Polymer film-based microwell array platform for long-term culture and research of human bronchial organoids. (April 2023)

• Record Type: Journal Article
• Authors: Baptista, Danielle; Tahmasebi Birgani, Zeinab; Widowski, Helene; Passanha, Fiona; Stylianidis, Vasili; Knoops, Kèvin; Gubbins, Eva; Iriondo, Cinta; Skarp, Kari-Pekka; Rottier, Robbert J.; Wolfs, Tim G.; van Blitterswijk, Clemens; LaPointe, Vanessa; Habibović, Pamela; Reynaert, Niki L.; Giselbrecht, Stefan; Truckenmüller, Roman
• Is Part Of: Materials today bio. Volume 19(2023)
• Publication Date: 2023-04
• Subjects: Bronchi/bronchioli -- Organoids -- Microwells -- Microthermoforming -- Organoid fusion -- (Organoid) microinjection; 2D Two-dimensional/two dimensions -- 3D Three-dimensional/three dimensions -- AM Acetoxymethyl -- AO Airway organoid -- ATP Adenosine triphosphate -- BME Basement membrane extract -- DAPI 4′, 6-diamidino-2-phenylindole -- DMEM Dulbecco's Modified Eagle Medium -- HMDS Hexamethyldisilazane -- PBS Phosphate-buffered saline -- PC Polycarbonate -- RH Relative humidity -- RT Room temperature -- SEM Scanning electron microscopy -- TEM Transmission electron microscopy; Materials science -- Periodicals; Biomedical engineering -- Periodicals; Biomedical materials -- Periodicals; 620.1
• DOI: 10.1016/j.mtbio.2023.100603
• Languages: English
• ISSNs: 2590-0064

Abstract

Abstract: The culture of lung organoids relies on drops of basement membrane matrices. This comes with limitations, for example, concerning the microscopic monitoring and imaging of the organoids in the drops. Also, the culture technique is not easily compatible with micromanipulations of the organoids. In this study, we investigated the feasibility of the culture of human bronchial organoids in defined x-, y- and z-positions in a polymer film-based microwell array platform. The circular microwells have thin round/U-bottoms. For this, single cells are first precultured in drops of basement membrane extract (BME). After they form cell clusters or premature organoids, the preformed structures are then transferred into the microwells in a solution of 50% BME in medium. There, the structures can be cultured toward differentiated and mature organoids for several weeks. The organoids were characterized by bright-field microscopy for size growth and luminal fusion over time, by scanning electron microscopy for overall morphology, by transmission electron microscopy for the existence of microvilli and cilia, by video microscopy for beating cilia and swirling fluid, by live-cell imaging, by fluorescence microscopy for the expression of cell-specific markers and for proliferating and apoptotic cells, and by ATP measurement for extended cell viability. Finally, we demonstrated the eased micromanipulation of the organoids in the microwells by the example of their microinjection. Graphical abstract: Polymer film-based microwell array platform for long-term culture and research of human bronchial organoids. Image 1