Abstract:
A biomedical device and method provide for decullularization, recellularization or other treatment of an organ of a human or animal. To keep pressures to a minimum and to ensure that the perfusion fluid uniformly perfused the organ, the organ is supported and rotated during the perfusion process. The organ is supported by a medium, which may comprise a liquid or pallets in a vessel, with a vessel mounted for limited rotation. The perfusion tubing, for supply of perfusion fluid to inform the organ can be mounted both to a support structure and the vessel. The perfusion system can include tubing for supply of air or an air substitute.

Description:
FIELD 
       [0001]    This invention relates to an apparatus for and a method of decellularizing, recellularizing or treating lungs or other organs, from a human or animal body. 
       BACKGROUND 
       [0002]    The following paragraphs are not an admission that anything discussed in them is prior art or part of the knowledge of persons skilled in the art. 
         [0003]    Recent work in the field of tissue engineering has led researchers to believe that it is possible to regenerate organs for use in transplantation. Achieving the goal of organ regeneration would greatly expand the donor pool for transplantation—at present a significant percentage of organs donated for transplantation are rejected due to concerns regarding the organ&#39;s suitability. 
         [0004]    Current state of the art involves harvesting an unsuitable organ. The unsuitable organ is then treated with a detergent solution—the compositions of which have been illuminated in a variety of scientific papers. The detergent removes the cells from the organ, but leaves behind the extra-cellular matrix (ECM). The ECM is made of proteins and glycosaminoglycans. This remnant structure is then used as a building block for regenerating a new organ. The matrix is infused with cells, which are then encouraged to grow around the matrix and re-form the lost organ. By starting with specific cell types that encourage growth, an entirely new organ—free of past defects—can be created. This work has been carried out successfully in small animal models for a variety of organs. 
       SUMMARY 
       [0005]    The invention provides a method to deliver perfusion fluid to an organ undergoing decellularization. A device or apparatus that makes use of this method is also presented. The device intends to achieve complete perfusion in human-scale organs using a combination of hydrostatic pressure and a pre-determined input pressure. The method in question rotates the organ undergoing decellularization such that all sections of the organ are exposed to a maximum pressure. The maximum pressure can be varied by the user and should be low enough to prevent damage to the extracellular matrix of the organ in question, while high enough to achieve adequate perfusion. 
         [0006]    The apparatus and method pertain to the fields of tissue engineering, pulmonary physiology, lung transplant, and organ generation and regeneration. 
         [0007]    The apparatus and method are specifically interested in the decellularization of lungs. In the pulmonary case, lung decellularization and recellularization has been carried out in mouse and rat lungs. Attempts to scale up the concept of decellularization to larger human-scale models have not been published in scientific literature. 
         [0008]    Decellularization of a large organ is not a trivial replication of the small animal case. In an aspect of this specification, a device that allows for complete decellularization of a human-scale lung is presented. 
         [0009]    In accordance with a first aspect of the present invention, there is provided: a biomedical device for use in decellularizing, recellularizing or otherwise treating human or animal organs, the device comprising: 
         [0010]    (a) a vessel to hold the organ being decellularized; 
         [0011]    (b) a support structure that holds the vessel during rotation; and 
         [0012]    (c) a mechanical component that rotates the vessel. 
         [0013]    In accordance with a second aspect of the present invention, there is provided a method of supplying perfusion liquid to an organ, the method comprising: 
         [0014]    (a) providing for a supply of a perfusion liquid to an organ; 
         [0015]    (b) providing for at least limited rotation of the organ; and 
         [0016]    (c) while rotating the organ, supplying perfusion liquid to the organ, to cause uniform perfusion of the liquid in the organ. 
         [0017]    Other aspects and features of the teachings disclosed herein will become apparent, to those ordinarily skilled in the art, upon review of the following description of the specific examples of the specification. 
     
    
     
       DRAWINGS 
         [0018]    The drawings included herewith are for illustrating various examples of articles, methods, and apparatuses of the present specification and are not intended to limit the scope of what is taught in any way. In the drawings: 
           [0019]      FIG. 1  is a perspective view of an apparatus for decellularizing lungs; 
           [0020]      FIGS. 2A ,  2 B,  2 C, and  2 D are perspective, top, side, and front views, respectively, of a vessel used to house the lungs during decellularization; 
           [0021]      FIGS. 3A ,  3 B,  3 C, and  3 D are side, front, top, and perspective views, respectively, of a central axis splitter; 
           [0022]      FIGS. 4A ,  4 B,  4 C, and  4 D are perspective, top, front, and side views, respectively, of a support that holds the central axis splitter and vessel above a surface; 
           [0023]      FIGS. 5A and 5B  are side and front views, respectively, of the vessel showing the tubing entering the vessel; 
           [0024]      FIG. 6  is a side of the apparatus; and 
           [0025]      FIG. 7  is a side view of an alternative embodiment of the apparatus 
       
    
    
     DETAILED DESCRIPTION 
       [0026]    It will be appreciated that for simplicity and clarity of illustration, where considered appropriate, reference numerals may be repeated among the figures to indicate corresponding or analogous elements. In addition, numerous specific details are set forth in order to provide a thorough understanding of the examples embodiments described herein. However, it will be understood by those of ordinary skill in the art that the example embodiments described herein may be practiced without these specific details. In other instances, well-known methods, procedures and components have not been described in detail so as not to obscure the example embodiments described herein. Also, the description is not to be considered as limited to the scope of the example embodiments described herein. 
         [0027]    Reference is now made to  FIG. 1 , in which an example of a device for decellularizing lungs is illustrated generally at  10 . Device  10  comprises a vessel  12  that houses a pair of lungs (not shown) during decellularization, a support structure  14 , a motorized rotation unit  16 , a central axis splitter  32 , and a perfusion circuit (not shown). Motorized rotation unit  16  rotates the vessel  12  using a gear  20  and a chain mechanism  22 . Chain mechanism  22  may be adopted from similar mechanisms used by bicycles. 
         [0028]    Reference is now made to  FIGS. 2A ,  2 B,  2 C, and  2 D in which a vessel that holds the lungs during decellularization is illustrated generally at  12 . Vessel  12  may be made of a transparent material, such as an acrylic plastic, so that the operator can view the lungs for the duration of the procedure. At each end, the vessel  12  is held at three points  26 A,  26 B, and  26 C for stability by central axis splitter  32 , described in  FIGS. 3A ,  3 B,  3 C, and  3 D. The holding or attachment points can be provided by three rods extending between the central axis splitters  32 . 
         [0029]    Vessel  12  comprises a rectangular prism  24  that houses the lung (not shown) during decellularization. Vessel  12  rotates under the influence of motorized rotation unit  16  of  FIG. 1 . The lung is attached to vessel  12  via cannulas (not shown) inserted into the pulmonary artery, pulmonary vein, and trachea of the lung. The cannulas are anchored to rectangular prism  24  as they pass through rectangular prism  24  to connect with the perfusion circuit, located outside of vessel  12 . One of the six faces of rectangular prism  24  functions as a lid  28 , and may be removed to allow for the insertion and removal of lungs. Lid  28  is held closed using conventional compression buckles  30 . 
         [0030]    During decellularization, the vessel  12  is filled with a liquid to support the lung. The liquid preferably has a density generally similar to lung tissue, so the lung is uniformly supported, and this may be saline solution or phosphate buffer solution (PBS). Alternatively, plastic or oil based pellets may be used, to fill the space not occupied by the lung, so as to support the lung. The pellets should be compressible under a load of no more than 20 kilograms. A further alternative is a gel that is formed to the shape and size of the lung. Agarose is a gel that may be suitable. 
         [0031]    Reference is now made to  FIGS. 3A ,  3 B,  3 C, and  3 D in which a central axis splitter is illustrated generally at  32 . Central axis splitter  32  allows vessel  12  (e.g. of  FIGS. 2A ,  2 B,  2 C, and  2 D) to rotate about a central axis  36  of the lung, without piercing the structure of the lung with an axle. Central axis splitter  32  allows the lung to rotate about axis  36  that approximately passes through its geometric centroid creating a hydrostatic advantage described below. 
         [0032]    Reference is now made to  FIGS. 4A ,  4 B,  4 C, and  4 D in which one of two support structures that stand at either end of device  10  is illustrated generally at  14 . Support structure  14  elevates vessel  12  (e.g. of  FIGS. 2A ,  2 B,  2 C, and  2 D) and central axis splitter  32  (e.g. of  FIGS. 3A ,  3 B,  3 C, and  3 D) above a surface such that they may rotate freely without obstruction. Additionally, support structure  14  transmits the weight of all the components of device  10  to the surface, much like the prier of a bridge. Support structure  14  includes a ball bearing  34  that may be commercially sourced for the axle  36 . The larger radius hole  38 , with a diameter of 31.75 [mm], reflects the outer diameter of sourced ball bearing  34 . 
         [0033]    Referring again to  FIG. 1 , motorized rotation unit  16  is connected to vessel  12  via an axle  40  and gearing mechanism  20 . Motorized rotation unit  16  rotates the lung throughout the duration of the decellularization process. The full range of optimal rotation speeds has yet to be determined; a range of 0.5 to 6 rotations per hour (rph) has been found to be effective. Motorized rotation unit  16  may not facilitate a continuous 360 degree rotation. Rather, it may allow vessel  12  to rotate 360 degrees in a clockwise direction, and then prohibit further rotation in the clockwise direction until a 360 rotation in the counterclockwise direction has occurred; other possible limits are 180 degrees and 90 degrees in each direction. This prevents the perfusion tubing from becoming kinked due to excessive twisting, whist still providing a complete rotation for the lung held within the vessel. 
         [0034]    In the embodiments shown, the angular rotation limits may be plus and minus 90 degrees from a central position.  FIGS. 1 and 5  show the vessel  12  at one limit position, i.e. the vessel  12  can be rotated through 90 degrees to a position with the lid  28  vertical, and through a further 90 degrees to the other limit position in which the lid  28  would be on the bottom of the vessel  12 . 
         [0035]    Various timing regimes may be used for the perfusion process or method depending on, for example, the nature and size of the organ subject to perfusion, the process being carried out (which may be other than decellularization), the size of the organ, and the liquid used for perfusion. For example, for a lung a single 15 minute cycle may be sufficient, with the vessel slowly rotated from one limit to the other limit during this period. For other applications, different time periods can be employed, and it may be beneficial to employ multiple cycles of rotating the vessel between the limit positions. It is also anticipated that the vessel and method of the present invention may be used for recellularization. 
         [0036]    The perfusion circuit (not shown) is an adaptation of perfusion circuits currently used in the pulmonary case. The perfusion circuit may be adapted from the XVIVO Lung Perfusion system developed by surgeons at the Toronto General Hospital, in Toronto, Canada. The difference between the perfusion circuit used and the XVIVO Lung Perfusion system being the decellularization perfusion circuit does not make use of a leukocyte filter or a gas exchange unit. 
         [0037]    Reference is now made to  FIGS. 5A and 5B  in which the tubing pathway from the outside of device  10  (external) to the inside of vessel  12  that holds the lungs to be perfused is illustrated generally at  42 . Tubing pathway  42  comprises venous return tubing  44 , pulmonary artery input line  46 , and tracheal return/input line  48 . 
         [0038]    Operational benefits of device  10  will now be discussed in more detail. Device  10  aims to achieve complete perfusion of a human-scale lung whilst using minimal pressure. This results in minimal damage to the extracellular matrix ECM. A more intact the ECM, the better the resulting mass will serve as a scaffold for regenerating organs. 
         [0039]    To achieve complete perfusion—that is, to deliver fluid to as many of small capillaries of the vasculature as possible—a minimum vessel pressure required. (Ideally, the fluid is delivered to every capillary, but it has to be accepted that this is usually not possible.) This pressure differs per lung. Initial experiments indicate the fluid pressure required to achieve complete perfusion is approximately 25.5 [mmHg]. Without rotation the lowest section of the lung will be about 7.5 [cm] below the pulmonary artery—the point at which fluid enters the lung. It should be noted this input channel is determined by the physiology of the lung and cannot be changed. The upper most part of the lung will be a further 7.5 [cm] higher than the pulmonary artery. To achieve complete perfusion at the lowest extreme of the lung, the input pressure should be 20 [mmHg]. This implies the pressure at the uppermost extremities of the lung will be a mere 14.5 [mmHg]. This will not achieve complete perfusion. This problem can be overcome by increasing the input pressure such that the highest extremity of the lung receives a fluid pressure of 25.5 [mmHg]. This will, however, expose the lower extremities of the lung to high pressure of approximately 36.5 [mmHg]. This high pressure may inadvertently damage the ECM of the remaining scaffold. Device  10  negates this issue by rotating the lung. This changes which section of the lung is the lower extremity. This exposes each section of the lung to an input pressure combined with the hydrostatic pressure of the decellularization fluid such that the total fluid pressure is 25.5 [mmHg], as desired. No part of the lung is ever exposed to a higher combined fluid pressure, minimizing damage to the ECM as desired. 
         [0040]    While the figures illustrate a single device that makes use of the principle described above, it is possible to design a device that makes use of the described principle but that does not resemble the device illustrated in the figures. 
         [0041]    A typical dimensions for the device, for use in treatment of lungs can include providing that the access of rotation, i.e. the center access, as located at 325 mm above the support surface. The rectangular prism  24  forming the vessel can have a length and width of approximately 500 mm, and a depth of approximately 200 mm. It will be understood that these dimensions can be varied depending upon the organ to be treated. 
         [0042]    While the above description provides examples of one or more processes or apparatuses, it will be appreciated that other processes or apparatuses may be within the scope of the accompanying claims. 
         [0043]    With reference to  FIG. 6 , this shows details of tubing for connecting perfusion circuits to a lung. In  FIG. 6 , the apparatus is indicated generally at  50 , and shows a vessel  52  having a lid  66 . The vessel  52  has supporting shafts  54  mounted by bearings in support structures  56 . 
         [0044]    Tubing for supplying perfusion fluids is mounted in one of the support structures  56 . The tubing comprises a tracheal tubing  58  for air, or air equivalent, that is mounted in the support structure  56 , and includes a section  58 A that is of sufficient length and flexibility to permit rotational movement between the vessel  52  and the support structure  56 . The tubing  58  also includes a section  58 B within the vessel  52 , again of sufficient length and flexibility to accommodate movement of the lung indicated at  68 . 
         [0045]    A further tubing comprises a pulmonary artery supply tubing  60  for supply of a perfusion fluid, e.g. a surfactant, that is also mounted in the support structure  56 . The tubing  60  includes a section  60 A of sufficient length and flexibility to permit rotational movement between the vessel  52  and the support structure  56 . The tubing  60  extends through the sidewall of the vessel  52  and includes a section  60 B within the vessel, that passes through a bottom of the vessel to a section  60 C extending outside the vessel and then returning through the vessel to a section  60 D extending upwards. This configuration has been adopted since it provides adequate control of the tubing, to avoid kinking and the like, while allowing for the necessary relative motion. The tubing  60 D extends upwards and is connected to the lung by a cannula (not shown). Also not shown, there would be a similar venous return tubing for return of perfusion fluid, following generally the same path as the tubing  60 . 
         [0046]    The configuration shown in  FIG. 6  is functional, but may have a disadvantage that the pulmonary arterial flow flow at the onset of flow into the lung or other organ  70  is a flow upwards. This may results in pushing air bubbles into the vasculature. Put another way, any venous return flow from the vasculature may not adequately remove air bubbles, since these will naturally want to move upwards. The arrangement in  FIG. 6  was adopted, since the lid  66  is provided at the top of the vessel  52  and is required to be removable. 
         [0047]    An alternative configuration is shown in  FIG. 7 , where the entire apparatus is indicated at  70 . The apparatus has a vessel  72  having support shafts  74 , again mounted by bearings in support structures  76 . 
         [0048]    In the embodiment in  FIG. 7 , the vessel  72  has a main vessel body  78 . Rather than providing a distinct and complete lid for the entire top surface of the vessel  72 , the vessel  72  has two separate openings  80  and  84 , each of which can be closed by a respective closure element  82  and  86 . The closure elements  82  and  86  are shown in an open configuration, with their closed configurations indicated by dotted lines in the openings  80  and  84 . 
         [0049]    The perfusion system for the lung or other organ, indicated at  88 , includes a tubing  90 , for supply of air or equivalent. 
         [0050]    As in  FIG. 6 , the tubing  90  is mounted in one of the support structure  76 , and includes a section  90 A, sufficiently long and flexible to enable relative rotation of the vessel  72 , and a section  90 B within the vessel, for connection to the lung  88 . 
         [0051]    For the supply of a perfusion fluid, a tubing  92  and a tubing  94  are provided. Each of these tubings  92 ,  94  includes sections  92 A and  94 A, also providing for rotation of the vessel  72  relative to the support structure  76 . Here, the tubings  92 ,  94  are shown including sections  92 B and  94 B mounted on the exterior of the vessel  72 . It is possible that the sections  92 B,  94 B could equally be mounted within the vessel  72 . 
         [0052]    The tubings  92 ,  94  also includes sections  92 C and  94 C, within the vessel  72  and connected by cannulas (not shown) to the lung or other organ  88 . 
         [0053]    It is anticipated that the advantage of the configuration of  FIG. 7  is that the cannulas for the perfusion fluid are then connected to the lung or other organ  88  from above flow at the onset of flow. Consequently, there will be natural tendency for any pockets of air, air bubbles and the like to flow out from the vasculature of the organ. It will be understood that, in the context of providing a flow of perfusion fluid to an organ, small bubbles of air and the like can serve to completely block off flow of the fluid, and it is usually important to ensure that the perfusion circuit is free of any air bubbles and the like. 
         [0054]    As the tubing  92  and  94  are now mounted to the vessel  72 , the openings  82 ,  84  are provided for access of the vessel, to enable the lung or other organ  88  to be placed in the vessel, and subsequently surrounded by fluid or other support medium, e.g. pellets and like. Once the lung or other organ  88  is in place and connected by cannulas then the closure elements  82  and  84  can be closed. As for the other embodiments, if the support medium is a fluid, then appropriate seals would be provided for the closure elements  82 ,  86 .
       While various embodiments of the invention have been described, the scope of the present invention is determined by the scope of the following claims. In the claims, a number of different features and aspects of the invention are defined and these can be combined together in any practical way having the necessary utility.