Name: Video: Setting Up the Pump-Free Multi-Channel Fluidic System and Loading the Tissue for Culturing
Uploaded: 2023-07-14T12:40:00
Duration: 4 min 36 s
Description: 314 Views. Setting Up the Pump-Free Multi-Channel Fluidic System and Loading the Tissue for Culturing

setting up the pump-free multi-channel fluidic system and loading the tissue for culturing

Pumpless Multi-Channel Flow Culture to Maintain and Assess Tissues and Cells

Perifusion systems have a long history in the life sciences. In particular, for the study of secretory function by islets, they have been used to characterize the kinetics of insulin secretion in response to secretagogues1. In addition to collecting outflow fractions for subsequent assay of hormones and metabolites, real-time sensors have been incorporated, predominantly for the detection of oxygen consumption2,3,4. Widespread efforts to better understand mechanisms mediating diseases of the eye have been limited by a lack of physiologically relevant methods to assess metabolic regulation and dysregulation of the various isolated components of the eye, including the retina, retinal pigment epithelial (RPE)-choroid-sclera and cultured RPE cells. Static systems designed for cultured cells have been adapted for tissue5, but tissue requires flow for adequate oxygenation. Flow systems have been successful at accurately and reproducibly measuring real-time responses in oxygen consumption rate (OCR) by the retina and RPE-choroid-sclera, and the tissues stay metabolically stable for more than 8 h allowing highly informative protocols involving multiple test compounds4,6,7,8,9. Nonetheless, the operation of fluidics systems has historically required a custom-made apparatus and trained technical staff in non-standardized methodologies. Such systems have not been adopted as the standard methodology in most laboratories. The BaroFuse is a newly developed fluidics system that does not rely on pumps, but rather on gas pressure to drive flow through multiple channels and tissue chambers (Figure 1). Each channel is continuously monitored for OCR, and the outflow is collected with a plate-based fraction collector for subsequent assay of contents. Importantly, the tissue perifusion chambers for the instrument are designed to accommodate tissues of various geometries and sizes.
The heart of the instrument is the fluidics system, where flow is driven from a sealed, pressurized reservoir through small inner diameter (ID) tubing (contributing the most significant flow resistance in the fluid circuit) up into the glass tissue chambers that house the tissue. Pressure to the media reservoir module (MRM) is supplied by low-pressure and high-pressure regulators connected to a gas cylinder containing a mixture of gases (typically 21% O2, 5% CO2, balance N2), and the reservoir is sealed from the top by the perifusion chamber module (PCM) that holds the tissue chamber assemblies (TCAs). Flow rate is controlled by the length and ID of the resistance tubes and the pressure setting of a low-pressure regulator. Outflow tubes connected to the top of tissue chambers deliver fluid to either a waste receptacle (that is continuously weighed for automatic determination of flow rate) or into wells of a 96-well plate controlled by the fraction collector. The O2 detection system measures the lifetime of an O2-sensitive dye painted on the inside of each of the glass tissue chambers downstream of the tissue. This information is then used to continuously calculate OCR. The entire fluidics system resides in a temperature-controlled enclosure and the gas tank, fraction collector and computer are the major components of the instrument (Figure 2A). Finally, software that runs the instrument serves to control its operation (including the preparation and timing of injected test&#160;compounds, flow measurement system, and fraction collector timing), as well as processing and graphing the OCR data and other supplemental measurements.
In this paper we describe the protocols for using the fluidics system to perifuse and assess OCR and lactate production rate (LPR) for various isolated components of the eye. LPR is a parameter reflecting glycolytic rate that is highly complementary to OCR, where the pair accounts for the two major branches of energy generation from carbohydrates in the cell10. As preparation of the tissue and loading it into the tissue chambers is best learned by watching the procedure, the video will help illustrate several of the critical steps that are performed during set up and operation that are not easily conveyed by text alone.
The description of the protocol is divided up into 8 sections that correspond to different phases of the experiment (Figure 2B): 1. pre-experimental preparation; 2. preparation/equilibration of the perifusate; 3. instrument set up; 4. tissue equilibration; 5. experimental protocol; 6. instrument break down; 7. data processing; and 8. assays of outflow fractions.

Author Spotlight: Advancing Eye Physiology Research via a Multi-Channel Flow Culture for Optimal Tissue Maintenance and Real-Time Assessment 

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

Watch this Scientific Journal Video about Setting Up the Pump-Free Multi-Channel Fluidic System and Loading the Tissue for Culturing at JoVE.com

Setting Up the Pump-Free Multi-Channel Fluidic System and Loading the Tissue for Culturing  

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Research

JoVE Journal

Bioengineering

314 Views. Setting Up the Pump-Free Multi-Channel Fluidic System and Loading the Tissue for Culturing

Video: Setting Up the Pump-Free Multi-Channel Fluidic System and Loading the Tissue for Culturing  

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.

Real-time analysis of live tissue yields important functional and mechanistic data. This paper describes the protocols and critical variables to ensure accurate and reproducible generation of data by a novel and pump-free multi-channel fluidics system that maintains and assesses a wide range of tissue and cell models.