Chemie | Biochemie | Medizin
Nathalie Weibel, 2000 | Oberbuchsiten, SO
The challenges of studying the function of the brain, one of the most complex organs in the human body, have increased the need for a new model system. The most promising technology uses human brain organoids. These 3D structures, derived from pluripotent stem cells, mimic an embryonic human brain and open up an impressive amount of possibilities for future research. This paper focuses on two different methods to prepare the organoids for immunofluorescent staining and imaging. The aim was to test a new method, so-called clearing, and compare it to the commonly used method based on sectioning. The conventional method requires thin sections of the organoids, which are stained afterwards. The new method clears the whole organoids, which makes them transparent. The experiments show that both methods allow the identification of different cell types, whereas the clearing rendered a better view of the whole organisation and allowed imaging of organoids in 3D. This study shows that clearing is a new method to assess the quality of organoids, which will help to improve this new model system for further applications, such as drug testing, personal medicine and the reduction of the amount of animal experiments in the future.
Fragestellung
Human brain organoids are self-organised 3D structures, which form different brain regions and contain different cell populations, such as stem cells and newly born neurons migrating away from the ventricular zone. It is crucial to develop new methods to assess the quality and organisation of organoids for further studies. Conventionally, organoids are sectioned to assess their inner organisation. In this study, a new method with promising applications for various kinds of tissues was used, making the organoids transparent. I compared these two methods and discussed which one is more efficient and attractive to use.
Methodik
The two different methods serve to prepare the organoids for an immunofluorescent staining. The commonly used method is a sectioning with a cryostat. After embedding and freezing the organoids into a solid block, 30 micrometer thin slices were cut and collected on a microscopy glass slide. The second, new method called clearing makes the whole organoids transparent, allowing the analysis of their organisation in three dimensions. After conducting an immunofluorescent staining against markers for several cell types, they were analysed with a confocal laser scanning microscope and the images were compared.
Ergebnisse
Analysing the sectioned organoid was unproblematic and the expected histological structures were present. The 28 to 30-day-old organoids developed several ventricles with prominent stem cell populations. These already started to differentiate into basal neural progenitors and a few newly born neurons were observed. Some foldings or cracks of the slices were visible and a black area in the middle of the organoids was noticed. The clearing of organoids worked surprisingly well the first time, and the same histological structures were observed as in the sections before. Additionally, thanks to the clearing, 3D-animated movies were made, giving a detailed insight into the whole organisation of an organoid. The dimensions of the different structures were easily detectible, and the surface of the organoids was also visible.
Diskussion
The self-organisation and differentiation of stem cells and the migration of newly born neurons away from the ventricular zone were visible using both methods. The observed black area in the middle of the organoids is a dead core, presumably a result of the lack of blood supply. The quality of the organoid slices was constant, with a few foldings or cracks happening while working with the cryostat. The clearing of organoids yielded very similar results. Additionally, it allowed more detailed observations of the dead core, the surface and the organisation and dimensions of the organoids. Both methods serve well for the analysis of human brain organoids. If the focus lies on checking the quality and growth, the sectioning is easier and faster, whereas the clearing is very time-consuming. However, it is better suited for a detailed analysis of the whole organoid and for 3D-imaging.
Schlussfolgerungen
The two methods compared in this paper help to assess the quality of human brain organoids. Using the clearing method renders a better overview of the whole structure and will therefore facilitate future attempts to improve this new model system and to use it to test various cellular properties, both in health and disease.
Würdigung durch die Expertin
Dr. Annina Denoth
Nathalie Weibel enthusiastically tackled the complex question of how the organization of human brain organoids can be studied best. She tried a completely novel method that clears organoids and compared it to the conventionally used method of sectioning. She carefully performed experiments, addressed experimental difficulties, and invested a lot of time and effort. She obtained fascinating results and thereby greatly helped to improve the assessment of organoids.
Prädikat:
hervorragend
Sonderpreis Gebauer Stiftung – Regeneron International Science and Engineering Fair (ISEF)
Kantonsschule Olten
Lehrer: Alexander Rauch
