Directional migration of neuronal PC12 cells in a ratchet wheel shaped microchamber

doi:10.1016/j.jbiosc.2009.02.020

Journal of Bioscience and Bioengineering

Volume 108, Issue 1, July 2009, Pages 76-83

https://doi.org/10.1016/j.jbiosc.2009.02.020 Get rights and content

Abstract

Directional migration of neuronal cells over long distances is critical for the developing and regenerating nervous system. A scaffold, which includes radial glia, provides a route between the germinal zone and the destination, with the direction of migration being attributed mainly to chemotaxis. However, the decrease in chemokine concentration with distance complicates cell guidance control over long distances. Our working hypothesis is that neuronal cells migrate directionally on an anisotropic and periodic scaffold. We tested this in a model involving neuronal PC12 cells cultured in a ratchet wheel-shaped microchamber. The microchamber was constructed by printing a patterned, thin film of polydimethylsiloxane (PDMS), to which the cells adhere weakly, onto a collagen-coated dish, to which the cells adhere strongly, using a microcontact printing technique. The cells can attach, extend neurites, and migrate on the anisotropic and periodic collagen-coated area between the ratchet wheel-shaped outer frame and round inner frame of the PDMS. We found that the microchamber geometry affected the direction of migration, even though the mean length of the longest neurite was independent of microchamber geometry. The time-course trace of cell body migration and neurite tips showed that the neurite tips remained around the tips of the ratchet teeth. These results suggested that neuronal cells migrate directionally on a scaffold, even in the absence of chemokine, and reveal a new conceptual framework for neuronal migration.

Section snippets

Microchamber fabrication

Culture dishes were coated with 30 μg/ml type I collagen (Cellmatrix Type I-A; Nitta Gelatin Inc, Osaka, Japan) in phosphate-buffered saline (PBS) for at least 3 h at room temperature, rinsed twice with deionized distilled water to remove excess type I collagen and ions, and then air-dried. PDMS (Sylgard 184, 10:1 mix; Dow Corning) stamps were made using a mold of stainless steel coated with a patterned resistance film (25-μm thickness; Hirai Seimitsu Kogyo, Osaka, Japan). The cured PDMS stamp

Trapping and observing PC12 cells in the PDMS-printed microchamber

The tracing of neuronal cells in a microchamber for long periods of time is difficult because the cells tend to escape from the chamber. The microchambers used in this study were constructed using the μCP technique 13., 14., as described in Materials and methods. First, to analyze the chemical and physical properties of the microchamber, we made a simple microchamber comprising circular collagen spots surrounded by a thin film of PDMS. Immunofluorescence staining of type I collagen clearly

Acknowledgments

This work was supported in part by Wako Pure Chemical Industries, Ltd, a Grant-in-Aid for Scientific Research on Priority Areas “System cell engineering by multi-scale manipulation” (20034015) to KO from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan, and a 21st century COE grant from the MEXT of Japan. The authors thank Dr. Keiji Naruse from Okayama University for helpful advice regarding the microcontact printing, and Mr. Yasuhiko Nagasaka (Beckman Coulter)

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Directional migration of neuronal PC12 cells in a ratchet wheel shaped microchamber

Abstract

Section snippets

Microchamber fabrication

Trapping and observing PC12 cells in the PDMS-printed microchamber

Acknowledgments

Neuron

Trends Neurosci.

Cell

Dev. Biol.

J. Neurosci. Methods

Trends Biotech.

Biomaterials

Curr. Biol.

Biomaterials

Cell

Developmental and evolutionary adaptations of cortical radial glia

Cereb. Cortex