Abstract:
In one aspect, a disposable centrifuge includes a container including an interior compartment for receiving the liquid and solids. The container is capable of rotating to urge the solids toward the periphery of the interior compartment. A fixed extraction conduit may be provided for extracting at least a portion of the solids from adjacent the periphery of the interior compartment of the container. A motive device may also be provided for forming a non-contact coupling with the container, which may rotate and/or levitate the container. A relatively low, continuous flow rate (about 250 ml/min to about 500 ml/min) may also be utilized. Related methods are also disclosed.

Description:
[0001]    This application claims the benefit of and incorporates by reference U.S. Provisional Patent Application Ser. No. 61/594,077. 
     
    
     TECHNICAL FIELD 
       [0002]    This disclosure relates generally to the fluid handling arts and, more particularly, to systems for separating solids, such as cells, from a liquid, using centrifugal force. 
       BACKGROUND 
       [0003]    The use of centrifugation to separate a solid fraction, such as cells, from a liquid fraction, of a suspension is well known. Typically, the centrifuges used for collecting cells from a bioreactor are not disposable components, and in any case require a halting of the centrifugation process in order to allow for cell recovery. Moreover, existing devices that attempt to achieve such semi-continuous centrifugation invariably require dynamic seals to introduce the cell suspension to and extract the supernatant from the centrifuge. This adds to the cost and complexity, risks breaching sterility, and also potentially results in the generation of heat and particles (which is deleterious in the case of autologous cell seperaration, and in many cases will necessitate a costly and time consuming added filtration step). These existing devices also typically rely on high flow rates and excessive g-forces, which may destroy fragile cells. 
         [0004]    Thus, a need is identified for a manner of providing an improved centrifuge. The centrifuge may at least rotate, and possibly levitate, as well, while the process of solids recovery is completed. Also, the arrangement may be such that the capacity of the separation compartment would be minimized to allow for a high separation efficiency at a relatively low flow rate (e.g., &lt;1 liter/minute, and possibly as low as 0.25-0.5 milliliters/minute), even with the use of dynamic seals. 
       SUMMARY 
       [0005]    According to one aspect of this disclosure, an apparatus for use in performing centrifugation with a liquid including solids is disclosed. In one embodiment, the apparatus comprises a container including an interior compartment for receiving the liquid and solids. The container is capable of rotating to urge the solids toward the periphery of the interior compartment. A fixed extraction conduit is provided for extracting at least a portion of the solids from adjacent the periphery of the interior compartment of the container. A motive device is also provided for forming a non-contact coupling with the container. 
         [0006]    The apparatus may also include a vessel for receiving the container. The vessel may have an inlet for introducing the liquid and solids to the container and a drain for draining at least liquid from the vessel. Any one of the inlet, the extraction conduit, or the drain may comprise a tube connected to a wall of the vessel by a static seal. 
         [0007]    The motive device may rotate the container via the non-contact coupling. The motive device may also be adapted to levitate the container via the non-contact coupling. The motive device may be adapted to levitate and rotate the container via the non-contact coupling. The motive device may comprise a magnet, a superconductor or an electromagnet. 
         [0008]    The container may include a bottom wall and an upstanding sidewall forming an at least partially open top. A lip may be provided adjacent the sidewall for assisting in retaining solids, such as cells, in the interior compartment during rotation. The container may comprise a rigid material, and may carry at least one magnet. 
         [0009]    A further aspect of this disclosure relates to an apparatus for use in performing centrifugation on a liquid including solids. The apparatus comprises a vessel and an open-ended container mounted for rotating within the vessel. The container includes an interior compartment for receiving the liquid and solids. A motive device is also provided for rotating the container by way of a non-contact coupling. 
         [0010]    In one embodiment, the motive device comprises a superconductor connected to a motor, and the container is adapted for forming a magnetic coupling with the superconductor. Alternatively, the motive device may comprise a magnet, and the container is adapted for forming a magnetic coupling with the magnet of the motive device. In the case where the motive device comprises a superconductor for levitating the container, a permanent magnet may be adapted for rotating to rotate the container via a magnetic coupling with the superconductor. A mechanical bearing may support the container for rotation relative to the vessel. 
         [0011]    The container may include a lip along an upper portion for assisting in retaining cells in the interior compartment during rotation. A fixed extraction conduit may also be provided for extracting at least a portion of the solid. The extraction conduit may be adjacent the periphery of the interior compartment of the container. 
         [0012]    Another aspect of this invention is an apparatus for use in performing substantially continuous centrifugation to separate cells from a liquid. The vessel is adapted for receiving the liquid, and a container is mounted for rotating within the vessel. The container includes an interior compartment for receiving the liquid, and the vessel includes an inlet for introducing the liquid to the interior compartment of the container, an extraction conduit from extracting cells from the interior compartment of the container, and a drain for draining at least liquid from the vessel. 
         [0013]    The arrangement may further include a motive device for rotating the container relative to the vessel. A motive device may also be provided for levitating the container relative to the vessel. The extraction conduit may comprise a partially non-linear tube in the container, which may have a substantially open top. 
         [0014]    A further aspect of this disclosure is an apparatus for use in performing substantially continuous centrifugation to separate cells from a liquid. The apparatus comprises a collapsible vessel and a container mounted for rotating within the vessel. The container includes an interior compartment for receiving the liquid. 
         [0015]    In one embodiment, the vessel comprises a flexible bag. A motive device may also be provided for forming a non-contact coupling with the container. In any of the foregoing situations, the liquid flow rate may be from about 250 ml/min to about 500 ml/min. 
         [0016]    A further aspect of the invention is an apparatus for use in performing centrifugation to separate cells from a liquid. The apparatus comprises a container mounted for rotation, the container including a first conduit for conveying the liquid to an interior compartment of the container and a second conduit for conveying liquid from the interior compartment, the first and second conduits each being connected to the container by way of a dynamic seal. A flow rate of the liquid is from about 250 ml/min to about 500 ml/min, and may be through one or more of the first conduit, the second conduit, or the interior compartment of the vessel. 
         [0017]    Yet another aspect of this disclosure relates to a system including a bioreactor and any of the above-described apparatuses. 
         [0018]    A method of centrifugation using a liquid including solids comprises rotating a container including the liquid, and during the rotating step, removing a major portion of the solids from the container. A method of centrifugation also comprises rotating a container including a liquid and cells, and during the rotating step, removing a major portion of the cells from the container. The method may further include the step of levitating the container within a vessel, and the removing step may comprise extracting the solids from adjacent the sidewalls of the container. The method may further include the step of conveying liquid from the container during the rotating step, which may involve overflowing a liquid fraction substantially free of cells from the container. 
         [0019]    Another method of centrifugation comprises rotating a container including a liquid and cells and, during the rotating step, transmitting liquid substantially free of cells from the container. The transmitting step may comprise overflowing the liquid from the container. 
         [0020]    In any of the foregoing methods, the liquid flow rate may be from about 250 ml/min to about 500 ml/min. In any of the foregoing cases, the container may have has a capacity of about 100 ml to about 300 ml, and possibly about 135 ml. 
         [0021]    A further method for performing centrifugation to separate cells from a liquid comprises providing a container mounted for rotation, the container including a first conduit for conveying the liquid to an interior compartment of the container and a second conduit for conveying liquid from the interior compartment, the first and second conduits each being connected to the container by way of a dynamic seal. The method further includes the step of flowing liquid through the container at a rate of about 250 ml/min to about 500 ml/min. 
         [0022]    An apparatus for use in performing centrifugation on a liquid including solids is also disclosed. The apparatus comprises a container including an interior compartment for receiving the liquid and solids, said container being capable of rotating, and a motive device for levitating the container. One of the motive device or the container comprises a magnet. One of the motive device or the container comprises a superconductor. The container may comprise an open-top bowl, and a fixed extraction conduit may extend into the container. 
         [0023]    A further aspect of the disclosure pertains to a centrifuge including a disposable bag for receiving the liquid and solids, and means for separating the liquid from the solids. The separating means may comprise a container for receiving the liquid and solids within the disposable bag, the container being coupled to a motive device (such as a motor for rotating a magnet). 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0024]      FIG. 1  is a schematic diagram illustrating a broad aspect of the disclosure; 
           [0025]      FIG. 2  is a schematic diagram illustrating a specific embodiment of the disclosure; 
           [0026]      FIG. 2   a  is a schematic diagram illustrating another specific embodiment of the disclosure; 
           [0027]      FIG. 3  is a schematic diagram illustrating an embodiment of a system including the disclose centrifuge; 
           [0028]      FIG. 4  is a partially cross-sectional, partially schematic view of yet another specific embodiment of the disclosure; 
           [0029]      FIG. 4   a  is a partially cross-sectional, partially schematic view of still another specific embodiment of the disclosure; 
           [0030]      FIG. 5  is a partially cross-sectional, partially schematic view of a further specific embodiment of the disclosure; and 
           [0031]      FIG. 6  is a schematic view illustrating a further aspect of the disclosure. 
       
    
    
     DETAILED DESCRIPTION 
       [0032]    Reference is now made to  FIG. 1 , which illustrates a centrifugation system  10  according to the basic concepts of the disclosure. This system  10  includes a vessel  12  including an interior compartment for receiving a container  14  capable of moving within the compartment as the result of a non-contact coupling. A motive device  16  external to the vessel  12  provides the forces for achieving the movement (which as discussed herein may be a combination of levitation and rotation), and an inlet I is provided for introducing the suspension to an interior compartment of the container  14 . An outlet O communicates with the container  14  along its periphery to recover the liquid dense with cells as the result of the centrifugal force created when the container  14  is rotated within the vessel  12 . The separated liquid may flow out from the container  14  into the interior compartment of the vessel  12 , and then be discharged through a drain D. A continuously operable and completely closed centrifugation system  10  thus results, without the need for dynamic seals or the like. 
         [0033]    Turning to  FIG. 2 , one particular embodiment of the centrifugation system  10  is shown. The vessel  12  is this embodiment comprises a housing, which may be formed of a rigid material, such as hard plastic or metal. The inlet I may be provided by a tube  12   a  through the upper wall, such as at or near the center, and a similar tube  12   b  mounted closer to the periphery provides the extraction conduit, or outlet O. A third tube  12   c  along the lower portion of the vessel  12  provides the conduit for discharging the media. 
         [0034]    The container  14  may also comprise a rigid or semi-rigid cup or bowl-shaped structure including a bottom wall  14   a  and an upstanding sidewall  14   b  forming an at least partially open top. The bottom wall  14   a  may support or carry one or more magnets  18 , which are arranged to interface with the external motive device  16 . The arrangement may be one that provides the container  14  with levitation and rotation in the absence of a physical bearing or the like. This may be achieved by using a field-cooled superconductor  20  as forming part of the motive device  16 , which when rotated may provide both the levitational and rotational force for the container  14  via the magnetic coupling or pinning with the magnets  18 . The details may be found in one or more of U.S. Pat. Nos. 6,416,215; 6,758,593; 6,837,613; 6,965,288 and 6,899,454, the disclosures of which are incorporated herein by reference. However, it is also possible to form other types of magnetic couplings, such as by using electromagnets or the like, that may achieve the levitation and rotation. Such systems are detailed in, for example, U.S. Pat. No. 5,141,327, the disclosure of which is incorporated herein by reference. 
         [0035]    When the suspension is introduced into the rotating container  14 , the interior compartment receives the liquid. The cells in this liquid are caused to move outwardly as the result of centrifugal force created by the rotation of the container  14 . The extraction conduit formed by tube  12   b  is mounted adjacent to the periphery of the container  14 , such as along the sidewall  14   b.  A pump (not shown) associated with the tube  12   b  may be used to apply a negative pressure and extract cell-rich liquid from the periphery of the container  14 . 
         [0036]    To provide continuous operation, it should be appreciated that the liquid will eventually line the vertical sides of the container  14  and may overflow from the open top. This liquid, which should be generally free of cells, flows into the interior compartment of the surrounding vessel  12 . This liquid may be drained from the vessel  12  through tube  14   c,  and may be discarded or subjected to further processing (such as by recycling it to the inlet I). In one particular embodiment, shown in  FIG. 2   a , the container  14  includes a lip along its upper portion to help contain the cell-laden liquid in the interior compartment. In this embodiment, the tube  14   c  is shown as having a non-linear portion in the interior compartment to assist in recovering the cells that have migrated toward the inner sides of the container  14  as the result of the centrifugal force created by rotation. 
         [0037]    Once processing is complete, the vessel  12  including the container  14  may be discarded. As should be appreciated, this single-use arrangement allows for these combined structures to be made of inexpensive disposable materials, which advantageously eliminates the risk of cross-contamination and cleaning costs. The vessel  12  including the container  14  along with the various connections for conveying fluid may also be provided as part of a cartridge for integrating with a system including other disposable components, such as perhaps a bioreactor or like cell culture device (see  FIG. 3 ). 
         [0038]    While the vessel  12  is described as being rigid or semi-rigid, it could take the form of a flexible bag  112  or the like, as shown in  FIG. 4 . The inlet  112   a,  outlet  112   b,  and drain  112   c  may be provided, as in the embodiment described above. The advantage is that the bag  112  may be folded and stored in a compact fashion prior to use, and then expanded. In this case, a support structure, such as a rigid container C may be provided for helping to ensure that the flexible walls of the bag do not collapse or interfere with the rotation of the container  114 . The bag  112  may also include a rigid portion  112   d  along all or a portion of the bottom thereof, which may further include a retainer (such as a projection or post  112   e ) for receiving and retaining the container  114 , such as by passing through an opening in magnet  118 . The arrangement may be such that the levitation and rotation of the container  114  via the external motive device  116  is not hampered (e.g., there is no direct engagement between the retainer  112   e  and container  114 , yet the structures remain coupled). 
         [0039]    In an alternative embodiment, the container  114  may be arranged to be supported by a physical or mechanical bearing. For example, a roller bearing  120  may be provided between the magnet  118  and the rigid portion  112   d  (or, alternatively, between the matrix material M and the rigid portion  112   d,  or with the magnet  118  or the matrix material M and the retainer  112   e ). The bearing  120  may comprise a race  120   a  for retaining a rolling element, such as a ball  120   b,  roller, or the like. In such case, the motive device  116  need not supply a levitative force, but may instead serve to transmit rotational torque only (and thus may comprise a rotating magnet or like structure forming a non-contact coupling through the vessel  112 ). Examples of such bearing arrangements may be found in U.S. Patent Application Publication No. 20100157752, the disclosure of which is incorporated herein by reference. A removable retaining element  122  may also be provided for retaining the container  114 . 
         [0040]    Another possible embodiment of a centrifuge system  200  is shown in  FIG. 5 . In this system  200 , the container  204  actually includes two magnetic subsystems: a first one that serves to levitate the container  204 , which includes a first magnet  206 , which may be in the form of a ring, and a second magnetic subsystem that includes at least two alternating polarity driven magnets  208   a,    208   b,  which may be positioned inside of the first, ring-shaped magnet  206 , to transmit driving torque. Polarization of the ring magnet  206  is vertical, and the driven magnets  208   a,    2086  are shown as being disk-shaped and having opposite or alternating polarities to form a magnetic coupling and transmit the torque to the levitating container  204 .  204 . Levitation magnet  206  and driven magnets  208   a,    208   b  may be integrated in one rigid structure such as by embedding or attaching all three to a lightweight, inert matrix material M, such as plastic or the like. 
         [0041]    To correspond to the ring-shaped levitation magnet, the motive device includes a superconducting element  210  that is generally annular. This element  210  can be fabricated of a single unitary piece of a high-temperature superconducting material (YBCO or the like), or may be comprised of a plurality of component parts or segments. Upon being cooled to the transition temperature in the presence of a magnetic field and aligning with the ring-shaped permanent magnet  206  producing the same magnetic field, the superconducting ring  210  thus provides the combined repulsive/attractive, spring-like pinning force that levitates the container  204  in the vessel  202  in an exceptionally stable and reliable fashion. In  FIG. 5 , the vessel  202  is shown as being supported on the outer surface of a special cryostat  220  designed for use with this system  200 . However, it is within the broadest aspects of the invention to simply support the vessel  202  on any stable support structure, such as a table (not shown), as long as it remains sufficiently close to the superconducting element  210  to induce the desired levitation in the container  204  therein. 
         [0042]    As in other embodiments described, a motive device is used to impart rotary motion to the container  204 , and may be positioned adjacent to and concentric with the annular superconducting element  210 . One example of a motive device for use in the system  200  of this third embodiment includes driving magnets  212   a,    212   b  that correspond to the driven magnets  208   a,    208   b  on the container  204  and having opposite polarities to create a magnetic coupling. The driving magnets  212   a,    212   b  may be coupled to a shaft  214  also forming part of the motive device. The driving magnets  212   a,    212   b  may be attached directly to the shaft  214 , or as illustrated in  FIG. 2 , may be embedded or attached to a matrix material. By positioning the driving magnets  212   a,    212   b  close to the container  204 , such as by inserting them in the opening  240  or bore defined by the annular superconducting element  210 , and rotating the shaft  214  using a motor  216  also forming a part of the motive device, synchronous rotation of the levitating container  204  is induced. 
         [0043]      FIG. 6  illustrates a centrifuge system  300  including a container  314  supported by a motive device  316  comprising a motor  316   a  and a rotating platform  316   b  for receiving the container  314 . The inlet and outlet for performing the substantially continuous flow of media is provided by conduits in the form of tubes  312   a,    312   b,  which are connected to the container  314  by dynamic seals  322 , and may be connected to a static support structure, such as a cap  324 . The arrangement thus allows for the container  314  to rotate to perform centrifugation. 
         [0044]    The arrangement is this embodiment may be used in connection with specific process parameters to ensure optimum efficiency (e.g., maximum cell separation with minimum destruction). The volume of the container may be between about 100 ml and about 300 ml, and in particular about 135 ml. The corresponding flow may be less than one liter per minute, and may be in the range of about 250 milliliters per minute (0.25 L/min) to about 500 milliliters per minute (0.5 ml/min). 
         [0045]    In the illustrated embodiment, no extraction conduit is located in the same position as the above-described arrangements. Accordingly, the segregated cells L may be collected at the end of the centrifugation process. This recovery may be aided by using a washing step (e.g., using a trypsinisation solution comprising 1.55 L tryposin (an enzyme) to release the cells and 7.45 L of a PBS buffer solution to keep the cells alive) that have accumulated on the container walls. The container  314  may be a single use component (e.g., a disposable bag or liner), and thus may be discarded after cell recovery. 
         [0046]    The foregoing descriptions of several embodiments made according to the disclosure of certain inventive principles are presented for purposes of illustration and description. The embodiments described are not intended to be exhaustive or to limit the invention to the precise form disclosed and, in fact, any combination of the components of the disclosed embodiments is contemplated. The term “flexible” as used herein in the context of the vessel refers to a structure of the vessel that, in the absence of auxiliary support, may conform to the shape of the fluid contained in the vessel, as contrasted with a “rigid” structure, which retains a pre-determined shape when the fluid is in the vessel. Modifications or variations are possible in light of the above teachings. For example, various materials may be used to form the vessel in any combination, including polymers (such as, for example, polypropylene for any flexible portions, and high density polyethylene for any rigid portions). The embodiments described were chosen to provide the best illustration of the principles of the invention and its practical application to thereby enable one of ordinary skill in the art to utilize the invention in various embodiments and with various modifications as are suited to the particular use contemplated. All such modifications and variations are within the scope of the invention when interpreted in accordance with the breadth to which it is fairly, legally, and equitably entitled.