4.6. Ex vivo gene transfer into kidney by the closed-circuit organ perfusion method

Based on the hypothesis that, by extending the incubation time of kidney cells with the virus, gene transfer could be accomplished, a closed‐circuit perfusion system for continuous circulation of the viral solution in the intact kidney ex vivo as well as in vivo was developed. This system consisted of a reservoir for the perfusate, a roller pump, an artificial membrane lung, and the kidney to be perfused, all connected by silicon tubing with a 3 mm inner diameter (Fig. l). The reservoir was a 300 ml glass bottle container placed in a thermostat‐controlled 37°C water bath. The pump had been modified from the portable organ perfusion machine PF‐3A model peristaltic pump (Gambro, Sweden) with roll per minute (rpm) control of the flow rate. The membrane lung consisted of 8 m of 1.47 mm inner diameter silicon tubing in a 2000 ml glass container gassed with carbogen gas (95% oxygen, 5% carbon dioxide) at a pressure of 15 mmHg, as described by Hamilton et al. in 1974. The kidney was attached to the perfusion system by cannulating the renal artery with a 14G PTFE cannula and the renal vein with a 12G PTFE cannula (Venflon^“, Sweden). The venous effluent was collected directly into the reservoir. The total volume of perfusate used was 350 ml, and it contained previously separated porcine red blood cells at a hematocrit value of 17% in Krebs‐Ringer solution (NaCl 20%, KCl 5%,CaCl₂ 5%, MgSO₄ 1%, NaHCO₃ 5%, KH₂PO₄ 1%, glucose 0.2%, H₂O 62.8%). Moreover, 25 000 IU heparin (Heparin LEO^®, Lövens, Denmark) and antibiotics were added. For ex vivo perfusion of the explanted kidneys, the perfusate also contained 20 000 IU of benzylpenicillinium sodium (Geepenil^®,Orion, Finland) and 20 000 µg of streptomycin (standard medium) as antibiotics and 5 ml of MEM amino acid solution (Gibco BRL, USA). The flow rate was adjusted to enable adequate diuresis. pH and oxygen saturation in the perfusate were measured in a laboratory blood gas analysis from the perfusate.

Before connecting the explanted kidney to the perfusion system, 10 ml of lidocain‐heparin solution {190 mg lidocain (Xylocain^“ 20mg/ml, Astra, Sweden) + 5 000 IU of heparin (Heparin Leo^“, Lövens, Denmark)} and physiological 0.9% saline were infused through the renal artery until the venous effluent was clear. The adenoviral preparation, 1 x 10¹¹ pfu in 20 ml of Krebs‐Ringer solution, was then infused into the arterial inlet, and perfusion was immediately initiated. The flow rate was set at 100‐120 ml min^–1 at the beginning of the perfusion.

At first, the ex vivo perfusion experiments were made at room temperature by using two explanted porcine kidneys, but as no reporter gene expression was seen in these kidneys, the perfusion temperature was raised to 37°C for the following experiments. A total of four explanted kidneys and one right lung were perfused ex vivo at 37°C, the average perfusion time being 12 hours. Following the experiments, tissue samples were taken for histological analysis, as it will be described below. During the kidney perfusions, diuresis and viral excretion into urine were monitored. The effluent flowing from the ureter was collected and passed into the recirculating reservoir to keep the amount of perfusate constant in the closed-circuit system.