Yu Laura M Y, Wosnick Jordan H, Shoichet Molly S
Department of Chemical Engineering & Applied Chemistry, University of Toronto, 200 College Street, Toronto, ON, Canada M5S 3E5.
J Neurosci Methods. 2008 Jun 30;171(2):253-63. doi: 10.1016/j.jneumeth.2008.03.023. Epub 2008 Apr 12.
Understanding the fundamentals of cell behaviour is imperative for designing and improving engineering strategies for regenerative medicine. By combining the precision of confocal microscopy with photochemistry, nerve growth factor (NGF) was chemically immobilized on chitosan films either in distinct areas or as concentration gradients. Using rhodamine as a proxy for NGF, a series of immobilized concentration gradients were created, using the number of rastering scans within a defined area and the distance between each area as a way to control the resulting gradient. The same photochemistry was applied to create NGF patterns on chitosan films which were visualized by immunostaining, and the immobilized NGF remained bioactive as demonstrated with a neuron survival assay. Neuron survival was 73.2+/-1.3% after 3 days of culture on chitosan films with 30 ng/cm(2) of homogenously immobilized NGF, which was comparable to 74.8+/-3.4% neuron survival on chitosan with 50 ng/ml of soluble NGF present. Interestingly, when neurons were plated on a chitosan film that had distinct immobilized NGF-patterned areas surrounded by unmodified chitosan, the neurons remained predominantly as single cells in the NGF-patterned regions, but formed aggregates outside of these patterns on the plain chitosan film. Thus, the immobilized NGF pattern influenced neuron behaviour and can be used to further probe mechanisms of other neuron behaviour such as axon guidance. Importantly, the versatility of the confocal laser patterning technique reported here can be extended to other factors to elucidate fundamental cell functions, and hence design strategies in regenerative medicine.
了解细胞行为的基本原理对于设计和改进再生医学的工程策略至关重要。通过将共聚焦显微镜的精确性与光化学相结合，神经生长因子（NGF）以不同区域或浓度梯度的形式化学固定在壳聚糖膜上。使用罗丹明作为NGF的替代物，通过在定义区域内的光栅扫描次数以及每个区域之间的距离来控制所得梯度，从而创建了一系列固定的浓度梯度。相同的光化学方法用于在壳聚糖膜上创建NGF图案，通过免疫染色进行可视化，并且通过神经元存活试验证明固定的NGF仍具有生物活性。在含有30 ng/cm²均匀固定NGF的壳聚糖膜上培养3天后，神经元存活率为73.2±1.3%，这与存在50 ng/ml可溶性NGF的壳聚糖上74.8±3.4%的神经元存活率相当。有趣的是，当神经元接种在具有由未修饰壳聚糖包围的不同固定NGF图案区域的壳聚糖膜上时，神经元在NGF图案区域中主要保持为单细胞，但在这些图案之外的普通壳聚糖膜上形成聚集体。因此，固定的NGF图案影响神经元行为，并可用于进一步探究其他神经元行为的机制，如轴突导向。重要的是，本文报道的共聚焦激光图案化技术的多功能性可以扩展到其他因子，以阐明基本的细胞功能，从而设计再生医学策略。
