Perfusion
System Design for Cell Studies Under Controlled Environment
Perfusion
System Design for Cell Studies Under Controlled Environment
Sponsor:
Ahmed Heikal, PhD
Associate Professor of Bioengineering
Functional Imaging and Biophysics of Biological Systems Laboratory
The Pennsylvania State University, University Park, PA
Design Team:
Daniel Beachler
Ji-Sung Kim
Trevor Ritz
Nathan Weidenhamer
Executive Summary | Design Overview | Design Criteria | Design Evaluation | Deliverables | Budget
For quantitative studies on cultured cells, it is critical to regulate the delivery of nutrients, growth factors. Such controlled regulation can be carried out using a perfusion system. Complete perfusion systems generally cost several thousand dollars, with limited flexibility. Our goal is to deliver a simplified, yet effective system that can be designed for much less.
A perfusion system was designed to allow real-time imaging of living cells and their response to chemical stimulation while being incubated in conventional Petri dishes. The perfusion chamber was constructed of anodized aluminum, and will use 1/16" inner diameter polyurethane tubing to deliver media, buffer, or chemical stimulus. Flow is driven by a Instech Laboratories P720/66 High Flow Peristaltic Pump (Plymouth Meeting, PA). The perfusion chamber includes four main ports (two to deliver and extract fluids, one for temperature measurement via thermocouple, and one for perfusion of CO2 or other gases). Additionally, the perfusion chamber contains an internal flow loop that will serve to circulate heated water, heating the chamber and maintaining a desired temperature. The system is specifically designed to be compatible with an Olympus IX81 inverted microscope (Melville, NY) and cells that are cultured within a 1.45 in. (3.68 cm) Petri dish. Additional testing was done to ensure that the perfusion chamber is watertight and can appropriately control temperature and flow rate. The entire system costs $187.
The perfusion chamber was constructed out of aluminum and consisted of two parts. The top part consists of a 3/4" inch diameter viewing window which can be covered with standard 22/32" round coverslips. It also contains four 11/64" inlet/outlet ports (two to deliver and extract fluids, one for temperature measurement via thermocouple, and one for perfusion of CO2 or other gases) which are accessible via a 1.45 inch diameter extruded cut. The bottom part consists of a water channel with two 11/64" inlet/outlet ports for water circulation and a Petri dish holder with a 3/4" viewing window. The bottom part is covered by a thin covering plate and sealed with two-part epoxy. The two parts are fastened together with four #8-32 socket head cap screws (see Figures 1,2).
Figure 1: Solidworks™ Model of Perfusion Chamber
Figure 2: Perfusion System Assembly
Flow will be supplied by an Instech P720/66 High Flow Peristaltic Pump (see Figure 3). This pump is capable of providing flow rates of 33 - 550 ml/hr (0.55 - 9.167 ml/min). Flow is driven using a rotating hexagonal roller that "pinches" the tubing, thus pushing the fluid forward. The pump was chosen based on its ability to supply a large range of flow rates.
Figure 3: Instech P720/66 High Flow Peristaltic Pump
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Compatible with 1.45 inch (3.68 cm) Petri dish and Olympus IX81™ inverted microscope |
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Two fastened parts constructed out of aluminum; watertight |
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Temperature controlled to 37±1°C |
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Four inlet/outlet ports (two to deliver and extract fluids, one for temperature measurement via thermocouple, and one for perfusion of CO2 or other gases) |
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Capable of delivering buffer and/or drugs at a flow rate of 2.5 ml/min |
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Dimensions: 4 inches X 5 inches X 1 inch |
The design was evaluated based on its compatibility with an Olympus IX81™ inverted microscope. HTB126 Breast Cancer Cells were cultured in a 1.45 inch (3.68 cm) Petri dish. The Petri dish was placed within the perfusion chamber and the perfusion chamber was placed in the optical system. Images were taken using confocal and differential interference contrast (DIC) techniques (see Figure 4).
Figure 4: Confocal (Left) and Differential Interference Contrast (DIC) (Right) Images of HTB126 Breast Cancer Cells
Temperature control was evaluated by circulating 60°C water from a water bath through the water channel. After about 15 minutes, the temperature equilibrated at 37°C and maintained at this temperature for another 15 minutes, after which the experiment was terminated (See Figure 5). These experiments were conducted with the pump operating at maximum speed. Therefore, further calibration will be necessary at different pump speeds and temperatures, but the system will be capable of maintaining temperature.
Figure 5: Validation of Temperature Control
Furthermore, the perfusion chamber was tested to ensure that it was watertight and could successfully deliver and extract fluids. Although not shown here, this was validated by operating the system with water and checking for leaks and Petri dish overflows.
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Anodized, watertight, temperature controlled perfusion chamber |
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Working perfusion system with tubing, connectors, and pump |
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Item (Description) |
Supplier |
Catalog Number |
Cost |
| 75' of 1/8" OD, 1/16" ID Polyurethane Tubing | Mc-Master Carr (Chicago, IL) | 5195T61 | $16.50 |
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2" X 2 1/8" O-ring (25 ct) not used |
Mc-Master Carr (Chicago, IL) | 9396K113 | $10.50 |
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Adhesive-backed Rubber (1/16" thick, 6" X 6") not used |
Mc-Master Carr (Chicago, IL) | 8463K411 | $4.45 |
| 1/16" ID Female Quick Turn Polypropylene Luer Lock (20 ct) | Mc-Master Carr (Chicago, IL) | 51525K261 | $7.38 |
| 1/16" ID Male Quick Turn Polypropylene Luer Lock (20 ct) | Mc-Master Carr (Chicago, IL) | 51525K131 | $8.24 |
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1/16" ID 90° Polypropylene Elbow (20 ct) not used |
Mc-Master Carr (Chicago, IL) | 53415K275 | $14.34 |
| Ounce of 22/32" Cover Glass |
VWR International (West Chester, PA) |
48382-041 | $45.23 |
| Aluminum Anodizing |
QMAC Quality Machining (State College, PA) |
N/A | $80.00 |
| Total | $186.64 | ||