JoVE Logo

Sign In

Abstract

Bioengineering

Maintaining and Assessing Various Tissue and Cell Types of the Eye Using a Novel Pumpless Fluidics System

Published: July 14th, 2023

DOI:

10.3791/65399

1EnTox Sciences, Inc, 2UW Medicine Diabetes Institute, University of Washington, 3Department of Ophthalmology, University of Washington, 4Department of Biochemistry, University of Washington

Abstract

Many in vitro models used to investigate tissue function and cell biology require a flow of media to provide adequate oxygenation and optimal cell conditions required for the maintenance of function and viability. Toward this end, we have developed a multi-channel flow culture system to maintain tissue and cells in culture and continuously assess function and viability by either in-line sensors and/or collection of outflow fractions. The system combines 8-channel, continuous optical sensing of oxygen consumption rate with a built-in fraction collector to simultaneously measure production rates of metabolites and hormone secretion. Although it is able to maintain and assess a wide range of tissue and cell models, including islets, muscle, and hypothalamus, here we describe its operating principles and the experimental preparations/protocols that we have used to investigate bioenergetic regulation of isolated mouse retina, mouse retinal pigment epithelium (RPE)-choroid-sclera, and cultured human RPE cells. Innovations in the design of the system, such as pumpless fluid flow, have produced a greatly simplified operation of a multi-channel flow system. Videos and images are shown that illustrate how to assemble, prepare the instrument for an experiment, and load the different tissue/cell models into the perifusion chambers. In addition, guidelines for selecting conditions for protocol- and tissue-specific experiments are delineated and discussed, including setting the correct flow rate to tissue ratio to obtain consistent and stable culture conditions and accurate determinations of consumption and production rates. The combination of optimal tissue maintenance and real-time assessment of multiple parameters yields highly informative data sets that will have great utility for research in the physiology of the eye and drug discovery for the treatment of impaired vision.

Explore More Videos

Keywords Tissue Culture

This article has been published

Video Coming Soon

JoVE Logo

Privacy

Terms of Use

Policies

Research

Education

ABOUT JoVE

Copyright © 2024 MyJoVE Corporation. All rights reserved