In vitro dermocosmetology : high content analysis approach using human primary keratinocytes
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In vitro dermocosmetology: high content analysis approach using human primary keratinocytes 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. At HCS Pharma, we are developing new dermocosmetology assays on human primary keratinocytes using our automated plateform and high content analysis suite. Assessment of compounds activity/safety can be performed on several parameters (wound healing, hydration, inflammation, toxicity, genotoxicity). Further parameters can be analyzed on demand using probes and immunocytochemistry. Through the use of high content analysis technologies, we can provide a wide range of results from phenotypes quantification cell by cell to live cell imaging movies. Materials and Methods Conclusions neonatal foreskin biopsy Caliper Sciclone robot Neonatal human primary keratinocytes are purchased frozen and thawed in 96 well- plates. One vial of primary keratinocytes allows to make several 96 well-plates. Cells can be used in proliferative or differentiated state. Several days of differentiation are necessary to obtain fully differentiated keratinocytes. Cell Culture Automation Image acquisition and high content analysis Cellular expansion 1-3 days Compound Exposure Automation of process in liquid handling are performed with a Sciclone ALH3000, Caliper™. 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 scratching •Immunocytochemistry ImageXpress, Mol. Dev. 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 simultaneously. Immunocytochemistry and specific probes are used to stain cells for automated imaging. 1-7 days 1-7 days 96 wells plates Results High content analysis requires stained, fixed, and imaged cells on a high-throughput microscope. Then, we characterize cell phenotypes with various measure (size, shap, signal quantification …). Figure 2 : Effects of reference compounds on keratinocytes wound healing after 24 hours of exposure. 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 efficiency on keratinocytes wound healing. Figure 1 : Time laps of wound healing. 4X light transmission. Wound is colored in red, keratinocytes in green. Proliferative keratinocytes were exposed to reference compounds exposed one day before the cell scratching. One day after cell scratching, Retinoic acid (RA) induced a significant increase of in vitro wound healing while EGF starving stopped significantly wound closure by the keratinocytes. Skin hydration process involves water transport through the derma. Transport of glycerol is also involved thought the presence of Aquaporin (AQP). AQP3 is the most abundant AQP in skin and closely links to skin hydration. Hydration Figure 3 : Induction of AQP3 in keratinocytes after 2 days of exposure. Nucleus in blue, AQP3 in green. Keratinocytes were exposed to compounds 2 days before differenciation. Both increased of AQP3 levels in keratinocytes membranes. As shown in figure 4, AQP3 induction is more important when cells are exposed longer to retinoic acid. Genotoxicity As animal testing has been banned in EU, genotoxicity potential of dermocosmetic compounds is 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 recombination. Figure 5 : Induction H2AX and 53BP1 spots in differentiated keratinocytes after 24 hours of exposure to benzo- a-pyren and aflatoxin b1. As γH2AX and 53BP1 are recruited at different steps of DNA double strand breaks reparation, responses are different between markers. BAP presents the highest genotoxic response when compared to Aflatoxin B1. Combined with S9 fraction, we can also assess the genotoxic potential of compound metabolites (in blue). Figure 4 : Effects of insulin and retinoic acid on AQP3 translocation to keratinocytes membranes. MetaXpress Software, Molecular Devices ™ • Primary human neonatal keratinocytes are sustainable for the development of dermocosmetology assay. • Development of automated wound healing assay shows consistent response to reference compounds in proliferative keratinocytes. • Induction of membrane aquaporin 3 shows good results in differentiated keratinocytes. Kinetic studies has to be performed. • Detection of γH2AX and 53BP1 spots are good markers of induced genotoxicity in differentiated keratinocytes. • All these assays can be customized on demand. HCS Pharma – French startup focused on in vitro preclinial research development with a specialisation in cell imaging – http://www.hcs-pharma.com To get this poster, please flash the QR- code You can use the I-NIGMA application from your store
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Transcript of In vitro dermocosmetology : high content analysis approach using human primary keratinocytes
In vitro dermocosmetology: high content analysis approach using human primary keratinocytes
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. At HCS Pharma, we are developing new dermocosmetology assays on human primary keratinocytes using our automated plateform and high content analysis suite. Assessment of compounds activity/safety can be performed on several parameters (wound healing, hydration, inflammation, toxicity, genotoxicity). Further parameters can be analyzed on demand using probes and immunocytochemistry. Through the use of high content analysis technologies, we can provide a wide range of results from phenotypes quantification cell by cell to live cell imaging movies.
Materials and Methods
Conclusions
neonatal foreskin biopsy
Caliper Sciclone robot
Neonatal human primary keratinocytes are purchased frozen and thawed in 96 well- plates. One vial of primary keratinocytes allows to make several 96 well-plates. Cells can be used in proliferative or differentiated state. Several days of differentiation are necessary to obtain fully differentiated keratinocytes.
Cell Culture
Automation
Image acquisition and high content analysis
Cellular expansion
1-3 days Compound Exposure
Automation of process in liquid handling are performed with a Sciclone ALH3000, Caliper™. 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 scratching
•Immunocytochemistry
ImageXpress, Mol. Dev. 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 simultaneously. Immunocytochemistry and specific probes are used to stain cells for automated imaging.
1-7 days 1-7 days
96 wells plates
Results
High content analysis requires stained, fixed, and imaged cells on a high-throughput microscope. Then, we characterize cell phenotypes with various measure (size, shap, signal quantification …).
Figure 2 : Effects of reference compounds on keratinocytes wound healing after 24 hours of exposure.
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 efficiency on keratinocytes wound healing.
Figure 1 : Time laps of wound healing. 4X light transmission. Wound is colored in red, keratinocytes in green.
Proliferative keratinocytes were exposed to reference compounds exposed one day before the cell scratching. One day after cell scratching, Retinoic acid (RA) induced a significant increase of in vitro wound healing while EGF starving stopped significantly wound closure by the keratinocytes.
Skin hydration process involves water transport through the derma. Transport of glycerol is also involved thought the presence of Aquaporin (AQP). AQP3 is the most abundant AQP in skin and closely links to skin hydration.
Hydration
Figure 3 : Induction of AQP3 in keratinocytes after 2 days of exposure. Nucleus in blue, AQP3 in green.
Keratinocytes were exposed to compounds 2 days before differenciation. Both increased of AQP3 levels in keratinocytes membranes. As shown in figure 4, AQP3 induction is more important when cells are exposed longer to retinoic acid.
Genotoxicity
As animal testing has been banned in EU, genotoxicity potential of dermocosmetic compounds is 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 recombination.
Figure 5 : Induction H2AX and 53BP1 spots in differentiated keratinocytes after 24 hours of exposure to benzo-a-pyren and aflatoxin b1.
As γH2AX and 53BP1 are recruited at different steps of DNA double strand breaks reparation, responses are different between markers. BAP presents the highest genotoxic response when compared to Aflatoxin B1. Combined with S9 fraction, we can also assess the genotoxic potential of compound metabolites (in blue).
Figure 4 : Effects of insulin and retinoic acid on AQP3 translocation to keratinocytes membranes.
MetaXpress Software, Molecular Devices ™
• Primary human neonatal keratinocytes are sustainable for the development of dermocosmetology assay.
• Development of automated wound healing assay shows consistent response to reference compounds in proliferative keratinocytes.
• Induction of membrane aquaporin 3 shows good results in differentiated keratinocytes. Kinetic studies has to be performed.
• Detection of γH2AX and 53BP1 spots are good markers of induced genotoxicity in differentiated keratinocytes.
• All these assays can be customized on demand.
HCS Pharma – French startup focused on in vitro preclinial research development with a specialisation in cell imaging – http://www.hcs-pharma.com
To get this poster, please flash the QR-
code
You can use the I-NIGMA application
from your store
