Controlled Adhesion and Growth of Long Term Glial and Neuronal Cultures on Parylene-C

Brennan

J Biomedical Materials Res

et al. 2013

Self Cite

Interfacing neurons with silicon semiconductors is a challenge being tackled through various bioengineering approaches. Such constructs inform our understanding of neuronal coding and learning and ultimately guide us toward creating intelligent neuroprostheses. A fundamental prerequisite is to dictate the spatial organization of neuronal cells. We sought to pattern neurons using photolithographically defined arrays of polymer parylene-C, activated with fetal calf serum. We used a purified human neuronal cell line [Lund human mesencephalic (LUHMES)] to establish whether neurons remain viable when isolated on-chip or whether they require a supporting cell substrate. When cultured in isolation, LUHMES neurons failed to pattern and did not show any morphological signs of differentiation. We therefore sought a cell type with which to prepattern parylene regions, hypothesizing that this cellular template would enable secondary neuronal adhesion and network formation. From a range of cell lines tested, human embryonal kidney (HEK) 293 cells patterned with highest accuracy. LUHMES neurons adhered to pre-established HEK 293 cell clusters and this coculture environment promoted morphological differentiation of neurons. Neurites extended between islands of adherent cell somata, creating an orthogonally arranged neuronal network. HEK 293 cells appear to fulfill a role analogous to glia, dictating cell adhesion, and generating an environment conducive to neuronal survival. We next replaced HEK 293 cells with slower growing glioma-derived precursors. These primary human cells patterned accurately on parylene and provided a similarly effective scaffold for neuronal adhesion. These findings advance the use of this microfabrication-compatible platform for neuronal patterning. © 2013 The Authors. Journal ofBiomedicalMaterials Research Part APublished byWiley Periodicals, Inc.Wiley Periodicals, Inc. J Biomed Mater Res Part A: 102A: 1350–1360, 2014.

Section: Luhmes In Isolationmentioning

confidence: 73%

Section: Introductionmentioning

confidence: 99%

Patterning human neuronal networks on photolithographically engineered silicon dioxide substrates functionalized with glial analogues

Brennan

J Biomedical Materials Res

et al. 2013

Self Cite

“…Once prepared, activation of chips in fetal bovine serum enables a wide range of cell types to be patterned in culture. Our group has successfully patterned primary murine hippocampal cells [14][15][16] , the HEK 293 cell line 17 , the human neuron-like teratocarcinoma (hNT) cell line 18 , primary murine cerebellar granule cells, and primary human glioma-derived stem-like cells. Figure 4 illustrates the potential to augment the patterning platform by using alternative activation solutions.…”

Section: Protocol Representative Resultsmentioning

confidence: 99%

Cell Patterning on Photolithographically Defined Parylene-C: SiO<sub>2</sub> Substrates

Brennan

et al. 2014

JoVE

Self Cite

Cell patterning platforms support broad research goals, such as construction of predefined in vitro neuronal networks and the exploration of certain central aspects of cellular physiology. To easily combine cell patterning with Multi-Electrode Arrays (MEAs) and silicon-based 'lab on a chip' technologies, a microfabrication-compatible protocol is required. We describe a method that utilizes deposition of the polymer parylene-C on SiO 2 wafers. Photolithography enables accurate and reliable patterning of parylene-C at micron-level resolution. Subsequent activation by immersion in fetal bovine serum (or another specific activation solution) results in a substrate in which cultured cells adhere to, or are repulsed by, parylene or SiO 2 regions respectively. This technique has allowed patterning of a broad range of cell types (including primary murine hippocampal cells, HEK 293 cell line, human neuron-like teratocarcinoma cell line, primary murine cerebellar granule cells, and primary human glioma-derived stem-like cells). Interestingly, however, the platform is not universal; reflecting the importance of cell-specific adhesion molecules. This cell patterning process is cost effective, reliable, and importantly can be incorporated into standard microfabrication (chip manufacturing) protocols, paving the way for integration of microelectronic technology.

“…Although we cannot as yet identify the key protein combinations in serum, we have developed the capacity to induce cell-tolerance to SiO 2 and cell repulsion from parylene (a complete reversal of the serum-induced parylene patterning effects reported previously. [6][7][8][9][10][11] This encourages the use of the parylene/SiO 2 platform for effective cell patterning with a standardized and consistent activation solution and protocol, without recourse to serum (which is poorly characterized and heterogeneous between batches).…”

Section: Rationalized Protein Activation Solutionsmentioning

confidence: 99%

Modulating patterned adhesion and repulsion of HEK 293 cells on microengineered parylene‐C/SiO₂ substrates

J Biomedical Materials Res

Cameron

et al. 2012

This article describes high resolution patterning of HEK 293 cells on a construct of micropatterned parylene-C and silicon dioxide. Photolithographic patterning of parylene-C on silicon dioxide is an established and consistent process. Activation of patterns by immersion in serum has previously enabled patterning of murine hippocampal neurons and glia, as well as the human hNT cell line. Adapting this protocol we now illustrate high resolution patterning of the HEK 293 cell line. We explore hypotheses that patterning is mediated by transmembrane integrin interactions with differentially absorbed serum proteins, and also by etching the surface substrate with piranha solution. Using rationalized protein activation solutions in place of serum, we show that cell patterning can be modulated or even inverted. These cell-patterning findings assist our wider goal of engineering and interfacing functional neuronal networks via a silicon semiconductor platform.