An Open-Source Normothermic Perfusion System Designed for Research Scientists

Human tumor microenvironment is a living, evolving complex that responds to drug perturbations in ways that are difficult to predict on a cellular level. Our goal has been to sustain the human tumor microenvironment ex vivo under near physiologic conditions for detailed interrogations. The machine presented here and addresses the lack of affordable normothermic profusion systems for research applications, enabling iterative studies for a large number of desired experimental endpoints.

Our laboratory will continue working to further scale the machine down for completely autologous profusion, enabling the system for immuno-oncology and cell therapy investigations. To begin, place the drum funnel on the right side of the workstation with the spout extending past the front edge of the table. Place a circular memory foam to fit the base of the drum with the diaphragm positioned underneath the foam.

Leave a four inch gap near the spout for outflow. Then place two water heating blankets on top of the foam insert with the connector tubing facing the top left corner and insert a 3/16-inch male Luer barb into each tube. Take a folded, sterile, warmer drape.

Cut two millimeters off the corner of the fold to create an outflow hole and place it on the drum. Next, obtain the 3D printed outlet connector and insert the magnet into its groove. Place this assembly beneath the drape through the outflow spout and align the cut hole with the hole in the connector and the magnet.

Raise the excess drape to cover the drum funnel and secure it using rubber bands or elastic ties. Fold the excess drape over the top to establish a sterile barrier. Now place the second water jacket on top of the full folded drape.

Then take a 30 inch piece of 1/4-inch tubing and connect the proximal end to the outlet at the base of the drum funnel spout and rest near the reservoir holder, which will be connected later. Now, place the pumps on a mobile cart positioned near the left side of the workstation. Position the stand in two clamps directly behind the pumps, so the clamps are elevated six inches above the pumps.

Further, obtain two pump heads and secure each into one of the pumps. Place two oxygenator into the clamps on the stand in a horizontal orientation. Then connect the arterial and venous 1/8-inch gas inflow tubing to the top right lower connector of their respective oxygenator.

Now, remove all tubing from the hemodialysis kit, except for the lines leading directly into the filter. Place one large roller pump immediately to the right of the hemodialysis filter for blood inflow. Then place two smaller peristaltic pumps to the right of the blood roller pump for dialysate inflow and outflow.

Obtain two 1/8-inch peristaltic tubings and insert them into the white pump clamp. Attach a female 1/8-inch Luer barb to both ends of each tubing. To connect the dialysate bag to the filter inflow, attach 1/8-inch tubing to the Luer connector on the bag.

Attach a male 1/8-inch Luer to the tubing's end and connect it to the inflow peristaltic pump tubing. Connect inflow and outflow circuits to the appropriate hemodialysis filter ports. Place the reservoir inside the reservoir holder with the exterior measurement markings and the yellow capped connector facing outward.

Next, cut a 36-inch piece of 5/16-inch tubing. Attach a Y connector to the distal end of this tubing and connect the proximal end to the outflow spigot at the bottom of the reservoir. Cut two 4-inch pieces of 5/16-inch tubing and attach each to the end of the Y connector.

Connect the other ends of these tubes to the horizontal prongs of the pump heads. Then cut two 4-inch pieces of 1/4-inch tubing and connect one to each vertical barb on the pump heads. Attach the other end to the left lower prong of each oxygenator.

Now take a 30-inch segment of 5/16-inch tubing. Cut the tubing in half and place a 5/16-inch Y connector over the distal cut end. Attach the proximal end to the outflow barb of the venous oxygenator.

Reattach the other half to the top barb of the Y connector. For the side sampling circuit, cut a 2-inch piece of 1/4-inch tubing. And using a needle driver, stretch the proximal end.

Attach this stretch tubing to the remaining barb of the 5/16-inch Y connector. After connecting the cuvette shunt sensor, connect the horizontal part of a female T connector to a 1/8-inch tubing fitted with a male Luer. Attach a sampling clave to the perpendicular aspect of the T connector.

Finally, connect a 1/8-inch male Luer to the other horizontal side of the T connector. Then cut a 12-inch length of 1/8-inch tubing and connect the proximal end to the sampling clave. Attach a male 1/8-inch Luer to the distal end and connect it to a second female T connector.

To one side of this T connector, attach a 1/4-inch male Luer and use a two inch piece of 1/4-inch tubing to connect it to a reservoir inflow barb. At the distal end of the 5/16-inch tubing, attach another 5/16-inch Y connector. Obtain approximately 55-inch of 1/4-inch tubing and connect it to the top barb of the Y connector.

For the hemodialysis blood circuit, attach a 1/4-inch male Luer to the distal end of the 1/4-inch tubing. Thread this tubing through the large roller pump and connect it to the blood inflow port at the top of the hemodialysis filter identified by red lined tubing. Next, take a 24-inch piece of 1/4-inch tubing and attach a male Luer to the proximal end.

Connect this tubing to the blue lined blood outflow port of the hemodialysis filter. Then attach the distal end to the inflow barb of the reservoir. Take a 36-inch piece of 1/4-inch tubing and stretch it to fit securely onto the barb of the 5/16-inch Y connector.

Next, obtain three four-way stopcocks and connect them in a linear sequence. Attach this assembly to the perpendicular port of a female T connector. Insert this component 12-inches from the left side of the 1/4-inch tubing.

Then attach external flow sensors to the 1/4-inch tubing immediately downstream of the drug infusion site. Make a cut two inches distal to the flow sensors and insert pressure sensors with barbed ends into the 1/4-inch tubing. Place the distal portion of the tubing into the center of the drum funnel.

Next, cut a 30-inch length of 5/16-inch tubing and connect its proximal end to the right outflow barb of the arterial oxygenator. Cut this tubing at the midpoint and insert a 5/16-inch Y connector. Then cut a 36-inch segment of 1/4-inch tubing and connect it to the barb of the 5/16-inch Y connector.

Reattach the external flow sensor. Make a cut two inches downstream and connect pressure sensors to the tubing end positioned at the drum funnel center for arterial inflow. Then cut a 30-inch piece of 1/4-inch tubing and attach a 1/4-inch male Luer to the proximal end for eventual connection to the specimen's inferior vena cava outflow.

Cut this tubing 6-inches from the reservoir and attach 1/4-inch male Luer to both ends. Insert a female T connector with a sampling clave on the perpendicular port. Introduce perfusate into the circuit following the previously described method.

Rewarm and connect the specimen and begin perfusion. In the first 24 hours of porcine liver perfusion, hepatic artery and portal vein flows averaged 107 and 501 milliliters per minute, respectively, with stable inflow pressures of 80 and 7.1 millimeters of mercury. Arterial, portal, and venous oxygen saturations in porcine livers averaged 95%85%and 74%with pH remaining near 7.2.

Glucose levels decreased steadily. Hematocrit and hemoglobin remained stable and potassium dropped slightly. Porcine livers produced 41 milliliters of bile and showed moderate lactate elevation in human perfusion.

Arterial and venous inflows averaged 113 and 317 milliliters per minute and oxygen saturation reached 98%92%and 86%in arterial, portal, and hepatic veins. Human livers maintained physiologic pH, stable hematocrit and hemoglobin, and showed glucose decline and lactate increase with bile production averaging 1.4 milliliters per hour. Histology confirmed viable hepatic parenchyma and tumor morphology after perfusion.

A mobile setup enabled CT imaging of perfused livers, yielding radiographic quality comparable to in vivo scans. The system also supported kidney and pancreas perfusion with preserved glomerular and Islets of Langerhans.