NEURON AGGREGATE CULTURE PLATFORM FOR IN VITRO CNS MYELINATION STUDY

J. Park1, H. Koito2, J. Li2 and A. Han1, 3* 1 Department of Electrical and Computer Engineering, 2 Department of Veterinary Integrative Biosciences, and

3 Department of Biomedical Engineering, Texas A&M University, College Station, TX, USA ABSTRACT

A cell culture platform for neuron aggregates has been designed and fabricated as an in vitro model for studying CNS myelination. The device is composed of nine culture chambers each of which has 55 cell trapping sites for culturing neuron aggregates in a spatially controlled environments in parallel. Neuron aggregates prepared from embryonic day 16 rat fetus were cultured with oligodendrocyte progenitor cells (OPCs) and showed successful growth inside the device. Neuron ag-gregates formed intense axonal network with neighboring aggregates and OPCs differentiated into mature oligodendrocytes, expressing myelin basic protein. KEYWORDS: Neuron culture microsystem, CNS myelination, Neuron aggregate, Co-culture microsystem

INTRODUCTION

Myelination in central nervous system (CNS) is a sequential and multi-step process that requires reciprocal signaling between neurons and oligodendrocytes (OLs). Myelin sheaths, formed by OLs that wrap around axons, allow rapid im-pulse signal transfer in the system, and dysfunction of myelin sheath results in many neurological disorders such as mul-tiple sclerosis or Alzheimer’s disease [1]. Nevertheless, due to the complexity of the process and lack of in vitro models that are easily accessible for experimental manipulations, the mechanism and myelin regulating signals remain largely unknown. Recently, Koito et al. introduced a novel CNS neuron aggregate culture system prepared from embryonic rat forebrains as an in vitro model system to study myelination [2]. Robust myelination that could not be found in conven-tional dissociated neuron and glia co-cultures was observed using this culture system. However, myelins expressed in the system showed dependency on distances between the neuron aggregates, a parameter that is difficult to control in conventional culture methods. Here, we introduce for the first time a poly(dimethylsiloxane) (PDMS) co-culture plat-form for neuron aggregates capable of simultaneously culturing 495 aggregates in a spatially controlled environment for potential screening of growth factors or drugs that promote CNS myelination.

EXPERIMENTAL

The neuron aggregate culture platform is composed of three PDMS layers; a culture chamber layer, a cell trap layer, and a cell loading layer (Figure 1A). Primary CNS neurons are prepared from forebrains of embryonic day 16 (E16) rats and aggregated by suspending them in culture media for three days. Suspended cells are then filtered with 70 µm and 40 µm cell strainers to obtain 40-70 µm diameter neuron aggregates. Aggregated cells are loaded into the nine culture chambers simultaneously via a single loading port on the cell loading layer. The radial distribution of the nine culture chambers allows cells in different chambers to be exposed to equal shear stress during the cell loading and culture media exchange process with the intention that more accurate chamber-to-chamber analysis could be achieved. Neuron aggre-gates loaded into the cell loading ports flow through the cell loading channels and are captured at the 495 trapping sites inside the nine culture chambers (Figure 1D). Cells are fed every 3-4 days by changing out culture media through the cell loading port. Cells that are not trapped at capturing sites and culture media waste from the nine culture chambers are collected from the waste reservoir. Arrays of trapping sites in the middle layer were designed to have three different row-to-row distances (500, 1000, 1500 µm) to investigate how different distances between the neuron aggregates affect myelin formation (Figure 2). The height of the culture chambers was 150 µm, while the pillar structures forming the traps attached to the top were only 100 µm long. This design lets trapping pillars to hang from the top part of the culture chamber with a 50 µm spacing between the bottom of the chamber and the pillars (Figure 1D – inset). This facilitates

Figure 1: (A) Three layers composing the neuron aggregate culture platform. (B) A photographic image of the PDMS device filled with color dyes (Red: Cell loading channels, Yellow: cell culture chambers). (C) Illustrations showing cross-sections of the device. (D) Close-up view of a cell culture chamber showing fluidic flow during the cell loading and culture media exchange process (Inset: A neuron aggregate captured at a trapping site).

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978-0-9798064-3-8/µTAS 2010/$20©2010 CBMS 46 14th International Conference onMiniaturized Systems for Chemistry and Life Sciences

3 - 7 October 2010, Groningen, The Netherlands

neuron aggregates to be captured more easily by reducing the fluidic resistance caused by the trapping structures as well as prevents the trapping structures from obstructing neurite outgrowth.

The cell loading layer and the culture chamber layer were replicated from SU-8 masters by conventional PDMS soft-lithography process and the middle layer that has the 100 µm high trapping structures was made by a two-step PDMS replication process. First, a SU-8 master with pillar structures were fabricated and a PDMS master was replicated from the SU-8 master. The final middle PDMS layer with pillar structures was then replicated from the PDMS master. Over-all fabrication steps of the device are shown in Figure 3. The three layers were treated with oxygen plasma and assembled under a microscope for precise alignment, followed by an autoclave sterilization. Prior to cell culture, culture chambers were coated with poly-d-lysine and matrigel® for enhanced cell adhesion and growth.

RESULTS AND DISCUSSION

The cell trapping structures obstruct the fluidic flow inside the culture chamber and can deteriorate the trapping effi-ciency when the fluidic resistances caused by the structures are too high. In order to optimize the size and the design of the trapping sites, a commercial finite element method (FEM) simulation tool (COMSOL Multiphysics®, Inc., MA) was used to analyze the fluidic flow inside the culture chamber. Simulation results show that the flow inside the chamber was minimally interfered by the optimized trapping structures and the flow velocity was uniform throughout the chamber during the culture media exchange process (Figure 4).

Neuron aggregates prepared from E16 rats were dispensed on the cell loading port and loaded into the nine culture chambers by applying negative pressure from the waste reservoir. Aggregated cells were trapped at each trap and showed successful neurite growth inside the device (Figure 5). After 10 days of culture in vitro (DIV 10), OL progenitor cells (OPCs), isolated from postnatal day 1-2 rat brains were added into the culture chambers uniformly through the cell loading port and co-cultured with neuron aggregates for six more days. At DIV 16, cells were fixed and immunostained for neurofi-lament (NF) and myelin basic protein (MBP). Figure 6 shows stained images of co-cultured neuron aggregates and OLs. Neuron aggregates formed intense axonal networks inside the culture chamber and thick bundles of axons connected neigh-

Figure 4: FEM simulation (COMSOL Multiphysics®) showing the velocity field inside a culture chamber. The fluidic flow inside the chamber was not obstructed by the trapping sites, indicating that cells will be captured at each trapping site during the cell loading process. Also, the uniform and low flow velocity field throughout the chamber shows that all captured cells will be equally ex-posed to minimum shear stress during the culture media exchange process.

Velocity Field (m/s)

Figure 3: Schematic illustration of the fabrication steps.

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Figure 2: (A) Cross-section of an assembled device showing trapping structures hanging from the top of the cham-ber. (B) Arrays of neuron aggregate trapping sites with different row-to-row distances. (C) Close-up view of a trapping structure.

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boring aggregates. MBP was observed both inside and outside of the neuron aggregates. Since MBP is expressed only in mature OLs, the result indicates that not only the additionally loaded OPCs but also the ones that were included in the neu-ron aggregates from the beginning successfully differentiated into mature OLs. This is a promising result showing that the device is capable of conducting co-culture experiments for CNS axon myelination studies. CONCLUSION

We have developed a PDMS neuron aggregate culture platform that can be used for in vitro CNS myelination studies. Neuron aggregates showed successful neurite outgrowth and formed intense axonal network inside the device. Co-cultured OLs also differentiated into mature OLs, expressing MBP. We expect that this microsystem will be a powerful tool for studying in vitro CNS myelination and for screening potential drug candidates that promote CNS myelination. ACKNOWLEDGEMENTS

This work was supported by the National Institutes of Health / National Institute of Mental Health (NIH/NIMH) grant #1R21MH085267. REFERENCES [1] D. Sherman, P. Prophy, Mechanisms of axon ensheathment, Nature Reviews, 6, pp. 683-690, (2005). [2] H. Koito, J. Li, Preparation of Rat Brain Aggregate Cultures for Neuron and Glia Development Studies, Journal of

Visualized Experiments, 31, (2009). CONTACT *A. Han, tel: +1-979-845-9686; arum.han@ece.tamu.edu

Figure 6: Immunostained images of co-cultured OLs and neuron aggregates inside the microdevice after 16 days of culture, including six days of co-culture period (Axon: NF-red, mature OLs: MBP-green).

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Figure 5: Phase contrast images of neuron aggregates inside the device. (A) Neuron aggregates captured inside the trapping sites after the cell loading process. (B) Neuron aggregates forming dense interconnecting axonal net-work with each other after 10 days of culture.

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