title: Guiding neuron development with planar surface gradients of substrate cues deposited using microfluidic devices.
abstract: Wiring the nervous system relies on the interplay of intrinsic and extrinsic signaling molecules that control neurite extension, neuronal polarity, process maturation and experience-dependent refinement. Extrinsic signals establish and enrich neuron-neuron interactions during development. Understanding how such extrinsic cues direct neurons to establish neural connections in vitro will facilitate the development of organized neural networks for investigating the development and function of nervous system networks. Producing ordered networks of neurons with defined connectivity in vitro presents special technical challenges because the results must be compliant with the biological requirements of rewiring neural networks. Here we demonstrate the ability to form stable, instructive surface-bound gradients of laminin that guide postnatal hippocampal neuron development in vitro. Our work uses a three-channel, interconnected microfluidic device that permits the production of adlayers of planar substrates through the combination of laminar flow, diffusion and physisorption. Through simple flow modifications, a variety of patterns and gradients of laminin (LN) and fluorescein isothiocyanate-conjugated poly-l-lysine (FITC-PLL) were deposited to present neurons with an instructive substratum to guide neuronal development. We present three variations in substrate design that produce distinct growth regimens for postnatal neurons in dispersed cell cultures. In the first approach, diffusion-mediated gradients of LN were formed on cover slips to guide neurons toward increasing LN concentrations. In the second approach, a combined gradient of LN and FITC-PLL was produced using aspiration-driven laminar flow to restrict neuronal growth to a 15 microm wide growth zone at the center of the two superimposed gradients. The last approach demonstrates the capacity to combine binary lines of FITC-PLL in conjunction with surface gradients of LN and bovine serum albumin (BSA) to produce substrate adlayers that provide additional levels of control over growth. This work demonstrates the advantages of spatio-temporal fluid control for patterning surface-bound gradients using a simple microfluidics-based substrate deposition procedure. We anticipate that this microfluidics-based patterning approach will provide instructive patterns and surface-bound gradients to enable a new level of control in guiding neuron development and network formation.
authors.affiliation: Department of Cell and Developmental Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA. mgillett@illinois.edu.
authors.forename: Larry J
authors.initials: LJ
authors.lastname: Millet
authors.forename: Matthew E
authors.initials: ME
authors.lastname: Stewart
authors.forename: Ralph G
authors.initials: RG
authors.lastname: Nuzzo
authors.forename: Martha U
authors.initials: MU
authors.lastname: Gillette
mesh_terms: D000818:Animals; D003585:Cell Biology / Q000295:instrumentation; D049109:Cell Proliferation; D003584:Cytological Techniques / Q000295:instrumentation*; D004058:Diffusion; D005898:Glass / Q000737:chemistry; D046210:Microfluidic Analytical Techniques; D016501:Neurites / Q000378:metabolism; D009474:Neurons / Q000166:cytology* / Q000378:metabolism; D051381:Rats; D013499:Surface Properties
references.citation: Electrophoresis. 2000 Jan;21(1):27-40
references.pmid: 10634468
references.citation: J Biomater Sci Polym Ed. 2003;14(4):383-94
references.pmid: 12747676
references.citation: J Neurobiol. 2006 Sep 15;66(11):1183-94
references.pmid: 16858695
references.citation: Lab Chip. 2007 Jun;7(6):681-94
references.pmid: 17538709
references.citation: Brain Res. 1986 Jul;393(1):121-6
references.pmid: 3730889
references.citation: J Biomed Mater Res A. 2004 Feb 1;68(2):235-43
references.pmid: 14704965
references.citation: Biomaterials. 2007 Jan;28(2):126-33
references.pmid: 16945407
references.citation: Ann Biomed Eng. 2008 Jun;36(6):889-904
references.pmid: 18392680
references.citation: J Cell Biol. 1983 Dec;97(6):1882-90
references.pmid: 6643580
references.citation: Electrophoresis. 2002 Oct;23(20):3461-73
references.pmid: 12412113
references.citation: Lab Chip. 2007 Nov;7(11):1454-60
references.pmid: 17960271
references.citation: J Neurosci. 1998 Jan 1;18(1):184-94
references.pmid: 9412499
references.citation: Dev Neurobiol. 2007 Nov;67(13):1765-76
references.pmid: 17659593
references.citation: Med Biol Eng Comput. 1998 Jan;36(1):135-41
references.pmid: 9614762
references.citation: Lab Chip. 2006 Mar;6(3):381-8
references.pmid: 16511621
references.citation: Lab Invest. 1995 Oct;73(4):565-76
references.pmid: 7474929
references.citation: Langmuir. 2003 Mar 4;19(5):1551-1556
references.pmid: 20725530
references.citation: Lab Chip. 2009 Mar 7;9(5):644-52
references.pmid: 19224012
references.citation: Lab Chip. 2008 Feb;8(2):227-37
references.pmid: 18231660
references.citation: Lab Chip. 2007 Aug;7(8):987-94
references.pmid: 17653340
references.citation: Proc Natl Acad Sci U S A. 1985 Apr;82(7):2183-7
references.pmid: 3856891
references.citation: Biosens Bioelectron. 2006 Jan 15;21(7):1093-100
references.pmid: 15961304
references.citation: J Neurosci Res. 1991 Oct;30(2):300-7
references.pmid: 1798054
references.citation: Lab Chip. 2008 Nov;8(11):1896-905
references.pmid: 18941691
references.citation: Langmuir. 2004 Sep 28;20(20):8805-11
references.pmid: 15379510
references.citation: In Vitro Cell Dev Biol Anim. 1995 Jan;31(1):45-54
references.pmid: 7704333
references.citation: J Neurosci. 1985 Nov;5(11):3030-8
references.pmid: 4056864
references.citation: Science. 2006 Dec 1;314(5804):1464-7
references.pmid: 17138901
references.citation: Biomed Microdevices. 2007 Feb;9(1):15-23
references.pmid: 17091393
references.citation: J Cell Biol. 1985 Sep;101(3):898-913
references.pmid: 4030898
references.citation: Lab Chip. 2005 Apr;5(4):401-6
references.pmid: 15791337
references.citation: Anal Chem. 2006 May 1;78(9):3084-9
references.pmid: 16642997
references.citation: Langmuir. 2006 Apr 25;22(9):4250-8
references.pmid: 16618172
references.citation: Lab Chip. 2010 Jan 21;10(2):235-9
references.pmid: 20066252
references.citation: Annu Rev Biophys Biomol Struct. 2007;36:213-31
references.pmid: 17269901
references.citation: Dev Biol. 1983 Jul;98(1):212-20
references.pmid: 6862106
references.citation: Angew Chem Int Ed Engl. 2006 May 26;45(22):3618-23
references.pmid: 16639763
references.citation: Proc Natl Acad Sci U S A. 2002 Oct 1;99(20):12542-7
references.pmid: 12237407
references.citation: Nat Methods. 2005 Aug;2(8):599-605
references.pmid: 16094385
references.citation: Tissue Eng. 2006 Feb;12(2):267-78
references.pmid: 16548685
references.citation: Anal Chem. 2001 Oct 1;73(19):4682-7
references.pmid: 11605847
references.citation: Cancer Sci. 2007 Sep;98(9):1311-7
references.pmid: 17627619
references.citation: Langmuir. 2006 Jun 20;22(13):5945-51
references.pmid: 16768534
references.citation: Neurochem Res. 2003 Nov;28(11):1639-48
references.pmid: 14584818
references.citation: Exp Cell Res. 1995 Aug;219(2):427-41
references.pmid: 7641794
references.citation: Lab Chip. 2009 Feb 7;9(3):417-26
references.pmid: 19156291
references.citation: Lab Chip. 2008 Jan;8(1):34-57
references.pmid: 18094760
references.citation: Anal Chem. 2007 Nov 15;79(22):8557-63
references.pmid: 17953452
references.citation: J Neurosci. 1999 Aug 1;19(15):6417-26
references.pmid: 10414970
references.citation: Dev Biol. 1988 Nov;130(1):232-6
references.pmid: 3181628
references.citation: Lab Chip. 2004 Apr;4(2):91-7
references.pmid: 15052346
references.citation: Nature. 2007 Mar 1;446(7131):46-51
references.pmid: 17310144
references.citation: EMBO J. 1984 Jul;3(7):1463-8
references.pmid: 6745238
references.citation: J Am Chem Soc. 2006 Mar 29;128(12):3880-1
references.pmid: 16551074
references.citation: Dev Biol. 1983 Jan;95(1):56-64
references.pmid: 6825932
references.citation: J Neurochem. 2007 May;101(4):865-82
references.pmid: 17254012
references.citation: J Neurosci. 1996 Jul 15;16(14):4389-401
references.pmid: 8699250
references.citation: Anal Chem. 2003 Nov 1;75(21):5775-82
references.pmid: 14588017
references.citation: J Biomech Eng. 1999 Feb;121(1):73-8
references.pmid: 10080092
references.citation: Angew Chem Int Ed Engl. 2004 Aug 13;43(32):4190-3
references.pmid: 15307084
references.citation: Biomed Microdevices. 2007 Oct;9(5):627-35
references.pmid: 17530414
references.citation: J Nanosci Nanotechnol. 2009 Dec;9(12):7330-5
references.pmid: 19908782
references.citation: Langmuir. 2007 Oct 9;23(21):10706-14
references.pmid: 17803329
references.citation: Eur Biophys J. 2009 Oct;38(8):1035-47
references.pmid: 19579023