Belkaid W et al. (2013), Cellular response to micropatterned growth prom...

XB-ART-49074

BMC Biotechnol 2013 Oct 11;13:86. doi: 10.1186/1472-6750-13-86.

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Cellular response to micropatterned growth promoting and inhibitory substrates.

Belkaid W , Thostrup P , Yam PT , Juzwik CA , Ruthazer ES , Dhaunchak AS , Colman DR .

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Normal development and the response to injury both require cell growth, migration and morphological remodeling, guided by a complex local landscape of permissive and inhibitory cues. A standard approach for studying by such cues is to culture cells on uniform substrates containing known concentrations of these molecules, however this method fails to represent the molecular complexity of the natural growth environment. To mimic the local complexity of environmental conditions in vitro, we used a contact micropatterning technique to examine cell growth and differentiation on patterned substrates printed with the commonly studied growth permissive and inhibitory substrates, poly-L-lysine (PLL) and myelin, respectively. We show that micropatterning of PLL can be used to direct adherence and axonal outgrowth of hippocampal and cortical neurons as well as other cells with diverse morphologies like Oli-neu oligodendrocyte progenitor cell lines and fibroblast-like COS7 cells in culture. Surprisingly, COS7 cells exhibited a preference for low concentration (1 pg/mL) PLL zones over adjacent zones printed with high concentrations (1 mg/mL). We demonstrate that micropatterning is also useful for studying factors that inhibit growth as it can direct cells to grow along straight lines that are easy to quantify. Furthermore, we provide the first demonstration of microcontact printing of myelin-associated proteins and show that they impair process outgrowth from Oli-neu oligodendrocyte precursor cells. We conclude that microcontact printing is an efficient and reproducible method for patterning proteins and brain-derived myelin on glass surfaces in order to study the effects of the microenvironment on cell growth and morphogenesis.

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Species referenced: Xenopus
Genes referenced: actl6a map2 mapt mbp

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	Figure 1. Microcontact printing guides neuronal morphogenesis (A) Shown on top is the silicon wafer with dimensions that are compatible with neuronal cell somata i.e., 10 μm wide lines separated by a pitch of 60 μm. Bottom half of panel shows an example of FITC-conjugated PLL lines printed onto coverglass. In all other cases unconjugated PLL was used. (B) Primary hippocampal neurons plated on micropatterned PLL and immunostained for neuron-specific beta-III tubulin. (C) Primary cortical neurons plated on micropatterned PLL and stained for F-actin. (D) Wafers with octagonal patterns designed to support a single neuronal cell body at each node. (E, F) Primary hippocampal neurons plated on micropatterned PLL and immunostained for beta-III tubulin. These neurons position their cell bodies at the nodes and their neurites extend outward, guided by the patterned PLL.
	Figure 2. Polarization of neurons growing on micropatterned substrates. (A) Hippocampal neurons cultured on micropatterned PLL and immunostained for axonal (Tau-1) and dendritic (MAP2) proteins. (B) Zoomed image of neuron in A. (C) Tau-1 immunostaining shows axons closely follow the patterned lines. (D) MAP2 immunostaining reveals that dendritic processes can be guided by the substrate, but are also regularly found extending on unpatterned glass. Pattern consists of intersecting 20 μm wide stripes spaced 420 μm apart.
	Figure 3. Morphological shaping of Oli-neu and COS7 cells by micropatterned PLL (A, D) High concentration (1 mg/mL) FITC-conjugated PLL lines were printed on top of a low-concentration (1 pg/mL) lawn of unconjugated PLL. (B, C) Oli-neu cells adhered preferentially to the high concentration lines, (E, F) COS7 cells showed a preference for the low-concentration PLL regions. C is a high magnification image overlaying FITC-PLL (green) from A and Oli-neu cells stained with rhodamine-phalloidin (red) for actin from B. F is a high magnification overlay of FITC-PLL and phalloidin-stained COS7 cells. Scale bars are 60 μm.
	Figure 4. Adhesion of COS7 cells on micropatterned PLL. Schematic representation (A) of the three different conditions used: (1) low concentration unconjugated-PLL lawn (1 pg/mL; green) and high concentration DyLight549-conjugated PLL lines (1 mg/mL; red), (2) high concentration DyLight549-conjugated PLL lines (1 mg/mL; red), and (3) low concentration lawn of unconjugated-PLL (1 pg/mL; green). (B) Cells seeded onto the first substrate adhere to the low-concentration lawn of PLL avoiding the high-concentration red PLL lines. (C) Cells seeded on the second substrate in the absence of a low concentration PLL lawn adhere to high-concentration red PLL lines. (D) Cells seeded onto low concentration PLL lawn (1 pg/mL) adhere and display a typical flat morphology. COS7 cells were stained for F-actin. 10 μm wide lines separated by a pitch of 60 μm. (E) Quantification of COS7 cell area overlapping printed PLL lines under the conditions schematized in A.
	Figure 5. Microcontact printed substrates demonstrate inhibition of Oli-neu process outgrowth by myelin. (A, B) CellTracker Green-positive Oli-neu cells plated onto DyLight549-conjugated-PLL lines alone (A) or covered with a myelin lawn (B). (C) In the presence of the myelin lawn, cells adhere to the PLL lines but exhibit a significant reduction in process outgrowth. (D) Microcontact printed myelin lines immunostained for myelin basic protein (MBP; red). (E) Control showing the lack of signal when the primary antibody was omitted. (F, G) CellTracker Green positive Oli-neu cells plated onto a PLL lawn printed with myelin lines. (G) Cells were treated with Y27632, a ROCK inhibitor. (H) Cells treated with Y-27632 show enhanced outgrowth compared to the untreated cells.

References [+] :

Baer, Myelin-mediated inhibition of oligodendrocyte precursor differentiation can be overcome by pharmacological modulation of Fyn-RhoA and protein kinase C signalling. 2009, Pubmed