Colloids as Mobile Substrates for the Implantation and Integration of Differentiated Neurons into the Mammalian Brain

Neuronal degeneration and the deterioration of neuronal communication lie at the origin of many neuronal disorders, and there have been major efforts to develop cell replacement therapies for treating such diseases. One challenge, however, is that differentiated cells are challenging to transplant due to their sensitivity both to being uprooted from their cell culture growth support and to shear forces inherent in the implantation process. Here, we describe an approach to address these problems. We demonstrate that rat hippocampal neurons can be grown on colloidal particles or beads, matured and even transfected in vitro, and subsequently transplanted while adhered to the beads into the young adult rat hippocampus. The transplanted cells have a 76% cell survival rate one week post-surgery. At this time, most transplanted neurons have left their beads and elaborated long processes, similar to the host neurons. Additionally, the transplanted cells distribute uniformly across the host hippocampus. Expression of a fluorescent protein and the light-gated glutamate receptor in the transplanted neurons enabled them to be driven to fire by remote optical control. At 1-2 weeks after transplantation, calcium imaging of host brain slice shows that optical excitation of the transplanted neurons elicits activity in nearby host neurons, indicating the formation of functional transplant-host synaptic connections. After 6 months, the transplanted cell survival and overall cell distribution remained unchanged, suggesting that cells are functionally integrated. This approach, which could be extended to other cell classes such as neural stem cells and other regions of the brain, offers promising prospects for neuronal circuit repair via transplantation of in vitro differentiated, genetically engineered neurons

Flexible and Hollow Micro Ring Electrode Arrays for Multi-Directional Monitoring of 3D Neuronal Networks

International audienceThree-dimensional (3D) cell culture based in vitro models shown great promise and able to replicate partially the development and complexity of in vivo nerve tissue. The functional characterization of 3D cell cultures is challenging. Because, most of the in vitro neural interfacing technologies are developed for 2D cell cultures, where it limits the access to the networks within the cultures. Hence, high-performance electrophysiological platforms that allow seamless integration with soft and stable tissue system, and multi-directional bio interfacing capabilities for long term are required.In this context, we report for the first time the fabrication of ‘Stargate’ look alike flexible and hollow micro-ring electrode array (SG MEA) with hole at the centre (40-μm to 150-μm diameter). The SG MEA was microfabricated on a 20-μm thick layer of flexible and biocompatible parylene C configured with 4 to 6 electrode sites. All gold electrode rings were coated with the conducting polymer poly(3,4-ethylenedioxythio-phene):poly(styrene-sulfonate) (PEDOT:PSS) to lower the impedance (5-8 kΩ) and obtain a better signal-to-noise ratio during extracellular recordings. The hollow ring shape of SG electrode architectures, with geometrical surface area of 289 μm2 to 1299 μm2 were designed specifically in such a way that it offers more freedom to the internal dynamics of cell cultures and multidirectional sensing of neuron activities within the 3D neuronal network, from both front and back-end of the SG MEA

Flexible and Hollow Micro Ring Electrode Arrays for Multi-Directional Monitoring of 3D Neuronal Networks

International audienceThree-dimensional (3D) cell culture based in vitro models shown great promise and able to replicate partially the development and complexity of in vivo nerve tissue. The functional characterization of 3D cell cultures is challenging. Because, most of the in vitro neural interfacing technologies are developed for 2D cell cultures, where it limits the access to the networks within the cultures. Hence, high-performance electrophysiological platforms that allow seamless integration with soft and stable tissue system, and multi-directional bio interfacing capabilities for long term are required.In this context, we report for the first time the fabrication of ‘Stargate’ look alike flexible and hollow micro-ring electrode array (SG MEA) with hole at the centre (40-μm to 150-μm diameter). The SG MEA was microfabricated on a 20-μm thick layer of flexible and biocompatible parylene C configured with 4 to 6 electrode sites. All gold electrode rings were coated with the conducting polymer poly(3,4-ethylenedioxythio-phene):poly(styrene-sulfonate) (PEDOT:PSS) to lower the impedance (5-8 kΩ) and obtain a better signal-to-noise ratio during extracellular recordings. The hollow ring shape of SG electrode architectures, with geometrical surface area of 289 μm2 to 1299 μm2 were designed specifically in such a way that it offers more freedom to the internal dynamics of cell cultures and multidirectional sensing of neuron activities within the 3D neuronal network, from both front and back-end of the SG MEA

Ordering of Water Molecules between Phospholipid Bilayers Visualized by CARS Microscopy

this paper, we report a multiphoton vibrational imaging study of onion-like structures which consist multiple alternate layers of phospholipid and water. We use coherent anti-Stokes Raman scattering (CARS) microscopy (16, 17) in which a pump laser beam at frequency p and a Stokes laser beam at frequency s are tightly focused into a sample to generate an anti-Stokes signal at frequency s p - 2 . CARS microscopy allows three-dimensional (3D) vibrational imaging with high sensitivity. The vibrational contrast arises from the enhancement of the CARS signal when s p - is tuned to a Raman band (18). The 3D sectioning ability results from the nonlinear dependence of CARS signals on the excitation field intensity, similar to two-photon fluorescence microscopy (19). The high-sensitivity is a consequence of the constructive addition of CARS signal from an ensemble of vibrational oscillators in the excitation volume. The multilamellar onions dispersed in deuterated dodecane, prepared via a selfemulsification process (20), provide an ideal system for investigating the properties of water between lipid bilayers. The water molecules in this preparation are only present between the bilayers for these onions. The use of a fully deuterated oil solvent allows selective imaging of lipids based on the vibrational signal from the C-H stretching mode, and of the interlamellar water based on the vibrational signal from the O-H stretching mode. The coherent addition of the CARS fields from multiple layers of water molecules produces a strong CARS signal, which allows us to study the properties of water confined between contacting lipid bilayers. 4 In particular, the relation between the excitation field polarization and the Raman tensor allows us to characterize the orientation o..

Colloids as mobile substrates for the implantation and integration of differentiated neurons into the mammalian brain.

Neuronal degeneration and the deterioration of neuronal communication lie at the origin of many neuronal disorders, and there have been major efforts to develop cell replacement therapies for treating such diseases. One challenge, however, is that differentiated cells are challenging to transplant due to their sensitivity both to being uprooted from their cell culture growth support and to shear forces inherent in the implantation process. Here, we describe an approach to address these problems. We demonstrate that rat hippocampal neurons can be grown on colloidal particles or beads, matured and even transfected in vitro, and subsequently transplanted while adhered to the beads into the young adult rat hippocampus. The transplanted cells have a 76% cell survival rate one week post-surgery. At this time, most transplanted neurons have left their beads and elaborated long processes, similar to the host neurons. Additionally, the transplanted cells distribute uniformly across the host hippocampus. Expression of a fluorescent protein and the light-gated glutamate receptor in the transplanted neurons enabled them to be driven to fire by remote optical control. At 1-2 weeks after transplantation, calcium imaging of host brain slice shows that optical excitation of the transplanted neurons elicits activity in nearby host neurons, indicating the formation of functional transplant-host synaptic connections. After 6 months, the transplanted cell survival and overall cell distribution remained unchanged, suggesting that cells are functionally integrated. This approach, which could be extended to other cell classes such as neural stem cells and other regions of the brain, offers promising prospects for neuronal circuit repair via transplantation of in vitro differentiated, genetically engineered neurons