3D-innervated epidermis grown on microfluidic chip
Podium 33
Presented by: Sébastien Cadau
Skin innervation is mandatory to maintain a healthy looking skin thickness and functionality with both essential sensing and neuro-induced skin reactions. Moreover the cosmetic industry is focusing on discovering novel Neurocosmetic Functional Ingredients that could improve the interactions between the skin and the nervous system to reduce skin aging or skin stress signs. Using innervated skin biopsies or classical skin reconstructed model coupled to non-human neurons, we previously demonstrated that the innervated skin network decreases with aging leading particularly to an epidermal thinning and and a lack of impregnation by neuromediators.
This work presents an advanced human based innervated skin models using the most advanced Organ-on-Chip technologies.
In this study, we developed a novel Neuroskin-on-chip Technology with the aim to, 1/ integrate hiPSC-derived sensory neurones in a growing 3D epidermal reconstruction, 2/ validate each biological functions independently and 3/ evaluate the performance of such model using neurocosmetic active ingredients. Compared to existing innervated epidermis or skin models made by classical coculture with or without insert, this model holds the promise to better mimick the real skin innervation physiology. Indeed, neuronal bodies will be fluidly isolated from the growing skin model thanks to microfluidic channels induced compartmentalization. Such connecting microchannels allow sprouting of axons from one compartment to the other, which opens innervation by terminal endings in the growing skin compartment.
Because of the uniqueness of the microfluidic design, we created a specific PDMS microfluidic device containing four major chambers connected together. First, in the center of the chip, a 8mm² large central deposition chamber had the capacity to accept primary keratinocytes. Second, two peripheric channels accepting hiPSC-derived neurons were place sideways of the central deposition chamber and were both connected to the central chamber by microchannels. Finally, the central chamber could be perfused by another chamber located underneath and physically separated by a porous polycarbonate membrane. Such compartmentalization opens perfusion capacity of the central upper chamber by the lower one using different culture media supplemented with specific additives allowing neuronal or epidermis maturation. Immunostaining on chip and image analysis were used to evaluate neuronal viability, maturation, neurite outgrowth and epidermal maturation.
The first results demonstrated a long-term viability until 4 weeks with a decrease of Nestin/Sox2 at 22 days in vitro of hiPSC-derived sensory neurons, indicating a proper differentiation process directly within the microfluidic device. Without keratinocytes in the central chamber, we observed after 7 days in vitro specific morphology of human sensory neurons in vitro with initial axonal projections in the lateral microchannels. Moreover after 21 days in vitro, neurites have grown up to 33% of the central chamber width from both side (2.4 mm). At 21 days in vitro, maturation markers contributing to neuronal functionality were observed and quantified: peripherin (intermediate filament mainly expressed in neurons of the peripheral nervous system), substance P (peptidergic nociceptor involved in neurogenic inflammation), TrKB (high affinity receptor for BDNF, NT-3, NT-4/5 as promoter of neuronal survival) and Nav1.8 (as pain sensor at low temperature). When keratinocytes were added, integration of axonal network within keratinocytes was observed with a positive impact on keratinocytes.
We succeed to develop and grow an innervated epidermis on chip for the first time. Pharmaceutical proof of concept will be done with neuropeptide releases (substance P, CGRP…) and epidermal thickness measurement. Ultimately, the model would be improved by innervating and growing a full skin model. These more physiological 3D-innervated skin models will be of real interest to screen Neurocosmetic Active ingredients targeting neurons and neuroskin interractions to better maintain and protect the neuroskin integrity from damages caused by aging or stresses.
This work presents an advanced human based innervated skin models using the most advanced Organ-on-Chip technologies.
In this study, we developed a novel Neuroskin-on-chip Technology with the aim to, 1/ integrate hiPSC-derived sensory neurones in a growing 3D epidermal reconstruction, 2/ validate each biological functions independently and 3/ evaluate the performance of such model using neurocosmetic active ingredients. Compared to existing innervated epidermis or skin models made by classical coculture with or without insert, this model holds the promise to better mimick the real skin innervation physiology. Indeed, neuronal bodies will be fluidly isolated from the growing skin model thanks to microfluidic channels induced compartmentalization. Such connecting microchannels allow sprouting of axons from one compartment to the other, which opens innervation by terminal endings in the growing skin compartment.
Because of the uniqueness of the microfluidic design, we created a specific PDMS microfluidic device containing four major chambers connected together. First, in the center of the chip, a 8mm² large central deposition chamber had the capacity to accept primary keratinocytes. Second, two peripheric channels accepting hiPSC-derived neurons were place sideways of the central deposition chamber and were both connected to the central chamber by microchannels. Finally, the central chamber could be perfused by another chamber located underneath and physically separated by a porous polycarbonate membrane. Such compartmentalization opens perfusion capacity of the central upper chamber by the lower one using different culture media supplemented with specific additives allowing neuronal or epidermis maturation. Immunostaining on chip and image analysis were used to evaluate neuronal viability, maturation, neurite outgrowth and epidermal maturation.
The first results demonstrated a long-term viability until 4 weeks with a decrease of Nestin/Sox2 at 22 days in vitro of hiPSC-derived sensory neurons, indicating a proper differentiation process directly within the microfluidic device. Without keratinocytes in the central chamber, we observed after 7 days in vitro specific morphology of human sensory neurons in vitro with initial axonal projections in the lateral microchannels. Moreover after 21 days in vitro, neurites have grown up to 33% of the central chamber width from both side (2.4 mm). At 21 days in vitro, maturation markers contributing to neuronal functionality were observed and quantified: peripherin (intermediate filament mainly expressed in neurons of the peripheral nervous system), substance P (peptidergic nociceptor involved in neurogenic inflammation), TrKB (high affinity receptor for BDNF, NT-3, NT-4/5 as promoter of neuronal survival) and Nav1.8 (as pain sensor at low temperature). When keratinocytes were added, integration of axonal network within keratinocytes was observed with a positive impact on keratinocytes.
We succeed to develop and grow an innervated epidermis on chip for the first time. Pharmaceutical proof of concept will be done with neuropeptide releases (substance P, CGRP…) and epidermal thickness measurement. Ultimately, the model would be improved by innervating and growing a full skin model. These more physiological 3D-innervated skin models will be of real interest to screen Neurocosmetic Active ingredients targeting neurons and neuroskin interractions to better maintain and protect the neuroskin integrity from damages caused by aging or stresses.