# Hippocampal Neurons' Alignment on Quartz Grooves and Parylene Cues on Quartz Substrate
## 4 Conclusions  
The following conclusions can be concluded from the obtained results:  
* Quartz grooves serve for neurons' alignment as topographical structures and parylene cues serve for neurons' alignment as structures with higher chemical affinity.
* Neurons patterned physically by isotropy grooves have a tendency to adhere to the bottom and stick to the grooves' walls.
* Neurons have a preferential tendency to grow in the longitudinal direction of the grooves, even if the neurons are not stuck to the grooves' walls.
* The most effective neuron alignment is for deep grooves, while the effectivity of grooves' widths varies according to the grooves' depth. For shallow grooves, a smaller width is better, while for deep grooves, a larger width is better.
* Neurons can be patterned chemically on parylene cues on quartz substrate due to their higher affinity to parylene treated by oxygen plasma.
* Neurons on quartz grooves and parylene cues on quartz substrate are aligned longitudinally according to groove/cue direction; however, neurons also present a small tendency of alignment in the perpendicular direction.
* Neurons' tendency to follow an individual parylene cue is much higher than neurons' tendency to follow an individual groove.
* The suggested best method for neurons' alignment would be to pattern neurons on low and thin parylene cues on quartz substrate, where there is a higher distance between parylene cues. Neurons presented a significantly higher affinity to parylene than quartz. The non-alignment is mainly caused by neurons crossing from one parylene cue to another, suggesting that the distance between parylene cues is more important than the parylene cues' width.  
**Supplementary Materials:** The following are available online at [[https://www.mdpi.com/2076-341/7/11/1/275/s1](https://www.mdpi.com/2076-341/7/11/1/275/s1)]([https://www.mdpi.com/2076-341/7/11/1/275/s1](https://www.mdpi.com/2076-341/7/11/1/275/s1)), Figure S1: Mechanism of Line Segments Direction Analysis (LSDA).  
**Author Contributions:** Conceptualization, [PERSON]; methodology, [PERSON]; software, J.S. and V.C.; validation, V.C., J.H., Y.Y., and T.-L.R.; formal analysis, [PERSON]; investigation, [PERSON]; resources, Y.Y. and T.-L.R.; data curation, J.S. and V.C.; writing--original draft preparation, J.S.; writing--review and editing, V.C., J.H., Y.Y., and T.-L.R.; visualization, [PERSON]; supervision, J.H., Y.Y., and T.-L.R.; project administration, J.H., Y.Y., and T.-L.R.; funding acquisition, J.H., Y.Y., and T.-L.R. All authors have read and agreed to the published version of the manuscript.  
Supported by the Ministry of Education, Youth and Sports project \"FIT\" (Pharmacology, Immunotherapy, nanoToxicology) CZ.02.1.01/0.0/0.15 003/0000495.  
**Institutional Review Board Statement:** Cell isolation underwent on redundant cadaver tissue from another rat experiments in accordance with the legal standards and animal welfare in European Union, especially Annex III of Directive 2010/63. Primary animal experiments were approved via Law on Animal Protection and the Ethical Committee for Laboratory Animal Treatment of the State Veterinary and Food Administration of the Slovak Republic (certification number 2791/18-221/3).  
**Informed Consent Statement:** Not applicable for studies not involving humans.  
**Data Availability Statement:** The data presented in this study are available on request from the corresponding author.  
This research has been financially supported by National Natural Science Foundation of China under Grant 51861145202, Independent Research Program of Tsinghua University under Grant 20193080047, and the Ministry of Education, Youth and Sports of the Czech Republic under the project CEITEC 2020 (LQ1601). We acknowledge CzechNanoLab Research Infrastructure supported by MEYS CR (LM2018110).  
The authors declare no conflict of interest.