A simple and inexpensive laser dissection of fasciculated axons from motor nerve organoids

Yasuhiro Ikegami; Tomoya Duenki; Ikuma Arakaki; Ryo Sakai; Tatsuya Osaki; Satoshi Ashihara; Tsuyoshi Furushima; Yoshiho Ikeuchi

doi:10.3389/fbioe.2024.1259138

A simple and inexpensive laser dissection of fasciculated axons from motor nerve organoids

Front Bioeng Biotechnol. 2024 Jan 29:12:1259138. doi: 10.3389/fbioe.2024.1259138. eCollection 2024.

Authors

Yasuhiro Ikegami^{1

2}, Tomoya Duenki^{1

2

3

4}, Ikuma Arakaki^{1

3}, Ryo Sakai^{1

3}, Tatsuya Osaki^{1

2}, Satoshi Ashihara¹, Tsuyoshi Furushima¹, Yoshiho Ikeuchi^{1

2

3

4}

Affiliations

¹ Institute of Industrial Science, The University of Tokyo, Tokyo, Japan.
² Institute for AI and Beyond, The University of Tokyo, Tokyo, Japan.
³ Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo, Tokyo, Japan.
⁴ Laboratory for Integrated Micro Mechatronic Systems, National Center for Scientific Research-Institute of Industrial Science (LIMMS/CNRS-IIS), The University of Tokyo, Tokyo, Japan.

Abstract

Motor nerve organoids could be generated by culturing a spheroid of motor neurons differentiated from human induced pluripotent stem (iPS) cells within a polydimethylsiloxane (PDMS) chip which guides direction and fasciculation of axons extended from the spheroid. To isolate axon bundles from motor nerve organoids, we developed a rapid laser dissection method based on localized photothermal combustion. By illuminating a blue laser on a black mark on the culture device using a dry-erase marker, we induced highly localized heating near the axon bundles. Moving the laser enabled spatial control over the local heating and severing of axon bundles. This laser dissection requires a black mark, as other colors did not produce the same localized heating effect. A CO₂ laser destroyed the tissue and the device and could not be used. With this simple, economical laser dissection technique, we could rapidly collect abundant pure axon samples from motor nerve organoids for biochemical analysis. Extracted axonal proteins and RNA were indistinguishable from manual dissection. This method facilitates efficient axon isolation for further analyses.

Keywords: PDMS; axons; laser; organoids; tissue engineering.

Grants and funding

The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This work was supported in part by a Grant-in-Aid for Challenging Research (Pioneering) from the JSPS (20K20643); a Grant-in-Aid for Transformative Research Areas (B) (20H05786); AMED-CREST; JSPS Core-to-Core Program (JPJSCCA 20190006); AMED (JP20gm1410001); The University of Tokyo GAP fund program; and the Institute for AI and Beyond (YoI). The study was also supported by Grant-in-Aid for Early-Career Scientists from JSPS (22K18167) (YaI), JST SPRING (JPMJSP2108) and the ANRI fellowship (TD).