was used to both seed cells and renew the growth medium. This system was equipped with a 96-channel pipetting head – using sterile 300 μl tips – and placed in a microbiological safety cabinet to ensure sterile cell handling. The initial cell suspension was homogenized by pipetting, then ~5,000 HepG2 cells/well were seeded into the ready-to-use BIOMIMESYS® Liver plate in 50 μl of culture medium (DMEM supplemented with 10 % FBS, 2 mM L-glutamine, non-essential amino acids (1X), 50 U/ml of penicillin and 50 μg/ml streptomycin).
  2. 2. HCS Pharma Biocentre Fleming Bat A 250 rue Salvador Allende 59120 LOOS BIOMIMESYS® is a registered trademark of HCS Pharma +33 769 999 137 www.biomimesys.com hello@biomimesys.com A groundbreaking 3D cell culture technology Accurate dispensing is crucial to ensure homogeneous seeding across the plate. Using the VIAFLO 96/384 allowed the cells to be seeded onto the center of each hydrogel, with a consistent pipetting height (z-height limit: 2.9 mm) and speed (set on 2) (Figure 2). Figure 2 :Using the VIAFLO 96/384 to seed HepG2 into a 96-well plate containing BIOMIMESYS Liver matrix. Following seeding, 150 μl/well of medium was added (z-height limit: 3.2 mm), speed (set on 2) and the plate was incubated at 37 °C under 5 % CO2. Seeding and media addition took under two minutes for a complete 96-well plate, indicating the potential of this set-up for easy scale-up. HepG2 viability assay The cell proliferation reagent WST-1 was used to determine the metabolic activity and viability of HepG2 cells. 100 μl/well of medium was removed, and replaced with 10 μl/well of WST-1. After a further 1.5 h incubation, OD440 measurements were performed using an Infinite® M200 PRO plate reader (Tecan). Chlorpromazine treatment of HepG2 cells The At Day 6, when the HepG2 cells have formed multicellular spheroids, the cultures were treated with the well-known hepatotoxic drug chlorpromazine (CPZ). 100 μl/well of medium was removed, and replaced with 100 μl/well of medium containing various amounts of CPZ to give final drug concentrations of 0,1, 10, 50, 100 and 250 μM (n=6/concentration; H=30 mm; S=1). Cells were then incubated at 37 °C under 5 % CO2 for 24 hours. Bile canaliculi activity of HepG2 cells Bile canaliculi activity of HepG2 cells treated with different concentrations of CPZ was observed using 5(6)-carboxy-2',7'- dichlorofluorescein diacetate (CDFDA), a synthetic substrate for bile canaliculi. After washing the hydrogels with PBS, cells were incubated with 200 μl/well of 3 μM CDFDA solution for 30 min. Cells were then washed in PBS and imaged using an ImageXpress® Micro Confocal High-Content Imaging System (Molecular Devices). RESULTS AND DISCUSSION HepG2 viability Thanks to the accuracy and ease of use of the VIAFLO 96/384 system, 3D cell cultures were successfully grown for six days after seeding without disturbing the hydrogels or forming multicellular spheroids. By enabling 96channel transfers in a single step, the VIAFLO 96/384 also significantly improves the reproducibility of the pipetting for both seeding and media exchange. This is demonstrated by the consistent cell viability between wells, with low CVs of just 4 % on Day 3 and 7 % on Day 6 (Figure 3). Figure 3 : Viability of HepG2 grown in BIOMIMESYS® liver plate (n=16), measured with WST-1 reagent.
  3. 3. HCS Pharma Biocentre Fleming Bat A 250 rue Salvador Allende 59120 LOOS BIOMIMESYS® is a registered trademark of HCS Pharma +33 769 999 137 www.biomimesys.com hello@biomimesys.com A groundbreaking 3D cell culture technology The translucence of BIOMIMESYS® hydroscaffold™ also means that HepG2 spheroid growth can be observed directly using bright field microscopy (Figure 4). Figure 4 : Bright field image of multicellular HepG2 spheroid six days after seeding. Chlorpromazine toxicity Thanks to the accuracy and ease of use of the The viability of HepG2 cells was measured after 24 hours treatment with the hepatotoxicity- inducing drug CPZ (Figure 5). The results indicate an IC50 of 34 μM, with excellent reproducibility at concentrations above 1 μM. Figure 5 : Viability analysis of HepG2 cultures treated with chlorpromazine (n=6). Bile canaliculi activity Direct fluorescence imaging and bright field microscopy showed that CDFDA efflux is efflux by cells could still be observed following treatment with low concentrations of CPZ. However, at higher CPZ concentrations (≥10 μM), it accumulates inside HepG2 cells, indicating that bile canalicular network formation and maintenance is disrupted (Figure 6). Figure 6 : Vizualization of bile canaliculi activity in CPZ- treated HepG2 spheroids, using CDFDA (green) and Hoechst staining (blue, for cell nuclei). Scale bar=100 μm.
  4. 4. HCS Pharma Biocentre Fleming Bat A 250 rue Salvador Allende 59120 LOOS BIOMIMESYS® is a registered trademark of HCS Pharma +33 769 999 137 www.biomimesys.com hello@biomimesys.com A groundbreaking 3D cell culture technology CONCLUSIONS The application described here demonstrates that HCS Pharma’s BIOMIMESYS® plate is fully compatible with INTEGRA’s VIAFLO 96/384 pipetting system. This combination provides a straightforward, compact and cost-effective solution for the generation of 3D multicellular spheroids suitable for a wide range of high content screening applications. The VIAFLO 96/384 ensures accurate and consistent cell seeding and medium exchanges, leading to very low variability in cellular activity between wells, which can be easily confirmed using bright field microscopy. This set-up simplifies maintenance and treatment of 3D cell cultures, offering researchers a straightforward workflow for performing reproducible viability and functional assays in a 96-well format. Throughput could be further improved by switching to a 384-well format, offering further productivity gains while minimizing the time spent pipetting. REFERENCE (1) Ryan SL, et al. Drug discovery approaches utilizing three-dimensional cell culture. Assay Drug Dev Technol, 2016, 14(1), 19-28.

    Als Erste(r) kommentieren

Growing interest in phenotypic screening, together with evidence that the drug response of cells grown in three-dimensional (3D) structures more closely resembles in vivo activity, has made high throughput, 3D fluorescence imaging an attractive screening option for drug discovery. However, creating 3D spheroids compatible with high content screening can be a difficult and expensive process. BIOMIMESYS® is a range of patented hyaluronic acid scaffolds for 3D cell culture. They are made of RGDS- and galactosaminegrafted hyaluronic acid, using an adipic acid dihydrazide crosslinker and extracellular matrix proteins (eg. type I, IV or VI collagen) to accurately mimic the in vivo extracellular environment. BIOMIMESYS® is suitable for automated testing thanks to the uniform thickness of the scaffold – around 650 μm – with an average porosity of 100 to 200 μm This application note describes a straightforward workflow for 3D cell seeding and spheroid formation using HCS Pharma’s BIOMIMESYS® plates in combination with INTEGRA’s VIAFLO 96/384 pipetting system. This process ensures rapid, controllable and reproducible 3D cell cultures, providing researchers with a highly efficient method to produce physiologically-relevant cellular models in a high throughput format

Aufrufe

Aufrufe insgesamt

27

Auf Slideshare

0

Aus Einbettungen

0

Anzahl der Einbettungen

1

Befehle

Downloads

0

Geteilt

0

Kommentare

0

Likes

0

×