Abstract:
According to various embodiments, a system can be provided to separate undifferentiated cells and/or stromal cells from a whole tissue sample. The whole tissue sample can be any appropriate tissue sample obtained directly from a patient. The tissue sample can be obtained during a selected operating procedure for immediate or quick application or re-application to the patient. Accordingly, autologous cells can be obtained intraoperatively for application to a patient substantially soon after obtaining a whole tissue sample.

Description:
FIELD 
       [0001]    The present disclosure is directed to a method and apparatus for separating biological materials, and particularly to a system for separating a selected fraction from a multiple-component biological material. 
       BACKGROUND 
       [0002]    This section provides background information that-is related to the present disclosure, but that is not necessarily prior art. 
         [0003]    Various cellular (or biological) materials can be used to assist a healing or recovery process in a human patient. Selected cell types, such as stromal cells, pluripotent or multipotent stem cells, or fully differentiated cells, can be applied therapeutically to the patient. For example, stem cells can be applied to an affected area of the patient, such as an area that may be damaged due to injury, chemotherapy, or radiation therapy, to assist in healing the area through differentiation of the stem cells and regeneration of the affected cells. 
         [0004]    In performing a therapeutic procedure on a human patient using undifferentiated cells, such as stem cells or stromal cells, the undifferentiated cells can be obtained from various sources, including the patient&#39;s own anatomy. Accordingly, certain autologous cells can be applied to or injected into various portions of the anatomy of the patient. Generally, a whole tissue or whole blood sample can be obtained from the patient during a first procedure, selected cells can be separated from the whole tissue or blood sample, and the selected, separated cells can be reapplied to or injected into the patient during a subsequent procedure. 
       SUMMARY 
       [0005]    This section provides a general summary of aspects of the invention, and is not a comprehensive description of its full scope or all of its features. 
         [0006]    According to various embodiments, a system can be provided to separate undifferentiated cells, including stem cells and/or stromal cells, from a whole tissue or whole blood sample. The sample can be any appropriate tissue or blood sample obtained directly from a patient. The sample can be obtained during a selected operating procedure with the object of separating undifferentiated cells from the sample for immediate or quick application to the patient. Accordingly, autologous undifferentiated cells can be obtained intra-operatively for application to a patient relatively soon after obtaining a whole tissue or whole blood sample. 
         [0007]    Further areas of applicability will become apparent from the description provided herein. The description and specific examples are intended for purposes of illustration only and are not intended to define the scope of the claimed invention. 
     
    
     
       DRAWINGS 
         [0008]    The drawings described herein are for illustrative purposes only of selected embodiments and not all possible implementations, and are not intended to limit the scope of the present disclosure. 
           [0009]      FIG. 1  is an elevation view of a separating system; 
           [0010]      FIG. 2A  is an exploded perspective view of a buoy system of the separating system of  FIG. 1 ; 
           [0011]      FIG. 2B  is a cross-section view of the buoy system of  FIG. 1  in plane  2 B- 2 B; 
           [0012]      FIG. 3  is an elevation view of a method of filling the separation system of  FIG. 1 ; 
           [0013]      FIG. 4  is a perspective view of a centrifuge system; 
           [0014]      FIG. 5A  is an elevation view of an operation of the buoy system of the separating system of  FIG. 1  shown partially in section; 
           [0015]      FIG. 5B  is a detail view of an operation of the buoy system, shown in section, of the separating system of  FIG. 1 ; 
           [0016]      FIG. 6  is an elevation view of a fractionated whole tissue or whole blood sample in the separation system of  FIG. 1 ; and 
           [0017]      FIG. 7  is an environmental view of an application of a selected fraction of the whole tissue or whole blood sample. 
       
    
    
       [0018]    Corresponding reference numerals indicate corresponding parts throughout the several views of the drawings. 
       DETAILED DESCRIPTION 
       [0019]    Example embodiments will now be described more fully with reference to the accompanying drawings. 
         [0020]    With reference to  FIG. 1 , a cell separating system  20  is illustrated. The cell separating system  20  can include a container  22  of any appropriate configuration, such as a substantially cylindrical container having a cylindrical outer wall  24  and closed by a substantially circular bottom wall  26  and a substantially circular upper wall or removable cap  28 . The container  22  extends along a longitudinal axis A and the top wall  28  and the bottom wall  26  are positioned separate from each other along and substantially perpendicular to the longitudinal axis A. It will be understood, however, that the container  22  can have any appropriate cross-section, such as a polygonal cross-section, a square cross-section, or any other appropriate shape. 
         [0021]    The separating system  20  can include a buoy system  30  positionable within the container  22  and able to move generally axially along the longitudinal axis A relative to the bottom wall  26  and the top wall  28 . Buoy system  30  can be configured to contact, in a desired, selected manner, an inner wall surface  32  of the wall  24 . For example, the buoy system  30  can be configured to frictionally engage the inner wall surface  32  while separating system  20  is in a substantially static state, and yet move axially relatively freely with respect to the container  22  while separating system  20  is being centrifuged, as described further below. According to various embodiments, the container  22  can be formed of a material selected such that wall  24  can flex outwardly and transversely to the longitudinal axis A, under a selected force. For example, as disclosed in U.S. Pat. No. 7,374,678, issued May 20, 2008, incorporated herein by reference, the container  22  can be positioned in a centrifuge and spun so that a large force, generally several times that of gravity, is applied towards the bottom wall  26  of the container  22  so that the wall  24  flexes outwardly and container  22  compresses. In the compressed state, the cylindrical outer wall  24  can expand in diameter such that inner wall surface  32  can move radially away from the buoy system  30 . Consequent clearance between buoy system  30  and inner wall surface  32  allows buoy system  30  to move axially within the container  22  and relative to the inner wall surface  32 . 
         [0022]    The buoy system  30 , as discussed in further detail herein, can include or define a separation or collection volume  40  between an upper buoy portion or member  42  and a lower buoy portion or member  44 . A withdrawal member or tube  46  can interconnect a withdrawal port  48  with an extraction port  50  associated with the buoy system  30 . The withdrawal port  48  can extend through the top wall  28  to an exterior of the container and the extraction port  50  can communicate with and allow access to the collection volume  40  within the buoy  30 . The withdrawal port  48  can include a connector, such as a luer connector  52 , that can be selectively capped or blocked with a cap or plug  54 . 
         [0023]    An introduction port  56  can also extend through the top wall or cap  28  and communicate with an interior volume  57  of container  22 . Introduction port  56  can also include a luer connector or an internal taper wall  58  that can interconnect with an introduction syringe  60 . A cap or plug  62  can be selectively interconnected with the introduction port  56 , after a material is introduced into the container  22 , to cap or block luer connector  58 . Also defined through the top wall or cap  28  can be a second fraction or plasma withdrawal port  64  that can include a luer lock or connector  65  and also be selectively capped or blocked with a cap or plug  66 . The second fraction or plasma withdrawal port  64  can be connected with a port or bore defined through the cap  28  to withdraw or remove a material that is positioned within volume  57  between the buoy system  30  and the cap  28  at any selected time, such as after a separation step formed with the separation system  20 . 
         [0024]    With reference to  FIGS. 2A and 2B , the buoy system  30  can include the extraction port  50 , the upper buoy portion  42  and the lower buoy portion  44 . The upper buoy portion  42  can have legs or arms  70  extending axially from an annular ring or member  72  to contact an upper transverse surface  74  of the lower buoy portion  44 . The legs  70  can extend generally parallel with the longitudinal axis A of the container  22 . The legs  70  can contact the upper surface  74  to help support the annular ring  72  of the upper buoy portion  42 . The legs  70  can be fixed or connected to the upper surface  74  or only contact the upper surface  74  in a non-fixed manner. 
         [0025]    The legs  70  can be formed in any appropriate configuration or number, including two legs as illustrated in  FIGS. 2A and 2B , four legs, any appropriate number of legs, or any other appropriate structure, shape or configuration. For example, a colonnade can be formed near an outer edge of the first buoy portion  42  and the second buoy portion  44  to assist in holding the two portions  42 ,  44  apart. Regardless, the legs  70  can support the first buoy portion  42  at a selected distance from the second buoy portion  44 . In yet another configuration, legs  70  can be omitted. 
         [0026]    The annular member  72  can connect to spokes or extension members  76  extending radially from a central post or tube  78 . The spokes  76  extend generally perpendicular to the axis A. The spokes, however, can extend at an angle relative to the central axis A for reduced drag, etc. The tube  78  defines an internal bore  80  that can communicate with a passage or internal bore  82  of the withdrawal port  50  to permit removal of material from the collection volume  40  of the buoy system  30 . The central tube  78  includes an external wall  84  that extends axially from radial spokes  76  at a center or middle of the annular ring  72  to contact the lower buoy portion  44 . 
         [0027]    A connecting or fusion portion  86  of the central post or tube  78  can extend into and be fixed to the lower buoy portion  44  in an internal well or bore  90 . The connecting portion  86  of the post  78  can be adhered, molded, fused, snap fitted, press fitted or interference fitted with the second buoy portion  44 . The connection portion  86  can hold the first buoy portion  42  to the second buoy portion  44 . Accordingly, the central post  78  can also assist legs  70  in holding the annular ring  72  a distance from the lower buoy portion  44  to define the collection volume  40 . Alternatively, central post  78  can hold the annular ring  72  spaced from lower buoy portion  44  in the absence of legs  70 . 
         [0028]    Fins or raised portions  92  can extend upwardly from the surface  74  of the lower buoy portion  44 . The fins  92  can include a ridge or notched portion  94 . The central post  78  also can be fixed to the fins  92  to assist in fixing or holding the first buoy portion  42  a selected distance from the second buoy portion  44 . 
         [0029]    Lower buoy portion  44  can include upper surface  74 , an annular or exterior wall  102  and a lower surface  45 . The upper surface  74  of the lower buoy portion  44  can be conical and angled or inclined downwardly and inwardly from an outer circumferential edge  100  defined by the intersection or connection of the annular or exterior wall  102  and the upper surface  74 . In an imaginary plane that includes longitudinal axis B of the central post  78  and that intersects the upper surface  74 , axis B and the line of intersection of upper surface  74  subtend an angle a. The angle a can be any appropriate angle such as about 45° to about 90°, preferably about 75°. Alternatively, upper surface  74  can be concavely spherical rather than conical. 
         [0030]    The central portion of the lower buoy portion  44  can define a sump or low portion  103  where a volume of material can be collected, as discussed further herein. The sump  103  can assist in forming a pellet of a selected portion of material, such as a cell fraction. The central post  78 , which defines an axial bore  80 , can also include a through passage that connects bore  80  in communication with an external receiving port  104  defined by outer wall  84  of post  78 . The port  104  can be positioned in or at the bottom of the sump  103  to allow withdrawal of the collected material through the tube  78  and, in turn, through extraction port  50 , withdrawal tube  46 , and the withdraw port  48 . 
         [0031]    Positioned around the central post or tube  78  can be a valve system  110 . The valve system  110  can include a sealing member  112  made of an appropriate material, such as a silicone rubber material. The sealing member  112  can be configured as a disc or washer including a central aperture, such as a round hole  113 , to receive and fit around the central tube  78 . A holding or valve actuation member  116  can include an upper apertured disc or washer  117  that is similar in surface area to the sealing member  112  and a mounting or holding cylinder or tube  118  that extends downwardly substantially perpendicularly from disc or washer  117 . Holding cylinder  118  and washer  117  receive and fit around central tube  78  below sealing member  112 . Holding cylinder  118  can also contact and be supported by the ledges  94  of fins  92  above surface  74 , thereby leaving a circumferential clearance around tube  78  relative to fins  92  and holding cylinder  118  to provide communication among the wedge shaped spaces between fins  92 . Briefly, the sealing member  112  and the disc  117  can form a flapper valve against the first buoy portion  42 . The upper disc  117  of holding member  116  can bend to allow the sealing member  112  to move away from the first buoy portion  42 , as discussed further herein. 
         [0032]    The height of the holding cylinder  118  can allow it to contact the ledge  94  and support the disc  117  at an appropriate elevation to hold the sealing member  112  against a bottom surface  120  of the annular member  72  when no opening force is acting on the valve portion  110 . The spokes  76  can interconnect the annular member  72  with the central tube or post  78  but also form passages  122  (i.e. openings) between the spoke member  76 . The passages  122 , when opened by the valve system  110 , allow material to move from an area above or exterior to the separation buoy system  30  into the collection volume  40 . 
         [0033]    The valve system  110  can be formed as one piece that includes both the sealing member  112  and the valve actuation member  116 . For example, the sealing member  112  can be a flexible material, such as a silicone rubber, spread or applied to the disc  117  of the actuation portion  116 . Alternatively, the sealing member  112  can be a separate piece that s fixed to the valve disc  117  with an adhesive or other connecting mechanism. Further, alternatively, the sealing member  112  can be separate from the valve actuation member  116  and only held in place with the biasing force of the valve actuation member  116 . In other words, the sealing member  112  can be manufactured separate from the actuation member  116  or assembled together as the valve system  110 . 
         [0034]    The valve actuation member  116  can be formed of a material that can flex when a force is applied to it, such as a centrifugal force or pressure differential force, as discussed in detail herein. The material selected for the valve actuation member  116  can include acrylic, polycarbonate, and any other appropriate resilient and substantially inert material The valve actuation member  116  can be formed of a resilient, yet flexible material. Thus, the actuation member  116  can provide a biasing or closing force on the sealing member  112  against the first buoy portion  42 . The valve actuation member  116 , therefore, can rebound from an open position to bias the valve  110  in the closed position. 
         [0035]    The buoy system  30  can be made of any appropriate material and can be made of a material selected according to the biological material to be placed and separated in the separation system  20 . The buoy system  30 , however, can be made of materials having a mean density in the range of about 1.03 g/ml to about 1.10 g/ml, preferably in the range of about 1.045 g/ml to about 1.07 g/ml. The density or specific gravity of the buoy separation system  30  can be selected to position the collection volume  40  at an area or position within the separation container  20  relative to the material positioned in the separation system  20 . The specific gravity of the buoy system  40  can be selected to move to a selected interface of two or more fractions of a separated multiple component material during separation. 
         [0036]    For example, the second buoy portion  44  can be made of a single material or multiple materials and the upper buoy portion  42  can also be made of a single material or multiple materials. The density can be selected by selecting appropriate portions of the materials from which each of the members is made. Selecting the density of the material from which each of the elements of buoy system  30  is made, together with selecting the relative volumes, locations and dimensions of the elements, can assist in achieving a selected placement of the buoy system  30  within the material. The materials can also be selected to be substantially non-reactive to the material being separated in the system  20 . 
         [0037]    With reference to  FIGS. 3-7  a method of using the separation system  20  is illustrated. Initially, a whole tissue sample  140  (e.g. bone marrow aspirate, whole blood, a combination of blood and bone marrow) is positioned within the separation system  20  via introduction from the filling syringe  60 . The buoy system  30  can be positioned near the bottom wall  26  of the separation container  22  during filling. The buoy system  30  can move during a centrifugation stage, as discussed further herein. 
         [0038]    The whole tissue sample  140  positioned in the separation system  20  can be any appropriate whole tissue sample. For example, the whole tissue sample can include whole blood, bone marrow aspirate or a mixture of whole blood and bone marrow aspirate. The whole tissue sample can include that described in U.S. Pat. No. 7,374,678, incorporated herein by reference above. The whole tissue same  140  positioned within the separation system  20 , however, can be separated into selected portions or fractions and selected fractions can be withdrawn from the separation system  20 . 
         [0039]    The separation system  20  can then be positioned in a centrifuge device  150 , as illustrated in  FIG. 4 . The centrifuge device  150  can include a centrifuge member or rotor  152  with separation wells that can hold the separation system  20  and appropriate blanks or other separation systems. The separation centrifuge  150  can include a centrifuge similar to the centrifuge sold by Biomet, Inc. with the GPS® Platelet Separation System. The separation system  20  can be spun in the centrifuge to direct a centrifugal force along the longitudinal axis A of the separation system  20  towards the bottom wall  26 . During centrifugation, the materials of a multi-component material can separate based on specific gravity and density of the components. As understood by one skilled in the art, denser materials will move towards the bottom wall of the separation system  20  and lighter materials move towards the top wall  28 . 
         [0040]    As illustrated in  FIGS. 5A and 5B , during centrifugation, as dense materials move towards the bottom wall  26 , the buoy system  30  can move towards the top wall  28  of the separation system  20 . As the buoy system  30  moves towards the top of the separation container  28 , generally in the direction of arrow X, the centrifugation force and the force of a material pressing on the valve system  110  can bend edges, such as outer or peripheral edges  112   a  and  117   a  of the valve system  110 . When the edges or outer portions  112   a ,  117   a  bend or move away from the under surface  120 , the valve is opened or unsealed. The sealing member  112  unseals from or moves to the open position relative to the annular member  72 , such as the bottom portion  120  of the annular member  72 . As illustrated in  FIG. 5A  and in detail in  5 B, when the edges  112   a ,  117   a  of the valve system  110  are bent, material can be move in the direction of arrow Y through the voids or passages  122  between the spokes  76  and into the collection volume  40 . 
         [0041]    Initially, the valve system  110  can be bent by the centrifugal forces and movement of the whole tissue sample  140  towards the collection volume  40 . Then the valve  110  can remain in the open position, bent, during centrifugation due to the density difference between the denser flexible material of discs  112  and  117  and the less dense biological material, such as plasma. 
         [0042]    Also, as discussed above, the separation container  22  can flex outwardly, allowing the buoy system  30  to move within the container  22  during centrifugation. The buoy system  30 , having the selected density or specific gravity, can move within the separation container  22  to a selected interface of appropriate materials or fractions of the whole sample  140  that is being separated by the centrifugal force. Due to the flex of the container  22  a space can form between the buoy system  30  and the interior wall  32 . Thus, material can also move in the direction of Y′ around the buoys system  30 . The lower buoy portion  44  can include a conical lower surface  45  to assist in movement of the buoy system  30  away from the lower wall  26 . 
         [0043]    As illustrated in  FIG. 6 , once centrifugation has slowed sufficiently, or stopped, the valve system  110  can close with the biasing force of the actuation member  116 , in the absence of the opening or external force (e.g. the centrifugal force). The buoy system  30 , due to its selected density, can allow the collection volume  40  to be positioned at a selected interface within the separated sample  140 . The separated whole tissue sample  140  can include at least a first fraction  140   a  that can be closest to the top wall  28  of the separation container  22  and a second fraction  140   b  that can be within the collection volume  40  of the buoy separation system  30 . The second fraction  140   b  can flow around the buoy system  30  when the container  22  flexes. The whole sample  140  can also be separated to include a third fraction  140   c  that can be closest to the bottom wall  26  of a separation container  22 . 
         [0044]    After the centrifugation has stopped, the valve system  110  can close or seal the collection volume  40  from the first fraction  140   a . Accordingly, during extraction or removal of the second fraction  140   b  within the separation or collection volume  40 , the first fraction  140   a  will not interfere or mix with the second fraction  140   b . Because the valve system  110  can assist in maintaining a physical separation of the selected fraction or second fraction  140   b  from the other fractions, the separated material can be maintained substantially pure and have a high yield. 
         [0045]    Once the separation has concluded, an extraction withdraw syringe or system  160  can be used to withdraw the material or selected fraction  140   b  from the separation volume  40  via the interconnection of the withdrawal port  48  via the tube  46 , the extraction port  50 , the central post or tube  78 , and the withdrawal hole  104 . Once the material is withdrawn from the separation system  20 , a user  170 , such as a surgeon, can apply the material to a patient  180  in any appropriate manner. For example, the extraction withdrawal syringe  160  can be fitted or interconnected with a needle  182  to allow injection of the selected fraction into the patient  180 . The selected fraction  140   b , as discussed above, can include undifferentiated and/or stromal cells that can be applied to the patient  180  for selected purposes, such as tissue re-growth, healing, or other appropriate purposes. 
         [0046]    Thus, the separation system  20  can allow for an introduction of autologous cells to the patient  180 . The whole tissue sample  140  can be withdrawn from the patient  180  with the delivery syringe  60  during a single operative procedure. The centrifuge system  150  can be positioned in an operating room or near an operating room for appropriately timed separation of the whole tissue sample to allow for extraction withdrawal of the selected fraction  140   b.    
         [0047]    The foregoing description of the embodiments has been provided for purposes of illustration and description. It is not intended to be exhaustive or to limit the invention. Individual elements or feature of a particular embodiment are generally not limited to that particular embodiment, but, where applicable, are interchangeable and can be used in a selected embodiment, even if not specifically shown or described. The same may also be varied in many ways. Such variations are not to be regarded as a departure from the invention, and all such modifications are intended to be included within the scope of the invention.