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    Tissue Engineering

    (H-319) A Simple 3D-Printed Bioreactor to Improve Oxygenation During Retinal Organoid Cultures

    Thursday, October 12, 2023
    2:00 PM – 3:00 PM PDT
    Location: Exhibit Hall - Row H - Poster # 319

      Presenting Author(s)

    • KS

      Kyle Schwab, MA

      Graduate Student
      Temple University
      Riverdale, Maryland, United States

      • Primary Investigator(s)

    • TL

      Tiansen Li

      Primary Investigator
      Nation Eye Institute, United States

      • Co-Author(s)

    • PH

      Phil Hwang

      Biologist
      Nation Eye Institute, United States

    Introduction::

    Retinal organoids, derived from pluripotent stem cells, closely replicate in vivo development and retinal morphology, providing a platform to study and develop treatments for hereditary degenerative retinal diseases, the leading causes of vision loss.


    However, researchers have encountered difficulty cultivating retinal organoids that fully recapitulate in vivo retinogenesis. Most current hPSC differentiation methods rely on static culture conditions, which are subject to variable oxygen tensions. During static differentiation of retinal organoids, the oxygen consumption rate may exceed the rate at which atmospheric oxygen can diffuse down through media, resulting in hypoxic conditions (< 1% O2). Excessive hypoxia is known to disrupt cell fate specification and leads to apoptosis, degrading complex neural structures.



    Materials and Methods:: To address this limitation, we have developed a novel, 3D-printed stirred bioreactor, which can be easily implemented in most labs, requiring only a 100mm Petri dish, magnetic stir plate, and small stir bar. This stirred bioreactor system provides a widely applicable and straightforward platform to increase and maintain dissolved oxygen concentrations leading to the generation of retinal organoids with greater size, yield, and improved differentiation. Stirred bioreactors were design using Fusion 360 computer aided design software and fabricated using a FormLabs Form 3B+ stereolithographic 3D-printer with BioMed Clear resin. We evaluated our system using both adherent and embryoid-body culture protocols with four human pluripotent stem cells lines (hPSCs), NRL-L75pfs, NEI-901a, 904b, and PEN-8E. For adherent cultures, once hPSCs reached 20-30% confluency in a 100mm Petri dish, differentiation was begun using neural-induction media. At day 6, stirred bioreactors fitted with small stir bars were inserted and dishes were returned to incubation on a magnetic stir plate set to 300 RPM. Culture proceeded until day 21 when sufficient maturation of optic vesicles was observed. Cultures were then scraped and transferred to low-adhesion dishes for continued free-floating culture. Measurements (n=3) of the partial pressure of oxygen in cell culture media were taken using contactless optical sensor spots placed on the bottom of the dish. On day 24, the number of retinal organoids from both static and stirred bioreactor conditions (N=3) were counted and measurements (n=20) of cross-sectional areas were calculated using ImageJ.

    Results, Conclusions, and Discussions::

    Figure 1. depicts 20-hour oxygen consumption profiles averaged over measurements taken on days 11, 15, and 18 of adherent cultures. These data suggests that under static conditions, the oxygen consumption rate of developing tissues reduces the amount of available oxygen to < 1% O2 within ~6 hours, inducing hypoxic conditions. In contrast, the stirred bioreactor (SBR) condition shows a more gradual decrease in oxygen concentration, but never dropping below ~4%. This suggests that our stirred bioreactor system can prevent the occurrence of hypoxic conditions between media changes.


    After day 21, we observed the formation of retinal organoids in both static and stirred bioreactor conditions from all four hPSC lines. Figure 2 shows a quantitative analysis of the number of ROs generated during adherent and embryoid body protocols. These results show a significant increase in number when comparing the static and stirred bioreactor conditions in 901, 8E, NRL-L75Pfs, and 901 EB cultures. Figure 3 shows a quantitative analysis of the cross-sectional area (µm2) of retinal organoids. These results show a significant increase in cross-sectional area retinal organoids from all cell lines and protocols when comparing the static and stirred bioreactor (SBR) conditions.


    Our research suggests that the stirred bioreactor platform maintains high oxygen concentrations ( >4% O2) during differentiation leading to greater size and yield of ROs compared to traditional static culture methods. The findings of this study support the claim that 3D-printed stirred bioreactors can provide a widely accessible tool to improve oxygen availability, and thus, increase the yield and size of retinal organoids with the ultimate goal of creating a more accurate physiological model.



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