Perfusion apparatus

The basic principles to consider in establishing an experimental system for lung perfusion experiments are considered with regard to the apparatus and the mode of ventilation and perfusion. 

Schnizler et al., 2003) permits the automation of both cRNA injection and two-electrode voltage clamp recordings from multiple oocytes in standard 96-well plates. Although the cost and maintenance of such an apparatus is likely to be restricted to industrial research environments, other automated oocyte perfusion systems have been described that would be suitable for smaller scale laboratories (e.g., Joshi et al., 2004). 

The apparatus is modified from that of Klerx et al. (1) and is represented diagrammatically in Fig. 1. High cell densities can be maintained by constant perfusion of medium through the hollow fibers of dialysis membrane (molecular weight cutoff usually 10,000) with minimal dilution of the MAb. The assembled apparatus, which can be moved readily around the laboratory on a small trolley, is designed for use m a controlled 37°C warm room, but can also be set up in an incubator that has the appropriate access ports. The equipment is inexpensive, and is simple to assemble and operate. 

Various other procedures have been devised to increase the surface area within a vessel upon which cells may grow. The problem is always to maintain an adequate supply of nutrients including oxygen and to remove waste products, particularly acid. The New Brunswick Scientific Co. (Appendix 3) produces a piece of apparatus which continuously perfuses roller bottles by means of a rotating cap through which the various feed tubes pass. 

A perfusion apparatus can be constructed and used to perfuse a rat liver with a solution of 0.05% collagenase in PBS-A (Seglen, 1976). Following treatment the released hepatocytes can be plated out onto collagen surfaces in the presence of 10% bovine serum, insulin and glucagon (10 /u,g/ml) and hydrocortisone (1 /iM). Attachment occurs within 3 h but no cell division occurs though liver specific enzymes... 

An apparatus suitable for use with isolated perfused organs is shown in Fig. 5.16. 

Besides the basic apparatus for microdialysis perfusions, fraction collection, and HPLC analysis, several additional instruments and devices are needed, depending on where the microdialysis probe is to be implanted. The most complicated instrumental setup is probably that required for brain dialysis. A stereotaxtic instrument and a stereomicroscope are necessary for precise positioning of microdialysis cannulae into various brain structures. Inhalation anesthesia is preferable and more convenient than injections. However, this type of anesthesia calls for additional equipment, such as air lines, valves, and mixing chamber for halothane or other anesthetic gases, as well as good ventilation of the operation theater. 

Lennemas et al. have developed a method for measuring human effective permeability (H-Peff) using a regional intestinal perfusion technique. In this method, a perfusion apparatus consisting of a multichannel tube with two inflatable balloons (10 cm apart) is swallowed by the patient and eventually located in the proximal jejunum. Dilute solutions of the test drag are introduced at the inlet located at the center of the 10 cm section, and the loss of drag is determined from the concentration in the outlet intestinal perfusate. In such a fashion, the H-Peff for 22 carefully selected drug molecules has been determined and a theoretical model of H-Peff has been developed. " The small size of the published H-Peff database is most likely due to the expense of the human measurement. 

Inject the heparin rapidly but steadily through the cannula Remove the syringe and attach the cannula to the outlet of the perfusion apparatus containing 500 mL of oxygenated HBSS at 37°C. Perfuse at 50 mL/min. Run the effluent to waste (ieeNote 5). 

Switch the perfusion apparatus over to recirculate and oxygenate 150 mL of HBSS at 37 C (containing 55.8 mg of EDTA, disodium salt) in a 250-mL beaker while the lungs are being trimmed. 

Fill the lungs with 9 mL of HBSS/EDTA through the tracheal cannula using a syringe. Immerse lungs in the beaker, attach the perfusion apparatus outlet to the cannula and add the subtilisin solution to the beaker. Continue the enzyme recirculating perfusion for 15 min. The temperature can be measured by use of a thermistor probe in the beaker of perfusate and should be reading 37°C. 

Fig. 1. Diagram of the perfusion apparatus used for the enzymic digestion of rat lungs.

The recirculadng perfusion apparatus consists of a double pump (Masterflex with 7016 heads, Cole-Parmer, Chicago, IL) to and from a falling film oxygenator (Fig. 1). One inlet tube comes from a botde of... 

Care has to be taken when switching the inlet tube through different solutions not to let air bubbles in. Best way is to switch off the pump, reset the flow and move the tube to the next solution. When more than one mouse is used, the perfusion apparatus has to be rinsed between mice with at least 100 ml of sterile GBSS. 

Figure 1. The apparatus for isolated perfusion of rat kidneys is shown schematically. The typical circuit for bench experiments is shown on the right, while the extension using thermostatted tubing which is required for experiments conducted in the bore of a high field superconducting magnet for either magnetic resonance spectroscopy (MRS) or magnetic resonance microscopy is shown on the left (not to scale). Modified with permission from Endre et al [301].

Ito 5 Characteristics of isolated perfused juxtaglomerular apparatus. Kidney Int SuppI 67 546-548,1998... 

Perfusion. Specially modified swivel caps have been developed that allow continuous perfusion of roller bottles (e.g. Bellco Autoharvester, New Brunswick apparatus). 

The isolated perfused kidney allows the investigator the ability to monitor the effects of chemicals on the intact kidney without the effects of extrarenal systems. Transport of chemicals occurs via normal mechanisms as vasculature and tubular lumen remain open in the perfused state, and renal handling and biotransformation of chemicals can be evaluated using this technique. However, to utilize an isolated perfused kidney preparation, a special apparatus must be used. 

Attach the heart by the aorta to the cannula of the Langendorff apparatus. The cannula is placed into the cut aortic stump of the isolated heart to perfuse the coronary arteries retro-gradely. (The valve separating the aorta and the left ventricle is closed by the pressure in the aorta, and the perfusion fluid is forced through the coronary arteries.)... 

Koppelstaetter C et al (2004) Assessment of a new cell culture perfusion apparatus for in vitro chronic toxicity testing. Part 1 technical description. ALTEX 21(2) 51-60... 

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