The overall goal of this procedure is to present a novel model of in vitro three dimensional neuronal networks coupled to micro electrode arrays. This is accomplished by first conditioning, the central part of the MEA with a mixed solution of poly de lysine and laminin then coat the microbeads with adhesion proteins, laminin and polylysine. The second step is to distribute the suspension of treated microbeads onto a multi-well plate where they will self-assemble and form a uniform layer.
The third step of the procedure is to plate the cells onto the active area of the MEA at a density of 2000 cells per square millimeter to create 2D neuronal networks. Six to eight hours after plating suspension is transferred from the multi-well plates to the MEA and the microbeads are allowed to self-assemble in a hexagonal compact structure. Ultimately, confocal microscopy is used to visualize the 3D networks, which are compared to the conventional 2D neuronal networks grown over MEA.
The main advantage of this technique over existing methods, like in vitro BDI dimensional cell cultures, is that in a BDI dimensional model, somata and growth cones are flattened, Andon and rise cannot spread in all direction. In addition, bursting activity dominates the electrophysiological dynamics of BDI dimensional networks while in a three-dimensional one, also a random specking activity coexists. We First had the idea of this method when we read the research paper of Sophie Potto published it in Nature Method in 2008, in which she used MICROBITS as a scaffold to build three dimensional ne networks.
We then adapted this method to me to make the first three dimensional in vitro recordings. To prepare the PDMS elastomer, mix the curing agent and a polymer by a volume ratio of one to nine in a Petri dish. Then shake the mixture and place it in the vacuum chamber for 10 minutes to eliminate air bubbles.
To construct the PDMS constraint, insert the PDMS material into the molder older after that, put it into the oven for 30 minutes at 120 degrees Celsius. The PDMS constraint should have the shape of a cylinder with an external and internal diameter of 22.0 and 3.0 millimeters respectively, and a height of 650 micrometers the day before plating. Couple the mask to the active area of the MEA under a stereo microscope, then sterilize the PDMS mask in the oven at 120 degrees Celsius.
Next, sterilize the glass microbeads in a conical vial of 70%Ethanol for two hours, rotated every 30 minutes to expose all the microbeads afterward. Remove the ethanol solution from the vial and rinse the microbeads two times with sterilized water. Then condition the central part of the MEA area delimited by the PDMS mask with 24 microliters of mixed solution of polylysine and laminin at 0.05 micrograms per milliliter.
Coat the microbeads with adhesion proteins, laminate and polylysine at 0.05 micrograms per milliliter. Subsequently, place them in the incubator overnight at 37 degrees Celsius to obtain a stable and long lasting neuronal network. Afterward, distribute the suspension of the treated microbeads onto the multi-well plate with a membrane insert where they will self-assemble and form a uniform layer.
Then fill each well of the membrane with 0.5 milliliters of medium. Put it in the incubator at 37 degree Celsius and 5%carbon dioxide until the neurons are ready to be plated in this step. After sacrificing an E 18 pregnant female rat, obtain the rat embryos, then remove the hippo campi from each rat embryo.
Place them into ice cold Hanks. Balance salt solution without calcium and magnesium. Next, dissociate the tissue in 0.125%of tryin Hanks solution containing 0.05%of DNAs for 18 to 20 minutes at 37 degrees Celsius.
After that, remove the supernatant with a peor pipette. Stop the enzymatic digestion by adding medium with 10%FBS for five minutes. Then remove the medium with FBS and wash once with the growth medium, its supplement 1%L-glutamine and Gentamycin at 10 micrograms per milliliter.
Subsequently, remove the medium. Refill it again with 500 microliters of growth.Medium. Its supplement 1%L-glutamine and Gentamycin at 10 micrograms per milliliter.
Dissociate the tissue pellet mechanically with a narrow PEs pipette until a milky suspension of cells is apparent. After that, dilute a small volume of cell suspension with the growth medium to obtain a final volume of 2.0 milliliters. Count the obtained cellular concentration with the hemo cytometer chamber.
Then dilute this concentration at a ratio of one to five in order to obtain the desired cell concentration of 600 to 700 cells per microliter. Plate the cells at a density of 2000 cells per square millimeter onto the active area of the MEA defined by the PDMS constraint to create a 2D neuronal network. Next, place the MEA device into the incubator with 5%humidified carbon dioxide atmosphere at 37 degrees Celsius.
To complete the preliminary step for the construction of 3D culture, distribute 160 microliters of the suspension with a cell concentration of 600 to 700 cells per microliter onto the surface of the microbeads monolayer positioned inside the multi-well plates. Then place the multi-well plates in the incubator with 5%humidified carbon dioxide atmosphere at 37 degrees Celsius, six to eight hours after plating transfer 30 to 40 microliters of the suspension with microbeads and neurons to the area of the MEA delimited by the PDMS constraint. After each transfer, wait about half a minute to allow the microbeads to self-assemble in a hexagonal structure.
Once all the layers are deposited and spontaneously assembled at 300 microliters of medium on the top of the area delimited by the PDMS constraint. Put the 3D structure coupled to the MEA in the incubator at 37 degrees Celsius and 5%carbon dioxide for 48 hours. After that, add one milliliter of growth medium with its supplement after three weeks in vitro, the 3D structure is stable also without the PDMS constraint shown.
Here is a DIC image of a single layer of beads coupled to the MEA surface. The position of the black dots represents the spatial layout of the electrodes. Immunofluorescent staining for map two, which is reflected by the red signal displays the distribution of dendritic arborization and ADA of the neurons around the microbeads directly coupled to the electrode plane of the MEA.
This image shows the maximum projection of a Zack sequence of A 3D culture displaying a highly interconnected network where neurons are labeled for new N expressed in mature post mitotic neurons. And for gaba, here's the representative Electrophysiological recording of the hippocampal networks in 3D configuration. And here's the representative electrophysiological recording of the hippocampal networks in 2D configuration.
Once mastered, this procedure can be done in seven hour. However, it is worth to notice that since the procedure has several steps that have been performed during different day, the time estimation to realize the entire procedure is merely an indication. While attempting this procedure, it's important to remember to first plate a 2D layer of neuron diuretic to the electrode of the mea, and then transfer very carefully the suspension of microbits and neuron from the multi-well plate to the mea in order to realize the 3D structure After its development.
This technique paved the way for researchers in the field of computational neuroscience to explore how network connectivity can shape the emergent network dynamics. After watching this video, you should have a good understanding of how to build an in vitro three dimensional neuronal network coupled to micro electrode arrays.
