June 2022
Volume 63, Issue 7
Open Access
ARVO Annual Meeting Abstract  |   June 2022
A Novel Tissue Bioreactor for Retinal Organoid Microenvironmental Control
Author Affiliations & Notes
  • Emma Drabbe
    University of Miami Health System Bascom Palmer Eye Institute, Miami, Florida, United States
    Biomedical Engineering, University of Miami College of Engineering, Coral Gables, Florida, United States
  • Timothy Arcari
    Biomedical Engineering, University of Miami College of Engineering, Coral Gables, Florida, United States
  • Emmanuel Rippes
    Biomedical Engineering, University of Miami College of Engineering, Coral Gables, Florida, United States
  • Ashutosh Agarwal
    Biomedical Engineering, University of Miami College of Engineering, Coral Gables, Florida, United States
    Sylvester Comprehensive Cancer Center, Miami, Florida, United States
  • Daniel Pelaez
    University of Miami Health System Bascom Palmer Eye Institute, Miami, Florida, United States
    Sylvester Comprehensive Cancer Center, Miami, Florida, United States
  • Footnotes
    Commercial Relationships   Emma Drabbe None; Timothy Arcari None; Emmanuel Rippes None; Ashutosh Agarwal None; Daniel Pelaez None
  • Footnotes
    Support  NIH Grant PAR-19-113
Investigative Ophthalmology & Visual Science June 2022, Vol.63, 1361 – F0292. doi:
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      Emma Drabbe, Timothy Arcari, Emmanuel Rippes, Ashutosh Agarwal, Daniel Pelaez; A Novel Tissue Bioreactor for Retinal Organoid Microenvironmental Control. Invest. Ophthalmol. Vis. Sci. 2022;63(7):1361 – F0292.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : In vitro culture systems generally apply homogeneous stimuli and rely on intercellular signaling to guide growth of tissues. However, to derive complex tissue structures such as the human retina, a gradation of certain stimuli is required. The inner retina resides in a hypoxic environment (2% O2) adjacent to the vitreous cavity. From there, oxygenation levels rapidly increase towards the outer retina (18% O2) at the choroid. Here we developed a novel tissue bioreactor allowing the maturation of inner and outer retinal cell phenotypes within an O2 gradient.

Methods : The bioreactor is assembled from a 75x25x3 mm acrylic slide, a PFA film, a cover glass, and double-sided adhesives, which were adjusted with computer numerical control milling and laser cutting (Fig. 1A). The 60 culture wells of 2 mm in diameter and 0.7 mm high each hold one retinal organoid. A nitrogen (N2) tank provides the bioreactor with 5 mL/min N2 gas and a dual syringe pump creates a 5 µL/min continuous flow of culture medium though the bioreactor (Fig. 1B). Gas diffusion through the PFA membrane and culture medium was predicted using computational modeling software for atmosphere (20.9% O2) and incubator (18.6% O2) conditions. O2 concentration measurements were performed with O2 sensors along the z-axis in 50 µm steps in atmospheric conditions.

Results : The gas diffusion throughout the culture medium resulted in an O2 concentration gradient along the z-axis (Fig 1C). The computational predictions in atmospheric conditions are in accordance with the measurements around the retinal organoid location in the bioreactor (Fig. 1D).

Conclusions : This open-well bioreactor is easily accessible for downstream analysis, establishes a steep O2 gradient and allows high-throughput retinal organoid culture. It will help retinal organoids mature into the complex structure to use them for disease modeling and drug testing.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.

 

(A) The bioreactor is fabricated from three main components. (B) The culture system provides the bioreactor continuously with fresh culture medium and N2 gas flow. (C) The cross section of the bioreactor shows (1) the N2 chamber under the cell culture wells where N2 diffuses upwards (2) to create an O2 concentration gradient (3) which is used for retinal organoid culture. (D) The computational O2 predictions throughout the culture medium compared to O2 sensor measurements in room air condition.

(A) The bioreactor is fabricated from three main components. (B) The culture system provides the bioreactor continuously with fresh culture medium and N2 gas flow. (C) The cross section of the bioreactor shows (1) the N2 chamber under the cell culture wells where N2 diffuses upwards (2) to create an O2 concentration gradient (3) which is used for retinal organoid culture. (D) The computational O2 predictions throughout the culture medium compared to O2 sensor measurements in room air condition.

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