Abstract:
A perfusion apparatus for perfusing an organ includes a pump for pumping perfusion liquid though a perfusion liquid conduit, the conduit having an outlet. A pressure sensor senses the pressure of the perfusion liquid downstream of the pump and upstream of the outlet. A controller controls the output of the pump responsive to the pressure, so as to keep the pressure of the liquid reaching the organ substantially constant.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
       [0001]    Not applicable. 
       STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT 
       [0002]    Not applicable. 
       BACKGROUND OF THE INVENTION 
       [0003]    There are currently promising applications for isolated cells from parent organs, such as the liver, spleen, kidney, adrenal, and pancreas. For example, therapies have been developed for the clinical application of isolated pancreatic cells called the Islets of Langerhans as a treatment for diabetic patients. Patients with diabetes either have Islets of Langerhans that do not function properly, and therefore, do not produce enough insulin, known as Type 2 Diabetes, or do not have Islet cells at all, Type 1 Diabetes. Therapies have been developed by which functioning Islets of Langerhans cells are transplanted into diabetic patients to restore the insulin producing ability of the pancreas. Such therapies require isolated Islet of Langerhans cells, but these cells must be isolated while still viable. Viable isolated cells are mostly obtained from organs of the very recently deceased. The apparatus and method for isolating the cells must be able to extract isolated cells with as little damage to the cells as possible. 
         [0004]    Many different methods and approaches have been attempted to isolate individual cells from their respective parent organs. Prior methods have produced isolated cells with some cell destruction. This cell destruction can result from the relatively severe mechanical stimulation that is used to isolate cells from an organ. For example, enzymatic digestion is used to separate cells of interest from an organ, such as Islet cells from a pancreas. The process can include the mechanical disruption of the organ, as by cutting or chopping, in order to harvest the cells of interest. The result can be damage and mortality to some portion of the cells of interest. 
         [0005]    One method that attempts to overcome the loss of damaged cells due to relatively severe mechanical stress is described in U.S. Pat. No. 5,079,160 to Lacy, et al. The method disclosed by Lacy, et al. comprises the steps of: placing an organ or a piece of an organ in a digestion chamber along with marble agitators; distending the organ or a piece of the organ with physiologically compatible medium containing a protease; continuously recirculating that medium; and separating the isolated cells. Agitators, such as those described in U.S. Pat. No. 6,833,270, greatly increase the amount of undamaged cells obtained through isolation without reducing the quality of the isolated cells obtained by gently agitating the organ. Moreover, the marbles are an appropriate size, weight, and density for obtaining beneficial results as compared to other agitators of varying size, weight, and density which can cause severe mechanical disruption of the organ tissue resulting in some cells being destroyed. 
         [0006]    The protease utilized by the Lacy, et al. method and other similar methods requires optimal temperatures for appropriate activity of the protease. This temperature depends on the particular protease and other operating conditions, however, the temperature is usually elevated relative to room temperature. Therefore the medium must be heated to the desired temperature, for example, 32° C. for some collegenases. Various devices for heating liquids are known, including heat exchange with other fluids and contact with heating elements. It is desirable that such devices for use in cell extraction provide very precise temperature control, sterility, and ease of use. 
         [0007]    It has been found that perfusion of organs is necessary prior to digestion of the organs by methods such as taught by Lacy et al. Perfusion is the flowing of a perfusion liquid through the organ. Perfusion distends the organ to open canals, pockets and spaces within the organ, to allow digestive enzymes to thoroughly contact the cells of interest within the organ during the enzymatic digestion step. Perfusion has been performed manually using a syringe. The perfusion liquid is placed into the syringe, and a catheter connected to the outlet of the syringe is placed into the organ. The operator then operates the syringe to inject perfusion liquid into the organ. The operator usually attempts to maintain a substantially constant fluid pressure during the perfusion process by applying a constant manual pressure to the plunger of the syringe. Due to the imprecise nature of the friction and fluid characteristics of a syringe, the inherent inaccuracies of human sensation, and the variable flow characteristics of the perfusion liquid through the organ, it is difficult to maintain a substantially constant fluid pressure during perfusion. 
       SUMMARY OF THE INVENTION 
       [0008]    A perfusion apparatus has a pump for pumping perfusion liquid though a perfusion liquid conduit. The conduit has an outlet. A pressure sensor is provided for sensing the pressure of the perfusion liquid downstream of the pump and upstream of the outlet. A controller provides for control of the output, such as flow rate, of the pump responsive to the fluid pressure, so as to keep the pressure substantially constant. 
         [0009]    The pump can develop pressures up to 500 mmHg or more depending on the application. The controller has a set point pressure, and controls the pump to maintain the perfusion liquid pressure substantially at the set point pressure. In one embodiment, the controller and the pump maintain the pressure ±5 mmHg from the set point pressure. The set point pressure can be between 80 mmHg and 180 mmHg. 
         [0010]    The controller can have a timer, and at least a second set point pressure. The controller causes the pump to maintain the perfusion liquid pressure at the first set point pressure for a time period, and following the time period causes the pump to maintain the perfusion liquid pressure at the second set point pressure. 
         [0011]    A method for perfusing an organ with a perfusion liquid includes the steps of providing a perfusion liquid conduit, the liquid conduit being connected to a pump; providing a pressure sensor for sensing the pressure of the perfusion liquid downstream of the pump; providing a controller for controlling the operation of the pump responsive to the pressure; positioning an outlet from the conduit in the organ; operating the pump to flow perfusion liquid through the organ; sensing the pressure of the perfusion liquid downstream of the pump, and controlling the operation of the pump responsive to the pressure so as to maintain the pressure of the perfusion liquid downstream of the pump and upstream of the organ substantially constant. 
         [0012]    An automatic fluid heating assembly includes a conductive conduit. A first clamp is provided for engaging the conduit at a first position, and a second clamp is provided for engaging the conduit at a second position. The clamps have respective first and second electrical contacts. A voltage source applies a voltage across the conduit from the first electrical contact and the first position to the second electrical contact and the second position, the voltage causing resistance heating of said conduit. The conductive conduit has an inlet opening and an outlet opening, whereby fluid flowing through the conductive conduit from the inlet opening to the outlet opening will absorb heat from the conduit between the first position and the second position. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0013]    There is shown in the drawings embodiments which are presently preferred, it being understood, however, that the invention can be embodied in other forms without departing from the spirit or essential attributes thereof. 
           [0014]      FIG. 1  is a perspective view of a perfusion apparatus according to the invention. 
           [0015]      FIG. 2  front elevation. 
           [0016]      FIG. 3  is a rear elevation. 
           [0017]      FIG. 4  is a left side elevation. 
           [0018]      FIG. 5  is a right side elevation. 
           [0019]      FIG. 6(A)  is a perspective view of a perfusion tray according to the invention. 
           [0020]      FIG. 6(B)  is a cross section of the perfusion tray of  FIG. 6(A) . 
           [0021]      FIG. 7  is a perspective view partially broken and partially in phantom, of a thermocouple according to the invention. 
           [0022]      FIG. 8  is a front view of a perfusion system according to the invention. 
           [0023]      FIG. 9  is a screen shot of a control display of pressure information. 
           [0024]      FIG. 10  is a screen shot of a control display of temperature information. 
           [0025]      FIG. 11  is a control display of time-programmed perfusion. 
           [0026]      FIG. 12  is a perspective view of a heating coil assembly according to the invention. 
           [0027]      FIG. 13  is a front elevation. 
           [0028]      FIG. 14  is a screen shot of a dual perfusion control display for a computer. 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
       [0029]    There is shown in  FIGS. 1-5  a perfusion apparatus  20  according to the invention. The perfusion apparatus  20  includes at least one pump assembly  24  and at least one heating assembly  28 . At least one pressure sensor is provided for sensing the pressure of liquid downstream of the pump assembly  24 . The pressure sensor provides data to at least one controller, the controller providing a control signal to the pump assembly  24  so as to maintain the pressure at substantially a constant value, which can be a set point value. The controller, or a separate controller, can also be provided to control operation of the heating assembly  28  so as to provide a desired fluid temperature. The operator can interface with the controller through suitable structure such as a touch activated control screen  30 . Computer control from a remote computer is also possible. A second pump assembly  36  can be provided to permit the perfusion of another organ, or another section of the same organ. While a single heating assembly  28  is shown, it is within the scope of the invention to have multiple heating assemblies. 
         [0030]    The pump assemblies  24  and  36  are connected by suitable conduits, such as flexible tubing, to the organ. A catheter can be used to provide infusion of the liquid into the organ. The catheter can be inserted in any suitable location. In the case of the perfusion of a pancreas, the catheter is inserted in the pancreatic duct. 
         [0031]    The organ can be placed on a suitable support which can also collect the perfusion liquid as it flows out of the organ, and preferably recycle this liquid to the pump assemblies  24  and  36 . A suitable perfusion tray  40  is shown in FIGS.  6 (A)-(B). The perfusion tray  40  has a base  44  and side walls  48 . A porous support surface  52  can be provided and rests over the base  44  as on suitable supports provided by the side walls  48 . The porous surface  52  permits perfusion liquid leaving the organ to pass through, where it is collected and directed by an incline surface  56  to an outlet  60 . A thermocouple port  64  can receive a probe  68  of a suitable temperature sensor such as the thermocouple  72  ( FIG. 7 ). The thermocouple input can be a type T thermocouple. The thermocouple  72  can be a disposable Luer lock thermocouple with a mini plug connector. A data lead  76  and plug  80  of the thermocouple  72  can be connected to a suitable input  84  on the perfusion apparatus  20 , such that temperature information is provided to the controller. The perfusion tray  40  can also have ports  90  connecting to one or more cooling channels  94 . Flow of a suitable cooling liquid such as water through the cooling channel  94  will conductively cool the perfusion liquid as it flows over surfaces of the perfusion tray  40 . This will keep the perfusion liquid cold, and any digestive enzyme in the perfusion liquid inactive. 
         [0032]    A typical system configuration is shown in  FIG. 8 . The perfusion apparatus  20  has connected thereto a perfusion liquid supply conduit  100 , which receives liquid from the heating assembly  28 . The supply conduit  100  can be routed through an infrared sensor  122  to sense the temperature of liquid leaving the heating assembly  28 . The supply conduit  100  is connected to the pump  24  and/or pump  36 . The pumps  24  and  36  can be a peristaltic pump, such that connection of the supply conduit  100  can be accomplished by threading the supply conduit  100  into the pump. Other pump designs are possible and the connections should be suited to the pump that is used. A branch  112  of conduit connects the supply conduit  100  to a pressure transducer protector  104  and a pressure input port  108 , and thereby to a pressure transducer that is located within the machine. The pressure transducer protector  104  provides an air reservoir to stabilize the pressure readings. The supply conduit  100  then is connected to the organ  120  such as a pancreas, by suitable structure such as a catheter. The organ can rest on a suitable support such as the perfusion tray  40 . A return conduit  118  is connected between the outlet  60  of the perfusion  40  and the heater assembly  28 . In the case where a second pump  36  is also used, the supply conduit  100  can branch at a Y-connector  124  to a second liquid supply conduit  126 , which can connect to another organ or another portion of the same organ as shown. The second liquid supply conduit  126  can also connect to a branch  112  of conduit which connects the second liquid supply conduit  126  to another pressure transducer protector  104  and another pressure input port  108 , and thereby also to a pressure transducer that is located within the machine. 
         [0033]    The perfusion apparatus can be operated in either a manual or automatic mode. As shown in  FIG. 9 , the touch screen  30  can be operated from either an AUTO or MANUAL mode by pressing the appropriate screen icon. In the MANUAL mode, the pressure is adjusted manually by controlling the flow rate through the pump responsive to pressure readings. In the AUTO mode, a pressure set point “PRESSURE SP” is used to set the desired pressure. The present value pressure “PRESSURE PV” is also indicated. The pump speed is increased by a controller when the PRESSURE PV reading is less than the PRESSURE SP, and the pump speed is decreased when the PRESSURE PV reading is greater than the PRESSURE SP reading. 
         [0034]    Temperature of the perfusion liquid can be controlled using the touch screen  30  display shown in  FIG. 10 . Alternatively, a different physical screen can be used to display the temperature set points at the perfusion tray  40 , the heating assembly  28  and the conduit  100 . Temperature at the perfusion tray  40  as measured by the thermocouple  72  is used to control the heating process. Temperatures at the heating assembly  28 , measured at the heating coil, and conduit  100 , measured by an infrared sensor  122 , are monitored for purposes of safety, for example, where the heater is on but no liquid is flowing these sensors will cause the controller to turn the heating assembly  28  off. An upper limit is determined and input as a set point SP, and should the present value PV exceed this set point, the heating assembly  28  is automatically turned off. Temperatures can also be monitored elsewhere in the system. 
         [0035]    The touch screen display  30  shown in  FIG. 11  is used in recipe mode to create time-varying protocols. Pressure and temperature set points can be entered as a function of time such that, for example, a cold perfusion liquid can be flowed through the organ at a temperature that will not activate a digestive enzyme in the liquid (2.0° C. for the first 300 seconds in  FIG. 11 ), and then the temperature can be raised to subject the organ to the activated enzyme for a given time (37.0° C. for the next 300 seconds in  FIG. 11 , which can be followed by another 60 seconds at 37.0° C. while pressure settings are changed). The recipe mode helps to ensure reproducibility with preset pressure and temperature settings. Different protocols can be created. For example, there could be a protocol for fatty pancreases, or for pancreases or other organs that weigh more than a certain weight, or less than a certain weight. Desired protocols for organs with certain characteristics could be programmed and used again for organs with the same or similar characteristics. As new protocols are discovered, or existing protocols are refined and improved, they can stored in the system and recalled for later use. 
         [0036]    The heating assembly can be of any suitable construction. A heating assembly  28  according to one aspect of the invention is shown in  FIGS. 12-13 . A heating coil  130  is constructed from a conduit having an inlet  134  and an outlet  138  and permits the passage of fluid through the interior of the conduit. The heating coil  130  is constructed from a conductive material. Electrical contacts are provided to cause an electrical current to flow through the conduit, which will cause the temperature of the coil to rise. Heat will be conducted from the coil to the perfusion liquid flowing within, causing the temperature of the perfusion liquid to rise. By controlling the applied voltage to the coil  130 , as by turning the applied voltage on and off, the heating of the coil and the perfusion liquid can be carefully controlled. Other means for controlling the heating of the coil, such as by varying current or applied voltage, can alternatively be used. 
         [0037]    The coil  130  can have any number of turns, or can be without turns. It is only necessary that the coil  130  have sufficient length to impart adequate heating to the perfusion liquid. The conduit  130  can be straight, curved or in any other suitable shape. A coil shape permits a length of coil to be provided in a confined space without angles or corners in the conduit such as might impede fluid flow. The coil characteristics can vary with such characteristics as the nature of the perfusion liquid, inlet liquid temperature and desired outlet temperature, the heat capacity of the liquid, the resistance of the coil material, flow rate of the liquid, the applied voltage, and the current flowing through the coil. The electrical contacts can be in any suitable form which both engage and hold the coil in place, and provide an electrical contact to a suitable voltage source. Suitable fastening structure can be used to adjust the clamping strength and to provide a good electrical contact. A heating coil thermocouple can also be located in at least one of the clamps to measure temperature at this location. 
         [0038]    The conduit  130  can be mounted on any suitable support, such as base  140 . The base  140  can be made from an insulating material so as not to conduct current away from the conduit  130 . Structure for engaging the conduit  130  is provided. The engagement structure can be engagement arms  142 ,  144  which detachably engage the heating conduit  130 . This will permit removal of the conduit  130  for replacement or cleaning. The engagement arms  142 ,  144  can have electrical contacts or can be made from an electrically conductive material such as metal. Fastening structure such as wing head thumbscrews  146  can be provided and securely tightened so there is good contact between the coil and the engagement arms  142 ,  144 . The surface of the metal coil makes contact with a spring-loaded thermocouple  138 . This thermocouple is used to monitor the surface temperature of the coil, which can be used to stop the heating process if the temperature exceeds a set point, such as a do-not-exceed safety set point. A cover  150  is provided. Suitable circuitry can be provided such that the conduit  130  will be OFF if the cover  150  is open and the conduit  130  is exposed. This will prevent accidental burns. 
         [0039]    The conducting material can be any suitable conducting material such as a metal. One such metal is stainless steel. Other conductive materials such as aluminum can be used, or non-conductive materials that are lined with a conductive material or have conductive materials secured thereto or embedded therein. 
         [0040]    A voltage source is connected to the contacts such that a potential difference will be applied to the contacts and across a length of the heating conduit  130 . The voltage that is applied across the conduit  130  can be varied. The applied voltage is generally low, between about 5 VAC and about 24 VAC. The applied voltage will depend operational parameters such as flow rate, inlet temperature, desired outlet temperature, the heat capacity of the fluid, the material resistance and the dimensions of the coil including inside diameter, outside diameter, and length. 
         [0041]    In one embodiment, the elements of the automatic heater are a stainless steel coil (0.150″ ID×0.180″ OD×10 ft), a buck and boost transformer (0.250 VA) (115 VAC in−12 VAC out), a relay (solid state for more accurate control period), and a temperature controller or PLC (programmable logic controller temperature control card). However, the conduit  130  could also be made of aluminum with other dimensions and thus with a different size transformer. 
         [0042]    A low voltage such as 12 VAC that is applied across the two ends of the coil can cause a high current of approximately 25 A to flow through the metal coil, and the temperature of the coil rises. By turning on/off the primary voltage of the transformer, the temperature of the fluid flowing through the coil can be accurately controlled. Since the coil has very little mass, its temperature rises and falls quickly response to heat transfer. A standard temperature controller is capable of turning the relay on/off. 
         [0043]    The heating assembly  28  can keep the temperature of the perfusion liquid constant at any temperature above room temperature. The heating assembly  28  can also be used to control the temperature of other fluids, such as fluid flowing through an organ digestion chamber. Any standard temperature controller can control the conduit  130  to maintain the fluid temperature at or near a desired set point, such as at 32.0° C., the optimal temperature for the enzyme to digest the pancreas during an islet isolation procedure. 
         [0044]    Silicone tubing or other conduit can be connected to the outlet of the heating coil  130 . From the outlet, the tubing can be routed through an infrared thermocouple bracket. This non invasive sensor will shut off the heater in case the temperature of the fluid has reached a maximum allowable limit. The temperature from the thermocouple  72  is used to control the process and the infrared temperature controller acts as a high limit temperature switch. The thermocouple  72  provides feedback to a suitable temperature controller and depending on this temperature, the heating assembly will turn ON and OFF accordingly. Therefore, the infrared sensor set point is set at the maximum allowable temperature. 
         [0045]      FIG. 14  is a screen shot on an interface for controlling the perfusion apparatus from a computer. This screen, suitable for a personal computer (PC), allows full control of the perfusion apparatus, and as shown, for dual perfusion control. Displays are provided for tray temperature  160 , loop A perfusion  162  and loop B perfusion  164 . Displays can also be provided for pressure A  172 , flow rate A  174 , pressure B  176 , and flow rate B  178 . Displays for the selection between auto-manual  180 , enable-disable protocol deviation  184 , and enable-disable report generation  188  can also be provided. Other displays are possible. The computer can be remotely located relative to the perfusion apparatus. Also, the recipe parameters can be inputted into the apparatus through standard spreadsheet programs suitable for PCs. Data acquisition similarly can be accomplished through PC based programming, and plotted, stored or otherwise processed by the PC. Changes in the protocols can be recorded and the results analyzed by PC based programs. 
         [0046]    In practice, the perfusion device  20  is used to automatically pump a fluid at a constant pressure. Typical pumping pressures are in the range of 0-300 mmHg. Flow rates can range from 0-300 ml/min. As the pressure in the organ starts to build up, the machine automatically adjusts the flow rate to maintain a constant pressure SP. The apparatus can be used in conjunction with a disposable perfusion tray  40 . The perfusion tray  40  and tubing set can be separately packed and sterilized. The perfusion tray  40  can be disposable and should then only be used once. 
         [0047]    The apparatus itself can contain two or more pumps, pressure sensors, touch screen, and a programmable logic controller (PLC) so as to simultaneously perfuse different organs or different portions of the same organ. Each pump can be run independently or together in manual or automatic mode. The heater assembly  28  can be manually controlled by pressing an on/off button on the screen. In manual mode, the flow rate is adjusted by turning the dial until the desired flow rate and/or pressure is achieved. In automatic mode, the desired pressure set point on the pressure monitor can be selected by pressing up or down on appropriate keys. The flow rate will automatically adjust itself to maintain a constant pressure. The user can increase or decrease the pressure set point at any time as desired. Flow rate can be controlled by an input on the touchscreen or by other means such as a pop-up keypad. 
         [0048]    The pump assemblies  24  and  36  are started after it is determined that the system is full of fluid. The heating conduit  130  should make good contact with the engagement arms  142 ,  144  and the heater cover  150  should be fully closed. The tubing extending from the outlet of the heating coil should go through the infrared sensor  122 . The thermocouple  72  is connected to the front panel. If the thermocouple  72  is not connected, the heating assembly will not operate. 
         [0049]    The thermocouple  72  is connected to the thermocouple fitting  64  of the perfusion tray  40 . The tubing is connected to the cooling ports  90  and cooling water is recirculated at low pressure. 
         [0050]    Starting from a cool temperature, the heater toggle switch is moved to the ON position. The temperature will now start rising in a controlled fashion and will reach the temperature set by the user on the controllers. Once this temperature is reached, the heater controller will cycle ON and OFF to maintain the desired temperature. There should be no cold air blowing on the chamber, heating coil, or tubing as this can adversely affect the heating time necessary to reach the set point. If for any reason the pump needs to be stopped, the heater should be turned off as well. If this step is not taken, the heating coil temperature will rise and reach 50.0° C. (or whatever set point is set on that thermocouple). This step can be programmed into a PLC to prevent the coil from overheating (if pump is off, then the heater is off). This is why it is important that the heating coil is in contact with the thermocouple. 
         [0051]    Similarly, the pressure set point can be adjusted using the screen of  FIG. 9 . The pressure sensor will provide to the controller a pressure reading PV which will be compared by the controller to the set point SP. The pumps operation will be adjusted, and the pressure will be adjusted accordingly to maintain pressure at or near, for example ±5 mmHg, from the set point pressure. 
         [0052]    This invention can be embodied in other forms without departing from the spirit or essential attributes thereof and, accordingly, reference should be had to the following claims rather than the foregoing specification as indicating the scope of the invention.