in vitro dermocosmetology : high content analysis approach using human primary keratinocytes and...
TRANSCRIPT
In vitro dermocosmetology: high content analysis approach using human primary keratinocytes and fibroblasts
Ferron PJ.*, Jarnouen K., Burzstyka J., Maubon N. *[email protected]; HCS Pharma, FRANCE
Abstract Cell biology is part of the R&D activity in dermocosmetology, as providers of active extracts or pure compounds have to demonstrate their efficacy and safety. At HCS Pharma, we are developing new dermocosmetology assays on relevant cellular models, like human primary keratinocytes and human primary fibroblasts, using our automated platform and high content analysis system. Through the use of 96 or 384 wells plates combined with process automation, we constantly increase the throughput in order to allow proof of concept assays of one or several compounds up to the screening of large compounds libraries, for single endpoint or live cells analysis to multiplexed phenotypic screening. Assessment of compounds activity/safety can be performed on several parameters in parallel with a multiplexed assay. Among them, we describe here wound healing, inflammation assay & extra cellular matrix analysis, which are routinely performed. Other parameters can be analysed on demand on live and fixed cells, using chemical probes or immunocytochemistry.
Materials and Methods
Conclusions
neonatal foreskin biopsy
Caliper Sciclone automated liquid handling system
Neonatal human primary keratinocytes and fibroblasts are purchased frozen. Cells are thawed and cultured in T75 flasks before being plated in several 96 or 384 wells plates. Cells can be used in proliferative or differentiated states.
Cell Culture
Automation
Image acquisition and high content analysis
Cellular expansion
Compound Exposure
Automation of process are performed with a Sciclone ALH3000 (Caliper™) liquid handling system. Mounted with a 96 or 384 well head, the Sciclone allows to work from medium to high throughput. Protocols are optimized and adapted to a wide range of cellular biology methods:
• Dilution and aliquoting of compounds
• Cell treatment
•Cell monolayer scratching
•Immunocytochemistry
•…
ImageXpress, Molecular Devices™
BD Pathway, BD
Columbus software, Perkin Elmer™
Images acquisition is driven by fully automated epifluorescence microscopes. Objectives from 4X to 60X allow to take the best images for phenotypic screening. Up to 4 fluorescence filters can be used allowing up to 4 different stainings on the same biological sample. Immunocytochemistry and specific probes are used to stain cells for automated imaging and further image analysis.
1-7 days 1-7 days
96 wells plates
Results
High content analysis requires staining, with or without fixation, and imaging cells on a high-throughput microscope. Then, cell phenotypes can be characterized using various features (size, shape, signal quantification, localization …).
Figure 2 : Wound healing of keratinocytes and fibroblast 24 hours after scratch.
Wound healing
Wounds can exhibit impaired healing, as a consequence of pathologic failure to process one of the normal stage of healing. Using automated cell scratching, we can study compounds efficacy on keratinocytes and fibroblasts migration.
Figure 1 : Time laps of wound healing. 4X light transmission. Wound is pseudocolored in red, keratinocyte monolayer in green.
Human primary keratinocytes and fibroblasts are mechanically “scratched” using an automated process. Wound reparation is followed using live cells microscopy. Results are expressed in percent of wound repair, and can be shown in final point or kinetics.
Following protein expression and localisation is commonly used to assess specific markers in response to compounds exposure. Targeted protein could be localized in the cytosol, the nucleus, the cytoplasmic membrane or in the extra cellular matrix. High content analysis allow to perform complex phenotypic screening, as well as simple features assessment, like protein translocation or protein expression.
Protein expression
Figure 5 : AQP3 in confluent human keratinocytes. 20X magnification, Nucleus in blue, AQP3 in yellow
Intra and extra cellular proteins are labeled using specific antibodies. Up to 3 different proteins can be imaged using multiplex labeling on the same biological sample.
Genotoxicity
As animal testing has been banned in EU for cosmetic safety assessment, genotoxicity potential of active compounds has to be performed on in vitro assays. In HCS Pharma, we developed a genotoxicity assay to assess DNA double strand breaks, through detection of phosphorylated H2AX, and 53BP1 which is activated in final steps of DNA repair.
Figure 6 : An example of high content imaging obtained with MMC at 6,25 µM on primary human keratinocytes, 20X magnification. Nucleus in blue, γH2AX in red, activated caspase 3/7 in green, and 53BP1 in yellow.
MetaXpress Software, Molecular Devices ™
• Primary human keratinocytes and fibroblasts are suitable for the development of automated dermocosmetology assay.
• Current validated & automated assays include the wound healing assay, NFkB & NRF2 translocation assays, Collagen I synthesis, and
genotoxicity assay through γH2AX assessment among other.
• These assays can be performed on several thousand experimental conditions (either compounds, fractions of extracts, and concentrations).
• All these assays can be customized on demand.
HCS Pharma – French startup focused on in vitro preclinical research development with a specialization in cell imaging – http://www.hcs-pharma.com
CTRL LPS
Figure 3 : Translocation of NF-kB (in green) from cytoplasm (CTRL), to nucleus (LPS) in fibroblast exposed to LPS.
Figure 4 : Fibronectin in confluent human fibroblasts. 20X magnification, Nucleus in blue, fibronectin in yellow
Hoescht
γH2AX
Caspase 3/7 Merge
53BP1
Figure 7 : gH2AX phosphorylation on primary human keratinocytes for 9 different compounds after 24h of exposure