RADNOTI 130003ISOLATED PERFUSED   LIVER SYSTEM
Key system Components
Key system components:•Primary reservoir
Key system components:•Primary reservoir•Secondary reservoir
Key system components:•Primary reservoir•Secondary reservoir•Membrane Oxygenator
Key system components:•Primary reservoir•Secondary reservoir•Membrane Oxygenator•Bubble Trap Compliance Chamber
Key system components:•Primary reservoir•Secondary reservoir•Membrane Oxygenator•Bubble Trap Compliance Chamber•Inflow Man...
Key system components:•Primary reservoir•Secondary reservoir•Membrane Oxygenator•Bubble Trap Compliance Chamber•Inflow Man...
Key system components:•Out Flow Bubble Trap
Key system components:•Out Flow Bubble Trap•Peristaltic Pump
Key system components:•Out Flow Bubble Trap•Peristaltic Pump•Liver Chamber
Key system components:•Out Flow Bubble Trap•Peristaltic Pump•Liver Chamber•3-Way Out Flow Manifold
Key system components:•Out Flow Bubble Trap•Peristaltic Pump•Liver Chamber•3-Way Out Flow Manifold•3-Way Overflow Manifold
Flow Path Overview
Flow Path:Perfusate is selectedeither from the primaryreservoir
Flow Path:Perfusate is selectedeither from the primaryreservoir or thesecondary reservoir
Flow Path:Perfusate is selectedeither from the primaryreservoir or thesecondary reservoirusing the three waystopcock locat...
Flow Path:Perfusate is selectedeither from the primaryreservoir or thesecondary reservoirusing the three waystopcock locat...
Flow Path:Perfusate is selectedeither from the primaryreservoir or thesecondary reservoirusing the three waystopcock locat...
Flow Path:and is pumped throughthe membraneoxygenating chamber
Flow Path:and up to the bubble trapcompliance chamber
Flow Path:and up to the bubble trapcompliance chamber          Tip: on initial priming or                            filli...
Flow Path:and up to the bubble trapcompliance chamber          Tip: on initial priming or                            filli...
Flow Path:and up to the bubble trapcompliance chamber          Tip: on initial priming or                            filli...
Flow Path:and up to the bubble trapcompliance chamber          When running in constant                            pressur...
Flow Path:and up to the bubble trapcompliance chamber          Generally you can leave this                            por...
Flow Path:and up to the bubble trapcompliance chamber          Generally you can leave this                            por...
Flow Path:and up to the bubble trapcompliance chamber                            The overflow manifold                    ...
Flow Path:and up to the bubble trapcompliance chamber                            Valve 1 directs flow                     ...
Flow Path:and up to the bubble trapcompliance chamber                            Valve 1 directs flow                     ...
Flow Path:and up to the bubble trapcompliance chamber                            Valve 1 directs flow                     ...
Flow Path:and up to the bubble trapcompliance chamberthe out flow of the bubbletrap then flows to theinflow manifold.
Flow Path:and up to the bubble trapcompliance chamberthe out flow of the bubbletrap then flows to theinflow manifold.     ...
Flow Path:The perfusate thenpasses through thecannulated organ (ortygon coupler whenpriming or flushing) andinto the outfl...
Flow Path:Perfusate then flows outto through the flow meter( if so equipped) to theoutflow bubble trap.
Flow Path:Perfusate then flows outto through the flow meter( if so equipped) to theoutflow bubble trap.                   ...
Flow Path:Perfusate then flows outto through the flow meter( if so equipped) to theoutflow bubble trap.The perfusate then ...
Flow Path:At this point theperfusate can be directedto :1. The primary reservoir
Flow Path:At this point theperfusate can be directedto :1. The primary reservoir2. The secondaryreservoir
Flow Path:At this point theperfusate can be directedto :1. The primary reservoir2. The secondaryreservoir3. Waste         ...
Flow Path:Example:If you are flushing thesystem close valve 1
Flow Path:Example:If you are flushing thesystem close valve 1 and2
Flow Path:Example:If you are flushing thesystem close valve 1 and2Leaving the valve open towaste will direct the flowout t...
Flow Path:ALERT!             SPECIAL NOTE:             when flushing the             organ you will             want to pr...
Flow Path:             SPECIAL NOTE:             when flushing the             organ you will             want to protect ...
Flow Path:             SPECIAL NOTE:             In this diagram the             flow meter would             need to be  ...
Flow Path:             SPECIAL NOTE:             In this diagram the             flow meter would             need to be  ...
Flow Path:             SPECIAL NOTE:             In this diagram the             flow meter would             need to be  ...
Flow Path:
Flow Path:Example:If the organ is flushedand you wish to have arecirculating path...
Flow Path:Example:If the organ is flushedand you wish to have arecirculating path...In this case back to theprimary reserv...
Flow Path:Example:If the organ is flushedand you wish to have arecirculating path...In this case back to theprimary reserv...
Flow Path:Example:If the organ is flushedand you wish to have arecirculating path...In this case back to theprimary reserv...
Flow Path:Example:If the organ is flushedand you wish to have arecirculating path...In this case back to theprimary reserv...
Flow Path:Example:If the organ is flushedand you wish to have arecirculating path...OrTo recirculate back to thesecondary ...
Flow Path:Example:If the organ is flushedand you wish to have arecirculating path...Orto recirculate back to thesecondary ...
Flow Path:Example:If the organ is flushedand you wish to have arecirculating path...Orto recirculate back to thesecondary ...
Flow Path:Example:If the organ is flushedand you wish to have arecirculating path...Orto recirculate back to thesecondary ...
INITIAL START UP
INITIAL START UP♦Fill the primaryreservoir with buffer
INITIAL START UP                        Make sure reservoir♦Fill the primary       selection stopcock is                  ...
INITIAL START UP                          WARNING: ALWAYS MAKE SURE                             THERE IS AN OPENING TO♦Fil...
INITIAL START UP♦Fill the primary       Note:reservoir with buffer   Adjust gas so that♦Turn on and adjust     a gentle st...
INITIAL START UP♦Fill the primaryreservoir with buffer♦Turn on and adjustgas from tank♦Open complianceport stopcock.
INITIAL START UP♦Fill the primaryreservoir with buffer♦Turn on and adjustgas from tank♦Open complianceport stopcock.♦Close...
INITIAL START UP♦Fill the primaryreservoir with buffer♦Turn on and adjustgas from tank♦Open complianceport stopcock.♦Close...
INITIAL START UP♦Fill the primaryreservoir with buffer♦Turn on and adjustgas from tank♦Open complianceport stopcock.♦Close...
INITIAL START UP♦Fill the primaryreservoir with buffer♦Turn on and adjustgas from tank♦Open complianceport stopcock.♦Close...
INITIAL START UP♦Close thestopcocks for theinflow and outflowpressuretransducers.
INITIAL START UP♦Close thestopcocks for theinflow and outflowpressuretransducers.♦Direct the outflowmanifold three waystop...
INITIAL START UP♦Close thestopcocks for theinflow and outflowpressuretransducers.♦Direct the outflowmanifold three waystop...
INITIAL START UP♦Close thestopcocks for theinflow and outflowpressuretransducers.♦Direct the outflowmanifold three waystop...
INITIAL START UP♦Close thestopcocks for theinflow and outflowpressuretransducers.♦Direct the outflowmanifold three waystop...
INITIAL START UPThe system will nowcirculate bufferdriven by theperistaltic pump.It will take severalmoments to purgeair f...
INITIAL START UP Once the system isprimed, Turn off theperistaltic pump.Close stopcocks atthe inflow andoutflow manifold.T...
Signal Generation
Signal Generation Now is a good timeto calibrate thepressuretransducers and ionselective electrodes(if so equipped.)
Signal Generation For the pressure                          Pressure                        Transducerstransducers typical...
Signal Generation Pressure Transducers For the pressuretransducers typicallythey will have to befilled with fluid andpurge...
Signal Generation Pressure Transducers For the pressuretransducers typicallythey will have to befilled with fluid andpurge...
Signal Generation Pressure Transducers Repeat theprocedure with theinflow pressuretransducer.
Signal Generation Pressure Transducers The pressuretransducers can becalibrated to yourdata acquisition atthis time. Set t...
Signal Generation Pressure Transducers The pressuretransducers can becalibrated to yourdata acquisition atthis time. Set t...
Signal Generation Pressure Transducers The pressuretransducers can becalibrated to yourdata acquisition atthis time. Set t...
Signal Generation Pressure Transducer Return thestopcocks to theirprevious position(accepting flow fromthe outflowmanifold...
Signal Generation Pressure Transducer                                        Note: Return the                             ...
Signal Generation Pressure Transducer                                        Note: Repeat the                             ...
Signal Generation                    pH ELECTRODES
Signal Generation pH ElectrodesThe pH electrodes areplugged directly fromthe mili volt adapter tothe data acquisitioninter...
Signal Generation                    O2 ELECTRODES
Signal Generation O2 ElectrodesPrior to calibrationof the oxygenelectrode, theelectrode should beexamined to insurethat th...
Signal Generation O2 electrodesTo obtain zerooxygen reading, thephysiologicalbuffer, placed in avented calibrationcontaine...
Signal Generation O2 ElectrodesTo obtain zerooxygen reading, thephysiologicalbuffer, placed in avented calibrationcontaine...
Signal Generation O2 ElectrodesThe electrode is thenremoved and insertedinto aerated containerof buffer. In this caseeithe...
Signal Generation O2 ElectrodesIt is recommendedthat the procedureis repeated threetimes in order toinsure the readingsare...
Signal Generation                    Temperature
Signal Generation TemperatureTemperature probesare preset and will notrequire calibration. Itis recommended thatthey be ve...
Signal Generation                    Flow Meter
Signal Generation Flow MeterThe Flow Meter ispreset and will notrequire calibration. Itis recommended thatit be verifiedpe...
Liver Preparation1. The animal is anesthetized and placed on its back; the anesthetic used may be a generalanesthetic such...
Liver Preparation6. The intestines are carefully moved to the left side of the animal, exposing the liver and surroundingv...
Liver Preparation12. Heparin (1000 units) is injected into the tail vein or into the vena cava, below the renal vein.13. N...
Liver Preparation20. The vena cava suture below the liver is secured and the stopcock to the atrial-thoracic vena cavacann...
Post Experimental Clean UpGlassware Maintenance & Post Experimental CleanupPost Experimental CleanupAfter the experiment h...
Post Experimental CleanupShared equipment is the most difficult to maintain properly. In order to maintain equipment prope...
Post Experimental CleanupUse of toxins, biohazardous materials and radiochemicals can present considerable complications t...
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Liver presentation

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Liver presentation

  1. 1. RADNOTI 130003ISOLATED PERFUSED LIVER SYSTEM
  2. 2. Key system Components
  3. 3. Key system components:•Primary reservoir
  4. 4. Key system components:•Primary reservoir•Secondary reservoir
  5. 5. Key system components:•Primary reservoir•Secondary reservoir•Membrane Oxygenator
  6. 6. Key system components:•Primary reservoir•Secondary reservoir•Membrane Oxygenator•Bubble Trap Compliance Chamber
  7. 7. Key system components:•Primary reservoir•Secondary reservoir•Membrane Oxygenator•Bubble Trap Compliance Chamber•Inflow Manifold
  8. 8. Key system components:•Primary reservoir•Secondary reservoir•Membrane Oxygenator•Bubble Trap Compliance Chamber•Inflow Manifold•Out Flow Manifold
  9. 9. Key system components:•Out Flow Bubble Trap
  10. 10. Key system components:•Out Flow Bubble Trap•Peristaltic Pump
  11. 11. Key system components:•Out Flow Bubble Trap•Peristaltic Pump•Liver Chamber
  12. 12. Key system components:•Out Flow Bubble Trap•Peristaltic Pump•Liver Chamber•3-Way Out Flow Manifold
  13. 13. Key system components:•Out Flow Bubble Trap•Peristaltic Pump•Liver Chamber•3-Way Out Flow Manifold•3-Way Overflow Manifold
  14. 14. Flow Path Overview
  15. 15. Flow Path:Perfusate is selectedeither from the primaryreservoir
  16. 16. Flow Path:Perfusate is selectedeither from the primaryreservoir or thesecondary reservoir
  17. 17. Flow Path:Perfusate is selectedeither from the primaryreservoir or thesecondary reservoirusing the three waystopcock located at theoutlet of the secondaryreservoir.
  18. 18. Flow Path:Perfusate is selectedeither from the primaryreservoir or thesecondary reservoirusing the three waystopcock located at theoutlet of the secondaryreservoir.
  19. 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. 20. Flow Path:and is pumped throughthe membraneoxygenating chamber
  21. 21. Flow Path:and up to the bubble trapcompliance chamber
  22. 22. Flow Path:and up to the bubble trapcompliance chamber Tip: on initial priming or filling of the system you will most likely need to close the compliance port stopcock
  23. 23. Flow Path:and up to the bubble trapcompliance chamber Tip: on initial priming or filling of the system you will most likely need to close the compliance port stopcock and open the bubble trap vent stopcock
  24. 24. Flow Path:and up to the bubble trapcompliance chamber Tip: on initial priming or filling of the system you will most likely need to close the compliance port stopcock and open the bubble trap vent stopcock so that the trap will have the opportunity to fill
  25. 25. Flow Path:and up to the bubble trapcompliance chamber When running in constant pressure mode, perfusate flow from the pump that is greater than the flow rate of the organ , will exit the bubble trap via the compliance port.
  26. 26. Flow Path:and up to the bubble trapcompliance chamber Generally you can leave this port open after the system has been primed.
  27. 27. Flow Path:and up to the bubble trapcompliance chamber Generally you can leave this port open after the system has been primed. Over flow exiting the compliance port is directed to the overflow manifold.
  28. 28. Flow Path:and up to the bubble trapcompliance chamber The overflow manifold will allow you to select where the overflowing perfusate is to be directed.
  29. 29. Flow Path:and up to the bubble trapcompliance chamber Valve 1 directs flow back to the primary reservoir.
  30. 30. Flow Path:and up to the bubble trapcompliance chamber Valve 1 directs flow back to the primary reservoir. Valve 2 directs flow back to the secondary reservoir.
  31. 31. Flow Path:and up to the bubble trapcompliance chamber Valve 1 directs flow back to the primary reservoir. Valve 2 directs flow back to the secondary reservoir. Valve 3 directs flow Out to Waste.
  32. 32. Flow Path:and up to the bubble trapcompliance chamberthe out flow of the bubbletrap then flows to theinflow manifold.
  33. 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. 34. Flow Path:The perfusate thenpasses through thecannulated organ (ortygon coupler whenpriming or flushing) andinto the outflow manifold.
  35. 35. Flow Path:Perfusate then flows outto through the flow meter( if so equipped) to theoutflow bubble trap.
  36. 36. Flow Path:Perfusate then flows outto through the flow meter( if so equipped) to theoutflow bubble trap. NOTE: The vent stopcock and the out flow stopcock of the bubble trap should be in the closed position.
  37. 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. 38. Flow Path:At this point theperfusate can be directedto :1. The primary reservoir
  39. 39. Flow Path:At this point theperfusate can be directedto :1. The primary reservoir2. The secondaryreservoir
  40. 40. Flow Path:At this point theperfusate can be directedto :1. The primary reservoir2. The secondaryreservoir3. Waste Sink or collection flask
  41. 41. Flow Path:Example:If you are flushing thesystem close valve 1
  42. 42. Flow Path:Example:If you are flushing thesystem close valve 1 and2
  43. 43. Flow Path:Example:If you are flushing thesystem close valve 1 and2Leaving the valve open towaste will direct the flowout to waste.
  44. 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. 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. 46. Flow Path: SPECIAL NOTE: In this diagram the flow meter would need to be protected.
  47. 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. 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. 49. Flow Path:
  50. 50. Flow Path:Example:If the organ is flushedand you wish to have arecirculating path...
  51. 51. Flow Path:Example:If the organ is flushedand you wish to have arecirculating path...In this case back to theprimary reservoir...
  52. 52. Flow Path:Example:If the organ is flushedand you wish to have arecirculating path...In this case back to theprimary reservoir…Close valve 3 (waste)
  53. 53. Flow Path:Example:If the organ is flushedand you wish to have arecirculating path...In this case back to theprimary reservoir…Close valve 3 (waste)Close valve 2
  54. 54. Flow Path:Example:If the organ is flushedand you wish to have arecirculating path...In this case back to theprimary reservoir…Close valve 3 (waste)Close valve 2.Open valve 1
  55. 55. Flow Path:Example:If the organ is flushedand you wish to have arecirculating path...OrTo recirculate back to thesecondary reservoir…
  56. 56. Flow Path:Example:If the organ is flushedand you wish to have arecirculating path...Orto recirculate back to thesecondary reservoir…Close valve 1
  57. 57. Flow Path:Example:If the organ is flushedand you wish to have arecirculating path...Orto recirculate back to thesecondary reservoir…Close valve 1Open valve 2
  58. 58. Flow Path:Example:If the organ is flushedand you wish to have arecirculating path...Orto recirculate back to thesecondary reservoir…Close valve 1Open valve 2Close valve 3 (waste)
  59. 59. INITIAL START UP
  60. 60. INITIAL START UP♦Fill the primaryreservoir with buffer
  61. 61. INITIAL START UP Make sure reservoir♦Fill the primary selection stopcock is in the off positionreservoir with buffer prior to filling.
  62. 62. INITIAL START UP WARNING: ALWAYS MAKE SURE THERE IS AN OPENING TO♦Fill the primary ATMOSPHERE ON THE PRIMARYreservoir with buffer RESERVOIR. IF FOR ANY REASON GAS PREASURE IS ALLOWED TO♦Turn on and adjust BUILD IN THE RESERVOIR RISK OFgas from tank SERIOUS INJURY OR FATALITY MAY OCCUR. From tank
  63. 63. INITIAL START UP♦Fill the primary Note:reservoir with buffer Adjust gas so that♦Turn on and adjust a gentle stream ofgas from tank bubbles flows from the gas dispersion frit in the primary reservoir. This will oxygenate the buffer and serve as a visual representation that gas is flowing From tank through the Membrane Oxygenator.
  64. 64. INITIAL START UP♦Fill the primaryreservoir with buffer♦Turn on and adjustgas from tank♦Open complianceport stopcock.
  65. 65. INITIAL START UP♦Fill the primaryreservoir with buffer♦Turn on and adjustgas from tank♦Open complianceport stopcock.♦Close valve 1 and 2on three way outflow manifold.
  66. 66. INITIAL START UP♦Fill the primaryreservoir with buffer♦Turn on and adjustgas from tank♦Open complianceport stopcock.♦Close valve 1 and 2on three way outflow manifold.♦Open valve 3(waste) on three wayout flow manifold
  67. 67. INITIAL START UP♦Fill the primaryreservoir with buffer♦Turn on and adjustgas from tank♦Open complianceport stopcock.♦Close valve 1 and 2on three way outflow manifold.♦Open valve 3(waste) on three wayout flow manifold♦Close valve 2 and 3on three-wayoverflow manifold
  68. 68. INITIAL START UP♦Fill the primaryreservoir with buffer♦Turn on and adjustgas from tank♦Open complianceport stopcock.♦Close valve 1 and 2on three way outflow manifold.♦Open valve 3(waste) on three wayout flow manifold♦Close valve 2 and 3on three-wayoverflow manifold♦Open valve 1(primary reservoirreturn) on three-wayoverflow manifold.
  69. 69. INITIAL START UP♦Close thestopcocks for theinflow and outflowpressuretransducers.
  70. 70. INITIAL START UP♦Close thestopcocks for theinflow and outflowpressuretransducers.♦Direct the outflowmanifold three waystopcock to passthrough themanifold.
  71. 71. INITIAL START UP♦Close thestopcocks for theinflow and outflowpressuretransducers.♦Direct the outflowmanifold three waystopcock to passthrough themanifold.♦Close the drain andrelief portsstopcocks on the outflow bubble trap.
  72. 72. INITIAL START UP♦Close 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. 73. INITIAL START UP♦Close 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. 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. 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. 76. Signal Generation
  77. 77. Signal Generation Now is a good timeto calibrate thepressuretransducers and ionselective electrodes(if so equipped.)
  78. 78. Signal Generation For the pressure Pressure Transducerstransducers typicallythey will have to befilled with fluid andpurged of bubbles.
  79. 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. 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. 81. Signal Generation Pressure Transducers Repeat theprocedure with theinflow pressuretransducer.
  82. 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. 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. 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. 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. 86. Signal Generation Pressure Transducer Note: Return the The pressure head isstopcocks to their determined by theprevious position elevation of the(accepting flow from bubble trapthe outflow compliancemanifold) and chamber. Thesetting the purge distance from theport stopcock (not chamber to theshown) to the closed pressure transducerposition. can be calculated to a known pressure.This will be yourehigh pressure Distance in mmcalibration point. divided by 13.6 = mm of mercury perfusion pressure should be 10-15 mm Hg (15-25cm above the liver.)
  87. 87. Signal Generation Pressure Transducer Note: Repeat the The pressure head isprocedure for the determined by theinflow manifold elevation of thepressure transducer. bubble trap compliance chamber. The distance from the chamber to the pressure transducer can be calculated to a known pressure. Distance in mm divided by 13.6 = mm of mercury.
  88. 88. Signal Generation pH ELECTRODES
  89. 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. 90. Signal Generation O2 ELECTRODES
  91. 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. 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. 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. 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. 95. Signal Generation O2 ElectrodesIt is recommendedthat the procedureis repeated threetimes in order toinsure the readingsare stable andreproducible.
  96. 96. Signal Generation Temperature
  97. 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. 98. Signal Generation Flow Meter
  99. 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. 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. 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. 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 (<5ml) into the liver. The liver should not beover expanded.16. The vena cava is immediately cut below the suture loop to permit blood to drain out.17. The stopcock is then re-opened and the liver perfused from the reservoir. A successful perfusion willhave the liver eventually blanching to an even beige color, without spotches or mottling.18. The mesenteric veins are tied off with the previously placed suture.19. The chest cavity is opened midline using scissors and the heart exposed. The atrial- thoracic venacava cannula is inserted through a cut placed in the right atria into the thoracic vena cava and securedwith suture. The cannula should fully dilated the vena cava to reduce backpressure.
  103. 103. Liver Preparation20. The vena cava suture below the liver is secured and the stopcock to the atrial-thoracic vena cavacannula is opened. This then forces fluid to exit from the liver into the atrial-thoracic vena cava cannula.21. The lines to the bile duct, portal vein and vena cava are held in positionThe liver is removed from the donor by supporting the diaphragm at the midline with forceps andcarefully cutting the diaphragm around the rib cage, first on side and then the other. Any attachment ofthe liver to surrounding tissue are carefully removed.22. The three lines are supported and the liver is then transferred to the organ chamber.23. As indicators of a successful perfusion, liver flow should be at least 4 ml/minute/gram liver (forperfusates without blood or other oxygen carriers) and bile flow 1-2 micro liters minute.
  104. 104. Post Experimental Clean UpGlassware Maintenance & Post Experimental CleanupPost Experimental CleanupAfter the experiment has been completed, the experimenter should take care to scrupulously clean theequipment. It is important to remember that the solutions that can sustain the heart and muscle will also provideexcellent media for bacteria. The cleaning procedures will be dependent upon the types of chemicals andbiological materials that are being used, the types of measurements that are being made and what substancescan interfere with those measurements and the frequency of the use of the equipment and number of operatorsinvolved. Non-phosphate soaps are preferred, since insoluble phosphates can form from calcium andmagnesium in physiological salt solutions. Note that bactericidal soaps may contain iodine or other materialswhich can affect isolated tissues and cells. Cleaning supplies and equipment, such as brushes, should be usedonly for cleaning this glassware and not used for other lab cleaning procedures. Questions and proceduresnoted here should be adjusted in accordance with your licensed procedures and the recommendations of yoursafety personnel.
  105. 105. Post Experimental CleanupShared equipment is the most difficult to maintain properly. In order to maintain equipment properly, it is generallybest (1) to assign the maintenance or the oversight of the equipment to one individual, who will monitorequipment and maintain cleaning supplies (2) to have written protocols posted with the equipment (3) to have alogbook where cleaning dates, as well as notification of problems, suggestions, etc., can be recorded.Often overlooked as a source of contamination is the water circulator supply. This should be kept clean and thebath rinsed and solution changed to reduce precipitate build up. Covering equipment to reduce air bornecontamination from microbes and spores is useful. Note that when baths are used intermittently, the lack offrequent cleaning and the lack of solutions rinsing out bacteria that are deposited in the tubing may result in acontamination problem when the system is finally used. A convenient rule of thumb for testing for contaminationin preparations that you have found reliable is that two consecutive experimental failures that are not explainedby an obviously damaged sample, poor surgical or dissection techniques or solution problems may be caused bybath contamination.GlasswareMuch of the Radnoti apparatus is borosilicate glass, which can be cleaned with a wide range of soaps, ethylalcohol, dilute HCl or HNO3 (0.1 M) or other solvents. Extensive flushing with distilled, deionized water to removeall traces of the cleaning agents and salts is recommended. Large glassware, such as reservoirs or assembliescan be flushed in place, but care must be taken to thoroughly clean aerators, stopcocks and associated parts.Aerators should be blown dry using gas or air at the final water rinse. If acid is used, the runoff water should notbe more acidic than the normal water pH. As with the use of any chemicals, proper protective gear and trainingare essential to reduce personnel hazards and experimental and environmental contamination. Heated acid orchromic acid is generally not recommended due to personnel hazards and possible heavy metal contamination ofthe system.If very lipohilic substances (prostaglandins, ionophores, certain dyes, etc.) are used, rinses with ethyl alcohol orthe most appropriate organic solvent can be used first, but this will necessitate thorough cleaning afterward toremove any traces of the organic solvent.
  106. 106. Post Experimental CleanupUse of toxins, biohazardous materials and radiochemicals can present considerable complications to ageneralized cleaning procedure. Having an apparatus and a contained area dedicated to these proceduresreduces problems. Diluted bleach can be used on glassware, but must be rinsed extensively. The use ofdisposable tubing and stopcocks will assist in cleanup, as will scheduling a run of these procedures, ratherthan intermittent experiments, if non-dedicated equipment must be used. Glassware can be sterilized but allfixtures, such as aerators, stopcocks caps, etc., should be removed prior to sterilization.The glass aerators can be cleaned with water, or dilute acid if clogged. The use of water or gas under highpressure can result in damage to the glassware and personnel and therefore is not recommended. After ageneral soap and water rinse to remove soluble materials, cleaning with 0.1M HCl or 0.1 M HNO3 for severalhours or overnight, followed by an extensive water rinse, will usually remove most contaminants. If this doesnot work, 1 M acid can be tried. Because the glass frit filaments are thin, high concentrations of acids, orespecially alkalis, can destroy them and are not recommended.Non-glass itemsInitial cleaning of non-glass items should be with aqueous soap solutions. Depending upon the chemicalresistance of the materials, the use of other solvents, cleaning procedures or sterilization may be possible.Areas and items to be especially well cleaned are the aerator, tubing, syringe ports, cannulae, pressuretransducer fittings, septa, balloon and other catheters and electrodes (oxygen, pacing, ion selective, etc.).Tubing should be inspected at the pump head for wear. Note that the interior of tubing can gradually beroughened during use and the abraded areas will form sites for bacterial growth. Tubing should be a highgrade with low plasticizer leaching. Note that silicone tubing is very permeant to gases, so it should not begenerally used to transport gassed solutions.

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