Design, Surface Treatment, Cellular Plating, and Culturing of Modular Neuronal Networks Composed of Functionally Inter-connected Circuits

Summary

This manuscript describes a protocol to grow in vitro modular networks consisting of spatially confined, functionally inter-connected neuronal circuits. A polymeric mask is used to pattern a protein layer to promote cellular adhesion over the culturing substrate. Plated neurons grow on coated areas establishing spontaneous connections and exhibiting electrophysiological activity.

Abstract

The brain operates through the coordinated activation and the dynamic communication of neuronal assemblies. A major open question is how a vast repertoire of dynamical motifs, which underlie most diverse brain functions, can emerge out of a fixed topological and modular organization of brain circuits. Compared to in vivo studies of neuronal circuits which present intrinsic experimental difficulties, in vitro preparations offer a much larger possibility to manipulate and probe the structural, dynamical and chemical properties of experimental neuronal systems. This work describes an in vitro experimental methodology which allows growing of modular networks composed by spatially distinct, functionally interconnected neuronal assemblies. The protocol allows controlling the two-dimensional (2D) architecture of the neuronal network at different levels of topological complexity.

A desired network patterning can be achieved both on regular cover slips and substrate embedded micro electrode arrays. Micromachined structures are embossed on a silicon wafer and used to create biocompatible polymeric stencils, which incorporate the negative features of the desired network architecture. The stencils are placed on the culturing substrates during the surface coating procedure with a molecular layer for promoting cellular adhesion. After removal of the stencils, neurons are plated and they spontaneously redirected to the coated areas. By decreasing the inter-compartment distance, it is possible to obtain either isolated or interconnected neuronal circuits. To promote cell survival, cells are co-cultured with a supporting neuronal network which is located at the periphery of the culture dish. Electrophysiological and optical recordings of the activity of modular networks obtained respectively by using substrate embedded micro electrode arrays and calcium imaging are presented. While each module shows spontaneous global synchronizations, the occurrence of inter-module synchronization is regulated by the density of connection among the circuits.

Introduction

Experimental and theoretical evidences support the possibility that the brain operates through coordinated activation of cell assemblies^1-5, which can be regarded as dynamic functional units that transiently interact with each other, shaping and underlying different brain states. Functional modularity is also dependent on and associated with the structural modular organization of the brain circuits^6,7. How function and structure of brain circuits mutually shape each other is still one of the main open questions in neuroscience. To provide a deeper understanding of this question, it is important to identify optimal experimental frameworks where it is possible to address, at least partially, those issues. Since controlled manipulation of the spatio-temporal dynamics of neuronal networks in in vivo experiments is challenging, the development of in vitro neuronal networks models is of significant interest due to their easy accessibility, monitoring, manipulation and modeling^8,9. In recent years, in vitro technologies supported by advanced substrate patterning methods have allowed to induce neuronal networks to develop a range of predefined modular structures³ and to study the functional properties of networks with imposed topologies¹⁰. In particular, methods were recently used to organize networks by imposing physical constraints^4,11. Indeed, to study the link between structure and function in neuronal networks and to provide a simplified but plausible representation of interacting neuronal assemblies, in vitro systems should provide inter-connected neuronal sub-populations. Widely studied 2D homogenous neuronal cultures do not impose any spatial constraints on the self-organized emergent wiring of the circuits. Therefore a possible approach to shape artificially interconnected cell assemblies is to position different neuronal populations in spatially distinct areas. The distance among these areas does not prevent the inter assemblies connections. This approach, while ensuring a considerable control over network complexity, has been shown to provide a richer repertoire of synchronization models^6,7,12.

In order to facilitate a reproducible culturing of modular neuronal assemblies, a protocol to assemble the self-organization of networks into neuronal clusters linked by axons and dendrites is presented and described. The polymeric structure for the physical confinement of neuronal cultures has been created from polydimtheylsiloxane (PDMS). PDMS is an elastomer widely used for biomedical applications owing to its biocompatibility, transparency and permeability to gases¹³. The PDMS is prepared and excluded from the micromachined SU8 2075^14,15 structures by spin-coating a liquid PDMS onto a "master" as described previously in Jackman et al.¹⁶ The achieved patterned neuronal networks are composed of inter-connected modules of different size and they have been successfully obtained on both coverslips and Micro Electrode Arrays (MEAs)^17-20. The density of connections between the modules can change the features of the network synchronization, from a fully synchronized network, typical of uniform cultures, to transient states of synchronization among modules.

Protocol

The procedure was done in accordance with the NIH standards for care and use of laboratory animals and was approved by the Tel-Aviv University Animal Care and Use Committee (permit number – L-14-019).

1. Preparation of Instruments and PDMS

1. Prepare the wafer (Table of Materials, or order the wafer from a microfabrication lab), a scalpel, and a pair of tweezers – sterilization is not needed.
2. Make poly-D-lysine (PDL) solution according to the following conditions: 4 mg/ml in 0.1 M borate buffer, pH 8, and store at -20 °C.
3. Prepare PDMS stencils according to the following procedure:
   1. Prepare polydimethylsiloxane (PDMS, silicone elastomer) by mixing together the base and the curing agent with a ratio of 10:1, then stir the 2 substances for approximately 5 min.
   2. Place in vacuum chamber twice for 15 min each time and verify bubbles are gone.
   3. When the PDMS is ready, prepare spin coater: open nitrogen knobs to allow gas usage, place the silicon wafer (Figure 1A) on spinner, and use the vacuum to prevent it from moving.
   4. Pour PDMS over wafer and activate the spinner for 1 min at 1,000 rpm (creates approximately 100 µm height).
   5. Place wafer on a hot plate at 100 °C for 30 min.
   6. When the PDMS has hardened, outline the stencil's borders with PDMS, using Pasteur pipette. Place wafer on a hot plate at 100 °C for 30 min.
   7. When the PDMS borders have hardened, use a scalpel to cut stencils according to the borders and peel off the silicon stencil from the wafer (a squared PDMS containing a single pattern).

2. Petri Dish and Cover Slip Preparation

1. Prepare 23 mm square glass cover slips and clean them according to the following order:
   1. Wash with distilled water, 70% ethanol, and acetone.
   2. Wash with isopropanol and fast dry with nitrogen.
2. Place patterned PDMS stencils on coverslip, and gently press to verify that they are firmly attached to the glass surface. Place in vacuum chamber for 15 min.
3. Drop 1 ml of PDL on stencil. Insert the coverslip into the vacuum chamber twice for 20 min each time, and leave PDL O/N to dry.
4. Prepare Petri dish to create a supporting cell network:
   1. Cover the surface of the dish (3.5 cm) with 1 ml of PDL for 2 hr in RT. Remove the PDL drop using a pipette.
   2. Wash with distilled water and leave to dry. Place a very small drop of silicone grease on each corner of the coverslip.
5. Place the coverslip on the center of the Petri dish (i.e., the PDMS should be facing up) and press gently to verify attachment.
6. Gently remove PDMS from coverslip using tweezers. For sterilization, expose to UV illumination for 7 min.

3. Multi-Electrode Array (MEA) Preparation

1. Clean MEA in this order (Table of Materials):
   1. Wash with water under tap and sonicate in a concentrated, enzymatic detergent 3 times.
   2. Sonicate in distilled water 3 times.
   3. Wash with distilled water (in hood, 1,000 µl tip) for 3 times and place under UV for 30 min.
2. Supporting network preparation:
   1. Prepare the designed support network mold.
      1. Pour PDMS into a 12-well plate (22 mm diameter).
      2. When the PDMS is hardened take out the mold and using a scalpel, cut a hole in the middle to create a ring shape.
      3. Place a designed support network mold on the center of the MEA (Figure 2A) and cover the rest of the surface with PDL for 2 hr at RT.
   2. Remove PDL using pipette and wash with distilled water.
   3. Remove the mold and leave to dry (Figure 2A).
3. Align stencil to MEA in the following way:
   1. Place stencil on designated micro manipulator. Use an inverted microscope to accurately align patterned structure to electrodes, and lower the stencil until it is placed on the MEA surface (Figure 2B).
      Note: Contact with a well cleaned MEA provides competitive adhesion between the PDMS and the MEA itself.
   2. Lift the micromanipulator and if needed, use tweezers to apply a small amount of pressure above the PDMS to prevent it detaching from the MEA.
   3. Gently press stencil to MEA surface, and use microscope to verify it is firmly attached, and well aligned. (Figure 2C-D).
4. Place MEA in vacuum chamber for 15 min; put a 1 ml drop of PDL on stencil.
5. Insert into vacuum chamber 2 times for 20 min each. Leave PDL to dry O/N in the incubator.
6. Before plating, remove the PDMS stencils from MEAs and wash using sterile water. Place under UV illumination for 7 min.

4. Dissection and Culture

1. Prepare cultures as described in Herzog et al. 2011 ²¹.

5. Plating

1. Calculate number of cells needed for plating, using a hemocytometer (optimal density according to the size of the circuit as discussed in the result).
   1. Make sure the counting chamber (hemocytometer) is clean and place a cover slip on it (use alcohol to clean).
   2. Dilute 10 µl of the cell suspension in 190 µl of plating medium (dilution 1:20).
   3. Load 10 µl of the diluted cells onto the edge of the counting chamber and slowly pipette the cells out allowing the chamber to fill itself.
   4. Using an inverted microscope, visualise the haemocytometer grid. Determine the number of cells in the chamber by direct counting (healthy cells should be round). Count the cells within the large square without those crossing the edges.
   5. Calculate the concentration of cells:
      Total number of cells/1000 µl = Total cells counted × dilution factor × 10⁴ (area of the hemocytometer).
      Note: For example, if the dilution factor was 20 and the total cells counted were 100:
      100 × 20 × 10⁴ = 0.1 x 10⁷ cells/1,000 µl or 1 x 10⁶/100 µl. In order to plate 0.75 x 10⁶ cells, take 75 µl out of the cells.
2. Take the number of cells needed for plating per single MEA or Petri dish, resuspend the cells to prevent aggregation, and plate them at the center, on top of the patternate area (if needed, it is possible to dilute the cells in the medium). For the MEA, place a 100 µl drop in the middle. For cover slip, place a 1,000 µl drop in the middle.
3. Incubate the plated cells at 37 °C for 40 min. Add plating medium to the plated cells, up to 1 ml per MEA and 2 ml per cover slip.
4. Keep at 37 °C and, every 4 days, dilute with fresh growth medium enriched with 0.5 Pen-Strep, 2% B-27 and 0.75% glutamax.
5. From 6/7 DIV, after seeing connections between the islands, dilute medium with 10 µl/ml FUDR (25 mg deoxyuridine + 62.5 mg uridine in 12.5 ml MEM (Minimum Essential Medium Eagle)) or any other anti-mitotic agent to prevent glial overgrowth.

Results

A SU8-2075 mold on a silicon wafer with a feature thickness of approximately 100 µm was used to shape the PDMS. The pattern was composed of squares of several dimensions, with a side length and distance varying between 200 and 700 µm (Figure 1B). The size of the square was chosen to fit the field of view of a 10X (for islands with a side length <800 µm) and of a 20X objective (for islands with a side length <400 µm). Three parameters, namely cell plating density, distance between circuits, circuits...

Discussion

A protocol to grow 2D modular neuronal networks in vitro composed of functionally inter-connected circuits is described. The procedure is based on patterning a cellular adhesive layer. Patterning is achieved with PDMS stencils reproducing the negative feature of the desired network architecture. PDMS stencils define the areas where the cellular adhesive layer is deposited. Once cells are plated, they spontaneously assemble to the coated islands and self-organize into active inter-connected circuits. Recordings o...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
PDMS, Sylgard 184	Dow Corning
Nalgene vacuum chamber	Thermo	5305-0609
Poly-D-lysine PDL	Sigma	P7886
Silicone grease - SILICAID 1010	aidchim Ltd	H3375
Spin coater	Laurell - Technologies Corporation	WS-650-23
12-well culture plate	Sigma	CLS3336
5-Fluoro-2’-deoxyuridine	Sigma	F0503
Uridine	Sigma	U3003
MEA1060-Inv-BC	Multi Channel Systems
TC02	Multi Channel Systems
Pen Strep	Biological Industries Beit Haemek	03-033-1c
B-27	Gibco	17504044
glutaMAX	Gibco	35050-038
MEM Minimum Essential Medium-Eagle	Biological Industries Beit Haemek	01-025-1B
Micro Electrode Arrays 4Q	Multi Channel Systems	60-4QMEA1000iR-Ti-pr	cleaning manual: http://www.multichannelsystems.com
Silicon wafer	microchem	SU8-2075	Preparation protocol: http://www.microchem.com