Spatially patterned gene delivery for localized neuron survival and neurite extension

Abstract

Natural tissues can have complex architectures, which arise in part from spatial patterns in gene expression. Regenerative strategies for damaged tissue must recreate these architectures to restore function. In this article, we demonstrate spatially controlled gene delivery from a substrate for directing cellular processes. Non-viral vectors were immobilized to substrates in linear patterns using microfluidic techniques, and cells cultured on the surface had localized gene expression within the cell population. Transfection was achieved in pattern widths as low as 100 mum, with efficiencies dependent on the microchannel treatment and vector concentration. The ability of patterned expression to localize cellular processes was investigated using a neuronal co-culture model. Patterned expression of the diffusible neurotrophic factor nerve growth factor initiated neuron survival and neurite out-growth primarily within the pattern, which decreased significantly in regions directly adjacent to the pattern. Primary neurite density was significantly greater on patterned substrates than on surfaces without patterns. This approach demonstrates the basic technology to create patterns of gene expression that can direct tissue formation and could be employed in regenerative strategies to recreate the complex cellular architectures observed in tissues.

Figure 1. Patterned lipoplex deposition

Immobilized lipoplexes (red) deposited in Pluronic-treated polydimethylsiloxane microchannels: (a) 1,000 µm, (b) 500 µm, (c) 250 µm, and (d) 100 µm. (e) Lipoplexes deposited using untreated microchannels (1,000 µm). Bars = 100 µm.

Figure 2. Patterned lipoplex binding efficiency

Quantification of lipoplex binding efficiency (total bound divided by total incubated in the channel) using polydimethylsiloxane microchannels, while varying (a) channel treatments (no treatment, O₂ plasma, Pluronic) and (b) channel widths (100, 250, 500, 1,000 µm). Values are reported as mean ± SEM (* P < 0.05).

Figure 3. HEK293T cells expressing the reporter gene enhanced green fluorescent protein (EGFP) in a pattern

EGFP expression (green) within cells cultured on substrates with patterned DNA complex deposition using Pluronic-treated microchannels: (a) 1,000 µm, (b) 500 µm, (c) 250 µm, and (d) 100 µm. Higher-magnification images present cell nuclei (blue) and transfected cells (green) overlaid: (e) 1,000 µm, (f) 500 µm, (g) 250 µm, and (h) 100 µm. White lines indicate pattern boundaries. Bars = 500 µm (a–d) and 100 µm (e–h).

Figure 4. Transfection efficiency was dependent on microchannel width and vector concentration

Transfection efficiency (percentage of transfected cells) in the pattern as a function of vector concentration and channel width. Lipoplex deposition was performed within Pluronic-treated microchannels. Values are reported as mean ± SEM.

Figure 5. Neurons cultured with cells expressing nerve growth factor (NGF) in patterns

Neurite extension (red) was observed at the region of transfected cells (green): (a, b) 250 µm width; (c, d) 100 µm width. pNGF to pEGFP ratio of 50:50. White lines indicate pattern boundaries. Cell nuclei (blue) are visible in (b, d). Bars = 100 µm.

Figure 6. Neuron survival and neurite outgrowth primarily observed within patterns of nerve growth factor (NGF) expression

(a) Neuron survival, normalized to surface area; (b) total neurite density quantified within and outside the region of patterned expression. Values are reported as mean ± SEM (** P < 0.01, *** P < 0.001).

Figure 7. Primary neurite density on patterned nerve growth factor (NGF) expression and non-patterned controls

The primary neurite density was greater on patterns of NGF expression than for cultures with NGF expression and no pattern or cultures with NGF added to the media. Values are reported as mean ± SEM (* P < 0.05, ** P < 0.01).

Figure 8. Predicted nerve growth factor (NGF) concentration gradients

(a) Mathematical model predictions of the NGF concentration profile for channels with widths of 250 and 100 µm. Note that the kinetic constant describing the rate of protein production (p) was estimated to be 1 ng/cm³/min. The production rate was varied and the concentrations modeled for (b) 250 and (c) 100 µm wide patterns. Gradients were modeled for 24 hours, and the reported concentrations are those at the material surface (z = 0).