Interfacing 3D Engineered Neuronal Cultures to Micro-Electrode Arrays: An Innovative In Vitro Experimental Model

Podsumowanie

In this work, a novel experimental model in which 3D neuronal cultures are coupled to planar Micro-Electrode Arrays (MEAs) is presented. 3D networks are built by seeding neurons in a scaffold made up of glass microbeads on which neurons grow and form interconnected 3D structures.

Streszczenie

Currently, large-scale networks derived from dissociated neurons growing and developing in vitro on extracellular micro-transducer devices are the gold-standard experimental model to study basic neurophysiological mechanisms involved in the formation and maintenance of neuronal cell assemblies. However, in vitro studies have been limited to the recording of the electrophysiological activity generated by bi-dimensional (2D) neural networks. Nonetheless, given the intricate relationship between structure and dynamics, a significant improvement is necessary to investigate the formation and the developing dynamics of three-dimensional (3D) networks. In this work, a novel experimental platform in which 3D hippocampal or cortical networks are coupled to planar Micro-Electrode Arrays (MEAs) is presented. 3D networks are realized by seeding neurons in a scaffold constituted of glass microbeads (30-40 µm in diameter) on which neurons are able to grow and form complex interconnected 3D assemblies. In this way, it is possible to design engineered 3D networks made up of 5-8 layers with an expected final cell density. The increasing complexity in the morphological organization of the 3D assembly induces an enhancement of the electrophysiological patterns displayed by this type of networks. Compared with the standard 2D networks, where highly stereotyped bursting activity emerges, the 3D structure alters the bursting activity in terms of duration and frequency, as well as it allows observation of more random spiking activity. In this sense, the developed 3D model more closely resembles in vivo neural networks.

Wprowadzenie

In vitro two-dimensional (2D) neural networks coupled to Micro-Electrode Arrays (MEAs)arethe gold-standard experimental model adopted to study the interplay between neuronal dynamics and the underlying connectivity. During the development, neurons recreate complex networks which display well definedspatio-temporalpatternsof activity^1,2 (i.e., bursts, network bursts, random spiking activity). MEAs record the electrophysiological activity from many sites (from tens to thousands of microelectrodes), allowing a detailed investigation of the expressed dynamics at the network level. In addition, the use of dissociated cultures makes possibile to design engineered-networks. It is easier to understand in this way the functional relationships between the recorded electrophysiological activity and the parameters of the network organization like cell density³, degree of modularity^4,5, presence of heterogeneous neuronal populations⁶, etc. However, all in vitro studies on dissociated cultured cells are based on 2D neuronal networks. This approach leads to oversimplifications with respect to the in vivo (intrinsically 3-dimensional, 3D) system: (i) in a 2D model, somata and growth cones are flattened and the axons-dendrites outgrowth cannot spread in all directions⁷. (ii) 2D in vitro networks exhibit stereotyped electrophysiological dynamics dominated by bursting activity involving most of the neurons of the network⁸.

Recently, different solutions have been developed to allow the construction of in vitro 3D dissociated neuronal networks. The common idea consists in creating a scaffold where neurons can grow in a 3D fashion. Such a scaffold can be realized with polymer gels and solid porous matrices^9-13. By exploiting the mechanical properties of the polymers, it is possible to embed cells inside these structures by defining a uniform block of 3D cultures of neurospheres¹¹. The main feature of this approach is the rigid mechanical property of the neurospheres^9,12. However, these materials have limited porosity, and they do not guarantee cell migration inside the matrix. To overcome this drawback, a possible solution consists in slicing the matrix into ‘unit’ modules. Unfortunately, the size and shape diversity of the particles could hamper the packing into regular layered structures. In⁷, Cullen and coworkers designed a 3D neuronal construct made up of neurons and/or astrocytes within a bioactive extracellular matrix-based scaffold. Such an engineered neural tissue allowed in vitro investigations to study and manipulate neurobiological responses within 3D micro-environments. This model consisted of neurons and glia distributed throughout the extracellular matrix (ECM) and/or hydrogel scaffolds (500-600 µm thick). In this condition, an optimum cell viability (greater than 90%) was found by plating cells at a final density of about 3,750 - 5,000 cells/mm³. It must be noted that such a density value is far lower than the one in the in vivo condition, where the cell density of the mouse brain cortex is about 90,000 cells/mm³¹⁴. To overcome this limitation Pautot and coworkers¹⁵ realized a 3D in vitro system where cell density and network connectivity are controlled to resemble in vivo conditions while enabling real-time imaging of the network. Practically, this method is based on the concept that dissociated cultured neurons are able to grow on silica microbeads. These beads provide a growth surface large enough for neuronal cell bodies to adhere and for their arborizations to grow, mature, extend, and define synaptic contacts to other neurons. This method exploits the spontaneous assembly properties of mono-dispersed beads to form 3D layered hexagonal arrays containing distinct subsets of neurons on different layers with constrained connectivity among neurons on different beads. The achieved cell density with this method was about 75,000 cells/mm³.

Recently, we have adapted Pautot’s method to MEAs¹⁶: the obtained results show that the 3D electrophysiological activity presents a wider repertoire of activities than the one expressed by 2D networks. 3D mature cultures exhibit an enhanced dynamic in which both network burst and random spike activity coexist. Similarly, Tang-Schomer and coworkers¹⁷ realized a silk protein-based porous scaffold which maintains a primary cortical culture in vitro for some months, and recorded the electrophysiological activity by means of a tungsten electrode.

In this work, the experimental procedures to build 3D neuronal networks coupled to MEAs will be described.

Protokół

The experimental protocol was approved by the European Animal Care Legislation (2010/63/EU), by the Italian Ministry of Health in accordance with the D.L. 116/1992 and by the guidelines of the University of Genova (Prot. N. 13130, May 2011). All efforts were made to reduce the number of animals for the project and to minimize their suffering.

1. Preparation of Materials and Supports

1. Construct the mold to build the PDMS (Poly-Dimethyl-Siloxane) constraint by means of a CNC (Computer Numerical Control) milling machine. Such a mold is realized in polycarbonate with the central cylinder in polytetrafluoroethylene (PTFE). This material allows an easier extraction of the mask once the PDMS has hardened.
   NOTE: The design of the mold has been performed by Computer-Aided-Design (CAD) and then delivered to the CNC milling machine.
2. Prepare the PDMS elastomer. PDMS is an organic polymer. It is composed of two elements, a curing agent and a polymer. Mix them by a volume ratio of 1:10, one part of curing agent and nine parts of polymer. Put the two components in a Petri dish, shake and insert the mix in the vacuum chamber for 10 min to eliminate air bubbles. 3 g of PDMS are sufficient for one constraint.
3. Construct the PDMS constraint. Insert the PDMS material into the molder and put it into the oven for 30 min at 120 °C. The PDMS constraint has the shape of an ideal cylinder with an external and internal diameter of 22.0 and 3.0 mm, respectively and a height of 650 µm.
4. The day before the plating, couple the mask to the active area of the Micro-Electrode Arrays (MEAs) by means of a tweezer under a stereomicroscope and sterilize the PDMS mask in the oven at 120 °C.
5. Sterilize the glass microbeads (nominal diameter of 40±2 µm; certified mean diameter of 42.3±1.1 µm) in 70% ethanol for 2 hr in a conical vial and rotate every 30 min to expose all microbeads. 
6. Remove the ethanol solution from the vial and rinse the microbeads two times with sterilized water.
7. Condition the central part of the MEA area delimited by the PDMS mask (diameter equals to 3.0 mm) with 24 µl of mixed solution of Poly-D-Lysine and Laminin at 0.05 μg/ml.
8. Coat the microbeads with adhesion proteins, Laminin and Poly-D-Lysine at 0.05 µg/ml and leave them overnight in the incubator at 37 °C, to obtain a stable and long-lasting neuronal network.
9. Remove the adhesion factors both from the glass surface of the MEA and from the glass microbeads. Aspirate approximately 95% of the coating solution with a pipette and apply a small volume- 24µl- of sterile water on the MEA surface. Aspirate again more than 95% of the water and let the MEA dry under the laminar hood for 1 hr before plating the cells.
   NOTE: In the case of microbeads instead, aspirate the coating solution and make a first wash with sterilized water and a second one with the basal medium and its supplement such as B27, in order to condition the glass surface where neurons will be plated. Do not let the microbeads dry. Leave them in suspension in the culture medium inside the vial.
10. Distribute, by means of a pipette -200 µl-, the suspension of treated microbeads (about 32,000) onto a multiwell plate with membrane insert (pore diameter 0.4 µm) where they will self-assemble forming a uniform layer.
11. Fill each well of the membrane with 0.5 ml of medium and put it in the incubator (T = 37.0 ^oC, CO₂ = 5%) until the neurons are ready to be plated (3.2).

2. Dissection of Embryos and Dissociation of Tissue

1. House adult female rats (200-250 g) at a constant temperature (22 ± 1 °C) and relative humidity (50%) under a regular light–dark schedule (light-on 7 AM–7 PM) in the animal facility. Ensure that food and water are freely available.
2. Anesthetize a pregnant female rat after 18 days of development (E18) using 3% isoflurane. Then, sacrifice the animal by cervical dislocation.
3. Remove hippocampi from each rat embryo and place them into ice cold Hank’s balanced salt solution without Ca²⁺ and Mg²⁺. At day 18 of development hippocampus and cortex tissues are very soft and do not need to be cut into small pieces. Further details can be found^18,19.
4. Dissociate the tissue in 0.125% of Trypsin/Hank’s solution containing 0.05% of DNAse for 18-20 min at 37 °C.
5. Remove the supernatant solution with a Pasteur pipette and stop the enzymatic digestion by adding medium with 10% fetal bovine serum (FBS) for 5 min.
6. Remove the medium with FBS and wash once with the medium with its supplement, 1% L-glutamine, gentamicin 10 µg/ml. Remove again the medium with a Pasteur pipette and refill it again with a small amount (500 µl) of growth medium with its supplement, 1% L-glutamine, gentamicin 10 µg/ml.
7. Dissociate the tissue pellet mechanically with a narrow Pasteur pipette until a milky suspension of cells is apparent. It is not necessary to centrifuge the cell suspension.
8. Dilute the small volume of cell suspension with the growth medium to obtain a final volume of 2.0 ml. Count the obtained cellular concentration with a hemocytometer chamber. Dilute this concentration at 1:5 in order to obtain the desired cell concentration of 600-700 cells/µl.

3. Cell Plating

1. Plate cells at a density of about 2,000 cells/mm² onto the active area of the MEA, defined by the PDMS constraint to create a 2D neuronal network.
   NOTE: Each hippocampus contains about 5 x 10⁵ cells, (1 x 10⁶ for a single embryo). Dissect 6 embryos to get a total amount of 6 x 10 ⁶ cells in 2 ml, and an estimated concentration of 3,000 cells/µl. Dilute this concentration at 1:5 in order to obtain the desired cell concentration and plate about 600-700 cells/µl. If the MEA area delimited by the PDMS constraint is about 7.065 mm², and the total number of plated cells 600 cells/µl x 24 µl = 14,400 cells, the final density of 2,038 cells/mm².
2. Place the MEA devices into the incubator with humidified CO₂ atmosphere (5%) at 37 °C.
3. Distribute 160 µl of the suspension with a cell concentration of 600-700 cells/µl (about 100,000 cells) onto the surface of the microbeads monolayer positioned inside the multiwell plates to complete the preliminary step for the construction of three-dimensional culture. Put the multiwell plates in the incubator with humidified CO₂ atmosphere (5%) at 37 °C.

4. 3D Neuronal Network Construction

1. 6-8 hr after the plating, transfer the suspension (microbeads with neurons) from the multiwell plates very carefully inside the area delimited by the PDMS constraint by using a pipette set for a volume of about 30-40 µl. After each transfer, wait for about half a minute, to allow the microbeads to self-assemble in a hexagonal compact structure.
2. Once all the layers are deposited and spontaneously assembled, refill with a large drop of about 300 µl of medium the top of the area delimited by the PDMS constraint.
3. Put the 3D structure coupled to the MEA in incubator (T = 37.0 ^oC, CO₂ = 5%) for 48 hr before adding a final volume (about 1 ml) of growth medium culture with its supplement.
   NOTE: Consider the total number of beads on the multiwell plates with membrane insert (30,000) and the number of beads on a single layer onto the MEA device (6,000). The resulting 3D structure is composed of 5 layers of microbeads and cells. Taking into account that the 3D neuronal network is not geometrically perfect, the resulting 3D structure could be composed of 5-8 layers.
4. The day after plating, carefully add the final volume of the medium (about 900 µl) inside the MEA ring. Maintain the 3D cultures in a humidified CO₂ atmosphere (5%) at 37 °C for 4-5 weeks. Replace half of the medium once a week.

5. Confocal Microscopy Acquisition

1. Fix the biological samples to the stage of the microscope in a holder. Set the laser (Argon, 496-555 nm), the scan speed (400 Hz) at which to acquire the image, the gain and offset (-0.3%) of PMT for each image to capture the correct bandwidth emission filter and in order to avoid saturation and to increase the signal to noise ratio. The Results Section reports the values used to acquire the images of Figure 4.
2. For the acquisition of the z-stack sequence, first select, the value of z position of the top and the bottom layer of the sample, and then select the step size to make the acquisition.

Wyniki

In this experimental procedure, a relevant role is played by the microbeads that define the mechanical scaffold for the growth of the 3D neuronal network. Figures 1A and B display the arrangement of the microbeads (nominal diameter of 40±2 µm; certified mean diameter of 42.3± 1.1 µm) in the plane. The peculiarity of such structures is that microbeads spontaneously self-assemble defining a compact hexagonal geometry (Figure 1B and C). The so generated scaffold guarante...

Dyskusje

In this work, a novel experimental in vitro platform made up of 3D engineered neuronal cultures coupled to MEAs for network electrophysiology has been presented. The use of microbeads as scaffold to allow the neuritic outgrowth along the z-axis has been tailored to be integrated with the planar MEA. In this way, the obtained micro-system results in a valid and reliable in vitro 3D model to study the emergent electrophysiological dynamics¹⁶.

MEA recording s...

Materiały

Name	Company	Catalog Number	Comments
Laminin	Sigma-Aldrich	L2020	0.05 µg/ml
Poly-D-lysine	Sigma-Aldrich	P6407	0.05 µg/ml
Trypsin	Gibco	25050-014	0.125% diluted 1:2 in HBSS w\o CA++, MG++
DNAase	Sigma-Aldrich	D5025	0.05% diluted  in Hanks solution
Neurobasal	Gibco Invitrogen	21103049	culture medium
B27	Gibco Invitrogen	17504044	2% medium supplent
Fetal bovine serum (FBS)	Sigma-Aldrich	F-2442	10%
Glutamax-I	Gibco	35050038	0.5 mM
gentamicin	Sigma-Aldrich	G.1272	5mg/liter
Poly-Dimethyl-Siloxane (PDMS)	Corning Sigma	481939	curing agent and the polymer
Micro-Electrode Arrays	Multi Channel Systems (MCS)	60MEA200/30-Ti-pr	MEA with: Electrode grid 8x8; Electrode spacing and diameter 200 and 30 µm, respectively; plastic ring without thread
Microbead	Distrilab-Duke Scientific	9040	1gr Glass Part.Size Stds 40 µm
Transwell	Costar Sigma	CLS 3413	multiwell plates with membrane insert (6.5 mm diameter porous 0.4 µm)
HBSS w\o Ca++ ,Mg++	Gibco Invitrogen	14175-052
Hanks Buffer Solution	Sigma	H8264
Teflon	Sigma	430935-5G	polytetrafluoroethylene
Rat	Sprague Dawley	Wistar Rat
Confocal Microscopy Upright	Leica	TCS SP5 AOBS
20.0x0.50 WATER objective	Leica	Leica HCX APO L U-V-I
40.0x0.80 WATER objective	Leica	Leica HCX APO L U-V-I
25.0x0.95 WATER objective	Leica	HCX IRAPO L