Fabrication of Magnetic Platforms for Micron-Scale Organization of Interconnected Neurons

Summary

This work presents a bottom-up approach to the engineering of local magnetic forces for control of neuronal organization. Neuron-like cells loaded with magnetic nanoparticles (MNPs) are plated atop and controlled by a micro-patterned platform with perpendicular magnetization. Also described are magnetic characterization, MNP cellular uptake, cell viability, and statistical analysis.

Abstract

The ability to direct neurons into organized neural networks has great implications for regenerative medicine, tissue engineering, and bio-interfacing. Many studies have aimed at directing neurons using chemical and topographical cues. However, reports of organizational control on a micron-scale over large areas are scarce. Here, an effective method has been described for placing neurons in preset sites and guiding neuronal outgrowth with micron-scale resolution, using magnetic platforms embedded with micro-patterned, magnetic elements. It has been demonstrated that loading neurons with magnetic nanoparticles (MNPs) converts them into sensitive magnetic units that can be influenced by magnetic gradients. Following this approach, a unique magnetic platform has been fabricated on which PC12 cells, a common neuron-like model, were plated and loaded with superparamagnetic nanoparticles. Thin films of ferromagnetic (FM) multilayers with stable perpendicular magnetization were deposited to provide effective attraction forces toward the magnetic patterns. These MNP-loaded PC12 cells, plated and differentiated atop the magnetic platforms, were preferentially attached to the magnetic patterns, and the neurite outgrowth was well aligned with the pattern shape, forming oriented networks. Quantitative characterization methods of the magnetic properties, cellular MNP uptake, cell viability, and statistical analysis of the results are presented. This approach enables the control of neural network formation and improves neuron-to-electrode interface through the manipulation of magnetic forces, which can be an effective tool for in vitro studies of networks and may offer novel therapeutic biointerfacing directions.

Introduction

Micropatterning of neurons holds great potential for tissue regeneration^1,2,3,4,5 and the development of neuro-electronic devices^6,7,8. However, the micron-scaled positioning of neurons at high spatial resolution, as in biological tissues, poses a significant challenge. Forming predesigned structures at this scale requires the guidance of nerve cell processes by locally controlling soma motility and axonal outgrowt....

Protocol

NOTE: Perform all biological reactions in a biosafety cabinet.

1. Magnetic platform fabrication

1. Lithography
   1. Cut glass slides into 2 x 2 cm² using a scriber pen. Clean the glass slides in acetone and then isopropanol for 5 min each in an ultra-sonication bath. Dry with ultra-high purity (UHP) nitrogen.
   2. Coat the glass with photoresist using spin-coating at 6,000 rpm for 60 s, to attain 1.5 µm thickness, and bake at 100 °C for 60 s. Expose the sample to a light source, using an appropriate wavelength for photoresist, with a desired pattern, using a photomask or mask-less lithography.
   ....

Results

Magnetic platforms with different geometric shapes were fabricated (Figure 1A). Magnetic patterns were deposited by sputtering: 14 multilayers of Co₈₀Fe₂₀ and Pd, 0.2 nm and 1 nm, respectively. Electron microscopy revealed the total height of the magnetic patterns to be ~18 nm (Figure 1B). This unique FM multilayer deposition creates a stable platform with perpendicular magnetization anisotropy (PMA) relativ.......

Discussion

The representative results demonstrate the effectiveness of the presented methodology for controlling and organizing neuronal network formation at the micron-scale. The MNP-loaded PC12 cells remained viable and were transformed into magnetic sensitive units that were attracted by the magnetic forces from the FM electrodes to specific sites. This is best demonstrated in Figure 5C, where the cells preferentially adhered to the larger vertices of the hexagons and not the thin lines. Moreover, b.......

Materials

Name	Company	Catalog Number	Comments
16% Paraformaldehyde (formaldehyde) aqueous solution	ELECTRON MICROSCOPY SCIENCES	15710
6-well cell culture plate	FALCON	353846
96-well cell culture plate	SPL life sciences	30096
Amphotericin B solution	Biological Industries	03-028-1B
AZ 1514H photoresist	MicroChemicals GmbH
AZ 351 B developer	MicroChemicals GmbH
Bovine serum albumin (BSA)	Biological Industries	03-010-1B
Cell and Tissue cultur flask	Biofil	TCF002250	75.0 cm^2 250 mL Vent cap, Non-treated
Cell culture dish	Greiner Bio-One	627-160	35 mm
Cell Proliferation Kit (XTT-based)	Biological Industries	20-300-1000
Centrifuge tube	Biofil	CFT021500	50 mL
Co80Fe20 at% sputter target	ACI Alloys		99.95%
Collagen type I	Corning Inc.	354236	Rat Tail, concentration range 3-4 mg/mL
Confocal microscope	Leica		TCS SP5
Cy2-conjugated AffiniPure Donkey Anti-rabbit secondary antibody	Jackson ImmunoResearch Laboratories, Inc.	711-165-152
DAPI fluoromount-G	SouthernBiotech	0100-20
Disposable needle	KDL		23 G
Disposable  syringe	Medispo	1160227640	10 mL
Donor horse serum	Biological Industries	04-124-1A
ELISA reader	Merk Millipore		BioTek synergy 4 hybrid microplate reader
Ethanol 70%	ROMICAL LTD	19-009102-80
Ethanol absolute (Dehydrated)	Biolab-chemicals	52505
Fetal bovine serum (FBS)	Biological Industries	04-127-1A
Fresh murine β-NGF	Peprotech	450-34
GMW C-frame electromagnet .	Buckley systems LTD		3470, 45 mm
Hydrochloric acid 32%	DAEJUNG CHEMICAL & METALS	4170-4100
ImageJ	US National Institutes of Health, Bethesda		NeuronJ plugin
Inductively coupled plasma (ICP)	Ametek Spectro		SPECTRO ARCOS ICP-OES, FHX22 MultiView plasma
Keithley source-measure	Keithley		2400
Keithley switching system	Keithley		3700
L-glutamine	Biological Industries	03-020-1B
Light microscope	Leica		DMIL LED
Maskless photolithography	Heidelberg Inst.		MLA150
Microscope Slides	BAR-NAOR	BN1042000C
Nitric acid 70%	Sigma-Aldrich	438073
Normal donkey serum (NDS)	Sigma	D9663
PBS 10x	hylabs	BP507/1LD
PC12 cell line	ATCC	CRL-1721
Pd sputter target	ACI Alloys		99.95%
Penicillin-streptomycin nystatin solution	Biological Industries	03-032-1B
PrestoBlue cell viability reagent	Molecular probes	A-13261	resazurin-based
Rabbit antibody to α-tubulin	Santa Cruz Biotechnology, Inc.
RF magnetron sputtering system	Orion AJA Int.		Orion 8
RPMI 1640 with l-glutamine	Biological Industries	01-100-1A
Sonication bath	KUDOS	SK3210HP	Frequency: 53 kHz. Ultrasonic power: 135 W
SQUID magnetometer	Quantum Design, CA
Triton X-100	CHEM-IMPEX INTERNATIONAL	1279	non-ionic surfactant
XTT cell viability reagent