Skip to main content
2024 BMES Annual Meeting Main banner
Icon Legend
This session is not in your schedule.
This session is in your schedule. Click again to remove it.

Neural Engineering

Neural Engineering - Poster Session E

Poster Z8 - Extended culture of sliced brain organoids using 3D-printed live tissue precision slicer

Saturday, October 26, 2024
10:00 AM - 11:00 AM EST
Location: Exhibit Hall E, F & G

Introduction:  Slicing tissue samples into thin pieces is commonly used in histology analysis and more recently for organotypic culture when tissue samples are sliced alive. Brain organoids derived from human pluripotent stem cells often have necrotic cores due to diffusion limits of oxygen and nutrient. Recent studies demonstrated that slicing brain organoids into slices significantly reduced necrotic cores and improved cortical layering. However, currently available devices for slicing tissue samples are either designed for fixed tissue samples at low cryogenic temperatures (e.g., Cryostats), or bulky and expensive (e.g., vibratome), preventing them from routine lab usage.

Here we report a cost-effective device designed to section brain organoids for subsequent culture. This device consists of components crafted from 3D-printed Nylon-12- a material suitable for autoclaving to ensure sterility. Its small footprint enhances portability, allowing for convenient placement within a biosafety cabinet for an added layer of sterility assurance. We demonstrated that the device both precisely and accurately makes slices. We further validate its suitability for long-term culture by extended tissue culture following slicing. Our results indicate that brain organoid slices are viable and show improved proliferation rate compared with unsliced organoids. This open-source hardware can be freely accessed and used broadly for other tissue types.

Materials and

Methods:  Device design: The slicer is structured around three key components: the base, the arm, and the box [Figure 1]. The machine's base functions as a motion stage, allowing for adjustments to achieve various cut sizes. Device fabrication: The base of the machine consists of a fixture plate attached to a motion plate. Once assembled the machine’s 3D printed Nylon-12 arm is interlock with screws and bearings to the base. The machine’s box is composed of acrylic and fixed upon the motion plate, and this is where the sample will be mounted to slice while embedded in agarose. Brain organoid culture: ESC line H1 were cultured on feeder-free condition using mTESR Plus medium on Matrigel. To form serum-free floating embryoid bodies (EB) cells were dissociated into single cells using Accutase and reaggregated in low adhesion u-bottom 96 well plates. EB’s are formed in mTESR plus and Y-27632 Rock inhibitor and after 24 hours are cultured in E6 medium for 6 days with SB431542 and dorsomorphin, promoting neural stem cell differentiation. EB’s are kept in 96 well plates for 14 days and then transferred to 10cm ultra low attachment dishes and placed on an orbital shaker. Organoids were matured following the protocol established in Sloan et al paper which uses the addition of Neurobasal and neurotrophic factors to promote mature cell populations. Proliferation assay: Slices were fixed and stained with Ki67 antibody to visualize cell proliferation, alongside DAPI staining. Ki67 and DAPI signals were quantified using Nikon Elements -GA3 software

Results, Conclusions, and Discussions: Slicing Quality: We tested the precision of the slice thickness by comparing the actual slice thickness with targeted thickness. Agarose gel block was sectioned at three distinct target thicknesses (0.250 mm, 0.350 mm, and 0.5 mm, respectively), which were typically used in slicing organoids considering diffusion limits. The averaged thickness of actual slices were 0.25, 0.33, and 0.50 mm respectively. No statistical significance between the targeted value and the slices sectioned were found for all three thicknesses. Organoid viability: To assess the effects after organoids slicing, we quantified the ratio of proliferating cells to total cells. Slicing was conducted on day 153 of organoid maturation, followed by a five-day post-slicing culture period. Staining revealed a significantly higher percentage of Ki67+ cells in sliced organoids compared to the unsliced controls [Figure 2]. This suggests that sliced organoids are actively proliferating, indicating that slicing does not compromise their viability, and improved nutrient and oxygen diffusion facilitate the development of organoids. In conclusion, we successfully designed and fabricated a tissue precision slicer for reproducibly producing brain organoid slices with minimal z-deflection. The sliced brain organoids showed good viability and significantly improved proliferation. We expect to disseminate this tool to the research community for improving routine organotypic culture workflow.

Acknowledgements (Optional):