1.RADNOTI 130003ISOLATED PERFUSED LIVER SYSTEM

2. Key system Components 3. Key system components:Primary reservoir 4. Key system components:Primary reservoirSecondary reservoir 5. Key system components:Primary reservoirSecondary reservoirMembrane Oxygenator 6. Key system components:Primary reservoirSecondary reservoirMembrane OxygenatorBubble Trap Compliance Chamber 7. Key system components:Primary reservoirSecondary reservoirMembrane OxygenatorBubble Trap Compliance ChamberInflow Manifold 8. Key system components:Primary reservoirSecondary reservoirMembrane OxygenatorBubble Trap Compliance ChamberInflow ManifoldOut Flow Manifold 9. Key system components:Out Flow Bubble Trap 10. Key system components:Out Flow Bubble TrapPeristaltic Pump 11. Key system components:Out Flow Bubble TrapPeristaltic PumpLiver Chamber 12. Key system components:Out Flow Bubble TrapPeristaltic PumpLiver Chamber3-Way Out Flow Manifold 13. Key system components:Out Flow Bubble TrapPeristaltic PumpLiver Chamber3-Way Out Flow Manifold3-Way Overflow Manifold 14. Flow Path Overview 15. Flow Path:Perfusate is selectedeither from the primaryreservoir 16. Flow Path:Perfusate is selectedeither from the primaryreservoir or thesecondary reservoir 17. Flow Path:Perfusate is selectedeither from the primaryreservoir or thesecondary reservoirusing the three waystopcock located at theoutlet of the secondaryreservoir. 18. Flow Path:Perfusate is selectedeither from the primaryreservoir or thesecondary reservoirusing the three waystopcock located at theoutlet of the secondaryreservoir. 19. Flow Path:Perfusate is selectedeither from the primaryreservoir or thesecondary reservoirusing the three waystopcock located at theoutlet of the secondaryreservoir. The perfusatethen travels to theperistaltic pump 20. Flow Path:and is pumped throughthe membraneoxygenating chamber 21. Flow Path:and up to the bubble trapcompliance chamber 22. Flow Path:and up to the bubble trapcompliance chamberTip: on initial priming orfilling of the system you willmost likely need to closethe compliance portstopcock 23. Flow Path:and up to the bubble trapcompliance chamberTip: on initial priming orfilling of the system you willmost likely need to closethe compliance portstopcockand open the bubble trapvent stopcock 24. Flow Path:and up to the bubble trapcompliance chamberTip: on initial priming orfilling of the system you willmost likely need to closethe compliance portstopcockand open the bubble trapvent stopcockso that the trap will have theopportunity to fill 25. Flow Path:and up to the bubble trapcompliance chamberWhen running in constantpressure mode, perfusateflow from the pump that isgreater than the flow rate ofthe organ , will exit thebubble trap via thecompliance port. 26. Flow Path:and up to the bubble trapcompliance chamberGenerally you can leave thisport open after the systemhas been primed. 27. Flow Path:and up to the bubble trapcompliance chamberGenerally you can leave thisport open after the systemhas been primed.Over flow exiting thecompliance port is directedto the overflow manifold. 28. Flow Path:and up to the bubble trapcompliance chamberThe overflow manifoldwill allow you to selectwhere the overflowingperfusate is to bedirected. 29. Flow Path:and up to the bubble trapcompliance chamberValve 1 directs flowback to the primaryreservoir. 30. Flow Path:and up to the bubble trapcompliance chamberValve 1 directs flowback to the primaryreservoir.Valve 2 directs flowback to the secondaryreservoir. 31. Flow Path:and up to the bubble trapcompliance chamberValve 1 directs flowback to the primaryreservoir.Valve 2 directs flowback to the secondaryreservoir.Valve 3 directs flow Outto Waste. 32. Flow Path:and up to the bubble trapcompliance chamberthe out flow of the bubbletrap then flows to theinflow manifold. 33. Flow Path:and up to the bubble trapcompliance chamberthe out flow of the bubbletrap then flows to theinflow manifold. Tip: When initially priming or flushing the system, the inflow and outflow cannulae will have to be coupled. This can be done by using a small section of tygon tubing and pushing the cannulae tips in either side. 34. Flow Path:The perfusate thenpasses through thecannulated organ (ortygon coupler whenpriming or flushing) andinto the outflow manifold. 35. Flow Path:Perfusate then flows outto through the flow meter( if so equipped) to theoutflow bubble trap. 36. Flow Path:Perfusate then flows outto through the flow meter( if so equipped) to theoutflow bubble trap.NOTE:The ventstopcock andthe out flowstopcock of thebubble trapshould be in theclosed position. 37. Flow Path:Perfusate then flows outto through the flow meter( if so equipped) to theoutflow bubble trap.The perfusate then isdrawn be the secondperistaltic pump headand pushed to the threeway outflow manifold. 38. Flow Path:At this point theperfusate can be directedto :1. The primary reservoir 39. Flow Path:At this point theperfusate can be directedto :1. The primary reservoir2. The secondaryreservoir 40. Flow Path:At this point theperfusate can be directedto :1. The primary reservoir2. The secondaryreservoir3. WasteSink orcollection flask 41. Flow Path:Example:If you are flushing thesystem close valve 1 42. Flow Path:Example:If you are flushing thesystem close valve 1 and2 43. Flow Path:Example:If you are flushing thesystem close valve 1 and2Leaving the valve open towaste will direct the flowout to waste. 44. Flow Path:ALERT! SPECIAL NOTE: when flushing the organ you will want to protect components that may be sensitive to the initial effluent by diverting flow around the component. 45. Flow Path: SPECIAL NOTE: when flushing the organ you will want to protect components that may be sensitive to the initial effluent by diverting flow around the component. 46. Flow Path: SPECIAL NOTE: In this diagram the flow meter would need to be protected. 47. Flow Path: SPECIAL NOTE: In this diagram the flow meter would need to be protected. This is done by using the three way stopcock on the outflow manifold to bypass the flow meter. 48. Flow Path: SPECIAL NOTE: In this diagram the flow meter would need to be protected. This is done by using the three way stopcock on the outflow manifold to bypass the flow meter. 49. Flow Path: 50. Flow Path:Example:If the organ is flushedand you wish to have arecirculating path... 51. Flow Path:Example:If the organ is flushedand you wish to have arecirculating path...In this case back to theprimary reservoir... 52. Flow Path:Example:If the organ is flushedand you wish to have arecirculating path...In this case back to theprimary reservoirClose valve 3 (waste) 53. Flow Path:Example:If the organ is flushedand you wish to have arecirculating path...In this case back to theprimary reservoirClose valve 3 (waste)Close valve 2 54. Flow Path:Example:If the organ is flushedand you wish to have arecirculating path...In this case back to theprimary reservoirClose valve 3 (waste)Close valve 2.Open valve 1 55. Flow Path:Example:If the organ is flushedand you wish to have arecirculating path...OrTo recirculate back to thesecondary reservoir 56. Flow Path:Example:If the organ is flushedand you wish to have arecirculating path...Orto recirculate back to thesecondary reservoirClose valve 1 57. Flow Path:Example:If the organ is flushedand you wish to have arecirculating path...Orto recirculate back to thesecondary reservoirClose valve 1Open valve 2 58. Flow Path:Example:If the organ is flushedand you wish to have arecirculating path...Orto recirculate back to thesecondary reservoirClose valve 1Open valve 2Close valve 3 (waste) 59. INITIAL START UP 60. INITIAL START UPFill the primaryreservoir with buffer 61. INITIAL START UPMake sure reservoirFill the primary selection stopcock isin the off positionreservoir with bufferprior to filling. 62. INITIAL START UPWARNING: ALWAYS MAKE SURE THERE IS AN OPENING TOFill the primary ATMOSPHERE ON THE PRIMARYreservoir with bufferRESERVOIR. IF FOR ANY REASONGAS PREASURE IS ALLOWED TOTurn on and adjustBUILD IN THE RESERVOIR RISK OFgas from tank SERIOUS INJURY OR FATALITY MAY OCCUR.From tank 63. INITIAL START UPFill the primary Note:reservoir with buffer Adjust gas so thatTurn on and adjust a gentle stream ofgas from tank bubbles flows fromthe gas dispersionfrit in the primaryreservoir. This willoxygenate thebuffer and serve asa visualrepresentation thatgas is flowingFrom tank through theMembraneOxygenator. 64. INITIAL START UPFill the primaryreservoir with bufferTurn on and adjustgas from tankOpen complianceport stopcock. 65. INITIAL START UPFill the primaryreservoir with bufferTurn on and adjustgas from tankOpen complianceport stopcock.Close valve 1 and 2on three way outflow manifold. 66. INITIAL START UPFill the primaryreservoir with bufferTurn on and adjustgas from tankOpen complianceport stopcock.Close valve 1 and 2on three way outflow manifold.Open valve 3(waste) on three wayout flow manifold 67. INITIAL START UPFill the primaryreservoir with bufferTurn on and adjustgas from tankOpen complianceport stopcock.Close valve 1 and 2on three way outflow manifold.Open valve 3(waste) on three wayout flow manifoldClose valve 2 and 3on three-wayoverflow manifold 68. INITIAL START UPFill the primaryreservoir with bufferTurn on and adjustgas from tankOpen complianceport stopcock.Close valve 1 and 2on three way outflow manifold.Open valve 3(waste) on three wayout flow manifoldClose valve 2 and 3on three-wayoverflow manifoldOpen valve 1(primary reservoirreturn) on three-wayoverflow manifold. 69. INITIAL START UPClose thestopcocks for theinflow and outflowpressuretransducers. 70. INITIAL START UPClose thestopcocks for theinflow and outflowpressuretransducers.Direct the outflowmanifold three waystopcock to passthrough themanifold. 71. INITIAL START UPClose thestopcocks for theinflow and outflowpressuretransducers.Direct the outflowmanifold three waystopcock to passthrough themanifold.Close the drain andrelief portsstopcocks on the outflow bubble trap. 72. INITIAL START UPClose thestopcocks for theinflow and outflowpressuretransducers.Direct the outflowmanifold three waystopcock to passthrough themanifold.Close the drain andrelief portsstopcocks on the outflow bubble trap.Couple the Inflowand Outflowcannulae. 73. INITIAL START UPClose thestopcocks for theinflow and outflowpressuretransducers.Direct the outflowmanifold three waystopcock to passthrough themanifold.Close the drain andrelief portsstopcocks on the outflow bubble trap.Couple the Inflowand Outflowcannulae.Turn on peristalticpump and turnreservoir selectionthree way stopcockto feed from primary ONreservoir. 74. INITIAL START UPThe system will nowcirculate bufferdriven by theperistaltic pump.It will take severalmoments to purgeair from the lines. You will most likelyhave to open andclose the vent andoverflow ports in thebubble trapcompliance chambertemporarily to buildup some perfusate.Once itapproximately twothirds full, return thevalves to theirprevious position.ON 75. INITIAL START UP Once the system isprimed, Turn off theperistaltic pump.Close stopcocks atthe inflow andoutflow manifold.This will trap thebuffer in the linesand keep the systemprimed. 76. Signal Generation 77. Signal Generation Now is a good timeto calibrate thepressuretransducers and ionselective electrodes(if so equipped.) 78. Signal Generation For the pressurePressureTransducerstransducers typicallythey will have to befilled with fluid andpurged of bubbles. 79. Signal Generation Pressure Transducers For the pressuretransducers typicallythey will have to befilled with fluid andpurged of bubbles. Set the three-waystopcock at theoutflow pressuretransducer so thatflow is acceptedfrom the outflowmanifold and thepurge port. 80. Signal Generation Pressure Transducers For the pressuretransducers typicallythey will have to befilled with fluid andpurged of bubbles. Set the three-waystopcock at theoutflow pressuretransducer so thatflow is acceptedfrom the outflow Vent portmanifold and the stopcockpurge port. (not shown) Fill a disposablesyringe with buffer.Open the transducerpurge port (one waystopcock notshown.) and gentlyfill the pressuretransducer domecausing air to bepurged. 81. Signal Generation Pressure Transducers Repeat theprocedure with theinflow pressuretransducer. 82. Signal Generation Pressure Transducers The pressuretransducers can becalibrated to yourdata acquisition atthis time. Set the three-waystopcock controllingflow to thetransducer to theclosed position. 83. Signal Generation Pressure Transducers The pressuretransducers can becalibrated to yourdata acquisition atthis time. Set the three-waystopcock controllingflow to thetransducer to theclosed position. Open the purgestopcock (notshown) on thetransducer. 84. Signal Generation Pressure Transducers The pressuretransducers can becalibrated to yourdata acquisition atthis time. Set the three-waystopcock controllingflow to thetransducer to theclosed position. Open the purgestopcock (notshown) on thetransducer.This will be yourzero pressurecalibration point. 85. Signal Generation Pressure Transducer Return thestopcocks to theirprevious position(accepting flow fromthe outflowmanifold) andsetting the purgeport stopcock (notshown) to the closedposition.This will be yourehigh pressurecalibration point. 86. Signal Generation Pressure TransducerNote: Return the The pressure head isstopcocks to theirdetermined by theprevious position elevation of the(accepting flow frombubble trapthe outflow compliancemanifold) and chamber. Thesetting the purge distance from theport stopcock (notchamber to theshown) to the closedpressure transducerposition. can be calculated toa known pressure.This will be yourehigh pressure Distance in mmcalibration point.divided by 13.6 = mmof mercuryperfusion pressureshould be 10-15 mmHg (15-25cm abovethe liver.) 87. Signal Generation Pressure TransducerNote: Repeat the The pressure head isprocedure for the determined by theinflow manifold elevation of thepressure transducer.bubble trapcompliancechamber. Thedistance from thechamber to thepressure transducercan be calculated toa known pressure.Distance in mmdivided by 13.6 = mmof mercury. 88. Signal GenerationpH ELECTRODES 89. Signal Generation pH ElectrodesThe pH electrodes areplugged directly fromthe mili volt adapter tothe data acquisitioninterface.Ideally a pH electrodewill output a voltage of0mV in a pH 7 buffer.This can very by +/-50mV based on theindividual pHelectrode.The Nernst equationtells us that a pHbuffer 4 should be160mV greater (morepositive) than thereading in a pH 7.The reading in a pH10should be -160mV less(more negative thanthat of a reading in apH7 buffer.The 160 comes frombeing 3(7-4)*59(Nernstvalue at 20degrees)=168mV for100% slope and 160 isslightly less than 100%theoretical. 90. Signal GenerationO2 ELECTRODES 91. Signal Generation O2 ElectrodesPrior to calibrationof the oxygenelectrode, theelectrode should beexamined to insurethat the electrodemembrane is intactand the interiorchamber is full ofbuffer. If theelectrode requiresmaintenanceplease refer to themanufacturersinstructions. 92. Signal Generation O2 electrodesTo obtain zerooxygen reading, thephysiologicalbuffer, placed in avented calibrationcontainer should begassed for at least10 to 30 minuteswith pure nitrogenat a rate of 3-6bubbles per/sec tomaintain a constanttemperature andgas saturation. 93. Signal Generation O2 ElectrodesTo obtain zerooxygen reading, thephysiologicalbuffer, placed in avented calibrationcontainer should begassed for at least10 to 30 minuteswith pure nitrogenat a rate of 3-6bubbles per/sec tomaintain a constanttemperature andgas saturation.The electrode isthen inserted intothe calibrationchamber andmonitored until thereading is stabile.Once the readinghas stabilized thereading can bet setto zero using theamplifier gain andoffset adjustments.The acquisitionsoftware can usethis as the lowpoint calibration. 94. Signal Generation O2 ElectrodesThe electrode is thenremoved and insertedinto aerated containerof buffer. In this caseeither the inflow or outflow manifold withperfusate beingpumped through. Afterstabilizing the readingwould be adjustedusing the amplifiergain and offset basedon the gas mixture(room air 21% oxygen,gas cylinder 100%oxygen, gas cylinder95% oxygen etc)This can be used asyour high pointcalibration in thedatacquisitonsoftware. 95. Signal Generation O2 ElectrodesIt is recommendedthat the procedureis repeated threetimes in order toinsure the readingsare stable andreproducible. 96. Signal GenerationTemperature 97. Signal Generation TemperatureTemperature probesare preset and will notrequire calibration. Itis recommended thatthey be verifiedperiodically measuringa known temperaturesuch as your heatercirculator bath and theresult compared to theread out of the bath. 98. Signal GenerationFlow Meter 99. Signal Generation Flow MeterThe Flow Meter ispreset and will notrequire calibration. Itis recommended thatit be verifiedperiodically measuringa known flow and theread out verified. 100. Liver Preparation1. The animal is anesthetized and placed on its back; the anesthetic used may be a generalanesthetic such as isoflurane or phenobarbital, depending upon the protocol requirements. Testfor the depth of anesthesia via toe pinch, eye reflex, etc.2. For best positioning, the limbs are retracted and secured with tape or string.3. The abdomen is wiped with 70% alcohol; the abdomen can be shaved, although this is notnecessary.4. A midline incision is made by lifting the skin with forceps and cutting the tissue. The abdomenis cut with the blunt end of blunt/sharp scissors from above the bladder to just below thediaphragm (rib cage). Care must be taken not to cut the abdomen or internal organs.5. The incision is extended into horizontally flaps on both left and right to expose the liver andintestines. The internal organ should be handled gingerly, especially the liver which is soft andeasily damaged. 101. Liver Preparation6. The intestines are carefully moved to the left side of the animal, exposing the liver and surroundingvasculature.7. The vena cava, portal vein, mesenteric veins and arteries and bile duct are located.8. Using a curved needle, non-cutting preferred and 00 or smaller silk suture, a suture is passed beneaththe portal vein, near the liver and past any branches. A second suture is passed beneath the vens cavadistal to this first suture(~5-10 mm). Note that the cannula will be inserted between the two sutures,moved forward towards the liver and its tip secured by the suture closest to the liver. The distal suturewill be used to occlude portal vein blood flow.9 A suture is passed beneath the vena cava, above the right renal vein. A suture is placed beneath themesenteric vein.10. A suture is passed beneath the bile duct, the bile cannula is inserted and the cannula secured viasuture. (bile duct cannula is PE 10 tubing with cuff).11. The appropriate sized portal vein cannula is selected by comparing the cannula tip to vein diameter.The cannula is then placed on the end of the perfusion line and the line flushed by opening the stopcockto clear air bubbles. The stopcock to the line is then closed. The pump should be cycling to permitperfusion to occur as soon as the stopcock is opened. 102. Liver Preparation12. Heparin (1000 units) is injected into the tail vein or into the vena cava, below the renal vein.13. Note that at this point the experimenter must work efficiently so that the liver is not ischemic; totaltime without blood flow to the liver should be less than 2 minutes from steps 14-17.14. The distal portal vein suture is tied to occlude blow flow, the vessel then nicked to permit insertion ofthe cannula, the cannula tip slid into the vein past the first suture and the cannula secured using the firstsuture. Blood will back flush into the cannula.15. The stopcock is opened to allow a small amount of fluid (