Patent Publication Number: US-9423327-B2

Title: Apparatus and method for processing biological material

Description:
This application is a divisional application of U.S. Application Ser. No. 12/326,061, filed Dec. 1, 2008, which is hereby incorporated by reference. 
    
    
     TECHNICAL FIELD 
     The present subject matter generally relates to an apparatus and method for processing biological material to concentrate and wash a biological component in the material. 
     BACKGROUND 
     Biological materials, such as cells, are used in numerous therapeutic, diagnostic and research applications. For example, stem cells may be administered to patients to obtain a desired therapeutic effect such as regeneration of tissue in vivo. In other situations, biological materials including cells may be administered for grafts, transplants, or other procedures. 
     To provide an effective preparation of the biological material, having sufficient concentration that may be administered to a patient or that may be useful for diagnostic and research purposes, it often is necessary to perform numerous and lengthy manipulations involving the material. For example, stem cells often are first separated and isolated from a tissue from which they are derived, such as muscle, blood or adipose (fat) tissue. The cells of such a composition then may have to be subjected to multiple rounds of purification, washing or other treatments before they can be introduced, such as by injection, into a patient. These procedures may require sequential transfer of the cells to different containers. They also may require further manipulations, such as to promote sedimentation. Each procedure preferably is performed aseptically or in a closed sterile system to limit or avoid the potential introduction of contaminating material or organisms into the composition. Alternatively, even if the cells will not be administered to a patient but, instead cultured in vitro, for example, they still may require extensive washing and concentration preferably in aseptic conditions. 
     Also, to be suitable for administration to a patient, it may be preferable for a preparation of biological material to be highly concentrated. This may permit a relatively small volume to be administered. For example, stem cell preparations of about 1×10 8  cells or more generally may be concentrated into a volume of less than five (5) mls for injection into a patient. 
     Although much work has been done in the field of tissue processing, there continues to be a need for advances in the field of processing biological material including in the areas of washing and concentrating material for subsequent therapeutic, diagnostic, research or other applications. 
     SUMMARY 
     In one example, the subject matter of this application is directed to a sedimentation assembly for concentrating cells in a suspension. The sedimentation assembly includes a first chamber for receiving the suspension including a cell population. The first chamber has a cell concentration zone for receiving a concentrated population of the cells upon application of a sedimentation force upon the chamber. The assembly also includes a second chamber that is adapted to be removably placed in fluid communication with a fluid destination or source, including the concentration zone of the first chamber. The first and second chambers as a unit are placeable in a sedimentation force field with the first and second chambers in fluid communication for flowing a portion of the suspension including a cell population into the second chamber. The chambers are preferably physically separable so that fluid communication is effected physically by joining the chambers or broken by physically separating the chambers. 
     In another example, the disclosed subject matter is directed to a sedimentation assembly for washing and concentrating a cell population in a suspension. The sedimentation assembly includes a first chamber for receiving a suspension including a cell population. The sedimentation assembly also includes a second chamber, adapted to be removably placed in fluid communication with a fluid destination or source, including the first chamber. The first and second chambers are placeable as a unit in a sedimentation force field with the first and second chambers in fluid communication, such that when the unit is subjected to the sedimentation force field at least a portion of the suspension flows from the first chamber to the second chamber, thereby forming a concentrated cell suspension in the second chamber. 
     The disclosure also is directed to methods of concentrating cells in a suspension. In one example, a method of concentrating cells in a suspension includes collecting a suspension including a cell population within a first chamber. The cell population is sedimented to obtain a concentrated cell suspension within the first chamber and the concentrated cell suspension is flowed into a second chamber under a sedimentation force field. 
     In a further example, a method of concentrating and washing cells in a suspension is disclosed. The method includes collecting a suspension including a cell population within a first chamber and sedimenting the cell population to obtain a concentrated cell suspension within the first chamber. The concentrated cell suspension is flowed into a second chamber under a sedimentation force field. The second chamber is detached from the first chamber and the concentrated cell suspension is flowed into a further fluid destination or source. The further fluid destination or source is placeable together with the second chamber in a sedimentation force field. 
     In a further example, an apparatus for reconstituting, washing or treating a cell preparation is described. The apparatus has a first chamber with at least one port. The apparatus also includes a second chamber that has at least one port and that is adapted to be repeatedly and removably placed in fluid communication with a fluid destination or source, such as the first chamber. At least one port of the first chamber has a resealable valve and at least one port of the second chamber has a member for opening the valve. 
     A method for reconstituting, washing or treating a cell preparation is also disclosed. The method includes placing a cell preparation within a first chamber and flowing the cell preparation from the first chamber into a second chamber which is adapted to be repeatedly and removably connected to and placed in fluid communication with the first chamber. One of the first and second chambers has a port having an automatically resealable valve and the other of the first and second chambers has a port having a member adapted to automatically open the valve when the chambers are connected. The second chamber is then disconnected from the first chamber and the valve automatically closed. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  is a partial cross-sectional view of one example of a sedimentation assembly according to the disclosure where first and second chambers are shown in a separated position and out of fluid communication; 
         FIG. 1 a    is an enlarged cross-sectional view of one example of a coupling between the first and second chambers of  FIG. 1 , with the chambers shown in a separated position; 
         FIG. 2  is a partial cross-sectional view of the of sedimentation assembly of  FIG. 1  with the first and second chambers shown in a connected position in fluid communication. 
         FIG. 2 a    is an enlarged cross-sectional view of the example of a coupling between the first and second chambers of  FIG. 2 , with the chambers shown in a connected position; 
         FIGS. 3 a -3 f    show one example of a method of using the sedimentation assembly of  FIG. 1  according to the disclosure; 
         FIG. 4  is a perspective view of one example of a holder, holding a modified sedimentation assembly for use in a sedimentation force field, specifically generated by a centrifuge; 
         FIG. 5  shows a further example of a sedimentation assembly with a holder, such as the holder of  FIG. 4 ; 
         FIG. 6  is a cross-sectional view of the example of the holder with the sedimentation assembly of  FIG. 4  located in the holder; 
         FIGS. 7 a -7 g    show an example of a method of using the sedimentation assembly of  FIG. 1  according to the disclosure; 
         FIGS. 8 a -8 h    show an example of a method of use of another sedimentation assembly according to the disclosure, where one chamber includes a plunger; 
         FIG. 9  is a cross-sectional view of a further example of a sedimentation assembly according to the disclosure. 
         FIGS. 10 a - d    are cross-sectional views of further examples of valves and connectors that may be used with an apparatus disclosed herein. 
     
    
    
     DETAILED DESCRIPTION 
     While detailed examples are disclosed herein, it is to be understood that these disclosed examples are merely exemplary, and various aspects and features described herein may have utility alone or in combination with other features or aspects in a manner other than explicitly shown but would be apparent to a person of ordinary skill in the art. 
     The subject matter of the present application is directed generally to an apparatus and method for processing biological material. In one example, the apparatus is a sedimentation assembly that may be used to concentrate biological material. In other preferred examples, the sedimentation assembly may be used to reconstitute, wash and/or otherwise treat the material with desired reagents and solutions. For example, the apparatus may be used to wash or treat cell preparations with selected buffers. In other examples, the apparatus may be used to treat a cell preparation with reagents such as serum, antibodies or growth factors. In further examples, the apparatus may be used to prepare cells for freezing and storage and may be used reconstitute a cell preparation that had been frozen and which may be required to be transferred to culture media. 
     In other preferred examples, the apparatus may be used to reconstitute, wash or otherwise treat a preparation of cells without necessarily sedimenting the cells. For example, the apparatus may be used to transfer a thawed cell preparation to tissue culture media so that the cells may be cultured. 
     Turning to the accompanying drawings,  FIG. 1  illustrates a sedimentation assembly generally at  10  that may be used in concentrating biological material, such as cells, from tissue. The sedimentation assembly includes a first chamber  12  that may receive biological material, such as a suspension of cells. The sedimentation assembly  10  also includes a second chamber  26  that may be placed in fluid communication with the first chamber  12 , for example, as seen in  FIG. 2 . That is, the first chamber  12  and second chamber  26  may be readily coupled together or connected to form a sedimentation assembly  10  as a stable, integrated unit. The chambers  12 ,  26  then may be separated and then reconnected, if necessary, so that fluid communication between the chambers may be repeatedly established, removed and re-established. For example,  FIG. 1  shows the sedimentation assembly  10  with the first and second chambers  12 ,  26  separated—and thus fluid communication has not yet been established or has been removed.  FIG. 2  shows the assembly  10  with the two chambers connected or having been reconnected and placed in fluid communication. As shown in  FIGS. 1 and 2 , a coupling  32  may be used to facilitate the connection, separation and reconnection of the two chambers. 
     In one example, the first chamber  12  is substantially rigid and the second chamber  26  may have the same or different degree of rigidity. The chambers, for example, may be generally be more rigid than bags commonly used in blood processing procedures, but may retain a degree of flexibility. Thus, in some examples, the chambers may be sufficiently pliable such that they may be manipulated by the application of no more than an average manual force. The chambers  12 ,  26  may be formed, at least in part, of substantially rigid transparent plastics such that the contents may be viewed during processing. Of course, the first and second chambers need not necessarily be made of the same materials or have the same degree of rigidity. In one preferred example, at least part of the second chamber  26  may be less rigid than the first chamber  12 , thereby permitting the volume of the second chamber to be manipulated or expelled by the application of force to the wall of the second chamber or by a change in pressure of the chamber. 
     The sedimentation assembly also is preferably disposable, and may be made from polyethylene, polypropylene or other materials that are suitable for use with biological material and that may be easily sterilized before use, or otherwise provided in a sterile form. Although typically not believed to be necessary, the chamber surfaces may be treated or coated with materials such as serum, albumin, polycations, polyanions, or other materials, as desired, using methods known in the art, to increase or decrease the adherence or affinity of selected biological material to the walls of the first and second chambers, or for other purposes. 
     The volumes of the first and second chambers  12 ,  26  may be selected depending on particular requirements. In one example, such as shown in  FIG. 1 , the second chamber  26  has a smaller volume than the first chamber  12 . This example may be used, for example, when the suspension of cells is to be concentrated into a smaller volume for administration to a patient or for further processing. The chambers  12 ,  26  also may assume numerous shapes, as desired. For example, as described further herein, one or both chambers may be in the form of a syringe with a moveable plunger therein. 
     In the example shown in  FIG. 1 , the first chamber  12  has an upper wall portion  14  which is cylindrical. The upper wall portion  14  of the first chamber  12  is closed at an upper end by a wall or base  15  and is joined at a lower end to a conical or tapered portion, forming a concentration zone or area  16  within the first chamber  12 , proximate its lower end. As shown in  FIG. 1 , an inlet tubing  20  may be attached to the first chamber  12  via an aperture  18  in the base  15 . The inlet tubing  20  may be used to introduce biological material including a suspension of cells into the first chamber  12 . The first chamber  12  also has an outlet  22  adjacent the lower end of the concentration zone  16 . The first chamber  12  further includes a vent  24  in the base  15  to permit venting of air as may be required when fluid is being added to or removed from the first chamber  12 . 
     In the example shown in  FIGS. 1 and 2 , the second chamber  26  is shown as having a substantially rigid spherical shape with a port  28  to permit the introduction and/or removal of fluid. Of course, the second chamber  26  may be constructed to be more or less flexible and to have a different shape, as desired. In this example, the second chamber  26  also includes a lower pocket or region  30  opposite the port  28 . The pocket  30  provides a space or zone where cells can accumulate during sedimentation, and may facilitate later removal of a fluid from the second chamber  26  with less disruption to the cells collected in the pocket  30 . Of course, the sedimented cells may be suspended within the second chamber and used directly as a final suspension for a desired purpose such as injection into a patient without further processing. 
     As noted above, in  FIG. 1 , the second chamber  26  is shown as physically separated from the first chamber  12 . Therefore, the second chamber  26  has not yet established or has been removed from fluid communication with the first chamber  12 .  FIG. 2  shows the second chamber  26  as connected to the first chamber  12 , so that the second chamber  26  is placed in fluid communication with the first chamber  12 . 
     As shown in  FIGS. 1 and 2 , a separable coupling  32  may be utilized to facilitate the connection, separation and reconnection of the first chamber  12  and second chamber  26 .  FIGS. 1 a  and 2 a    show cross-sectional, enlarged views of an example coupling  32 .  FIG. 1 a    shows an arrangement of the coupling when the chambers  12 ,  26  are not connected and not in fluid communication with each other.  FIG. 2 a    shows an arrangement when the chambers  12 ,  26  are connected and fluid communication between the chambers may have been established. 
     As shown in  FIGS. 1 a  and 2 a   , the illustrated coupling  32  includes two mating elements. A first mating connector or element  34  of the coupling  32  is shown as being externally threaded at its upper end, and engaged with the first chamber  12  via complementary threads in the outlet  22 . It will be appreciated that the first mating element  34  may be constructed in other ways to engage the first chamber  12  or may be molded with or otherwise connected to the first chamber  12 . The first element  34  shown in  FIGS. 1 a  and 2 a    also includes an outer collar  35  that is internally threaded, a blunt cannula  36 , located within the collar. 
     A second mating connector or element  38  of the coupling  32  may be threaded, molded or otherwise connected to the second chamber  26  at its port  28 . In the example illustrated in  FIG. 1 a   , the second mating element  38  is shown with internal threads at its lower end that engage complementary external threads extending from the port  28  at the top of the second chamber  26 . The second mating element  38  also includes at its upper end an external thread or flange  37  for mating with the internally threaded collar  35  of the first mating element  34 . 
     In this illustrated example, the second mating element  38  of the coupling  32  further includes a flexible pre-slit, re-sealable septum valve  40 . As seen in  FIG. 1 a   , the septum valve  40  is biased towards a closed position. Therefore, the septum valve  40  automatically closes and seals the second chamber  26  from the environment when the first and second chambers  12 ,  26  are separated. As seen in  FIG. 2 a   , the septum valve  40  also automatically seals against the cannula  36  when the chambers  12 ,  26  are connected. 
     The disclosed apparatus is not limited to a particular connector or valve construction shown. For example, the above elements may be otherwise constructed or reversed in their placement, if desired. It also will be appreciated that other examples may include valves on both chambers, as desired. 
     To join the two chambers  12 ,  26  and place them in fluid communication, the first and second mating elements  34 ,  38  of the coupling  32  are connected together. This causes the cannula  36  to pass through the re-sealable septum valve  40 , as indicated in  FIG. 2 a   . In this arrangement, the connector provides a closed passageway or channel  42  in the sedimentation assembly  10  that is sealed from the environment. In this regard, the septum valve is preferably elastically stretched about the penetrating member. In this example with the first and second chambers  12 ,  26  connected as a unit, fluid including cells i.e a cell suspension (or liquid alone), may flow in either direction (first chamber  12  to second chamber  26  or second chamber  26  to first chamber  12 ) depending on the direction and magnitude of forces applied to the sedimentation assembly  10 . To remove the fluid communication between the chambers  12 ,  26 , the cannula  36  is withdrawn from the septum valve  40 , which automatically re-seals instantaneously. 
       FIGS. 3 a -3 f    illustrates generally a method of use of a sedimentation assembly  10 . As shown in  FIGS. 3 a  and 3 b   , the first chamber  12 , which has received a suspension of cells, may be connected to a second chamber  26  and fluid communication between the chambers may be established. A coupling  32  may be used to facilitate the connection of the two chambers, creating a sedimentation assembly  10  in the form of an integrated unit, with the chambers  12 ,  26  rigidly connected together by the coupling  32 , as seen in  FIG. 3   b.    
     The sedimentation assembly  10  may be placed in a sedimentation force field, such as a centrifugal force field, although a simple gravitational force field, i.e. normal gravitational force, may be sufficient to promote sedimentation in certain circumstances. The sedimentation force field, such as developed by centrifugation in  FIG. 3 c   , should be sufficient to cause desired cells of the suspension to become concentrated in the concentration zone  16  of the first chamber  12  and, optionally, to flow from the first chamber  12  to the second chamber  26 . 
     After the second chamber  26  receives a quantity of the desired suspension of cells, the second chamber  26  may be separated from the first chamber  12 , as illustrated in  FIGS. 3 d  and 3 e   . Thus, the sedimentation assembly  10  may be inverted, as shown in  FIG. 3 d   , to reduce potential spillage as the cannula  36  is removed from the septum valve  40 . The second chamber  26  then may be disconnected at the coupling  32  from the first chamber  12 , such as by disengaging the internal threads of the collar  35  from the flange  37  on the second chamber  26 , and withdrawing the cannula  36 . 
     With the second chamber  26  disconnected and separated from the first chamber  12 , as indicated in  FIG. 3 f   , the concentrated suspension of cells may be removed from the second chamber  26  such as by use of a syringe  41 . If desired, the cells also may be maintained in the second chamber  26 , such as for further processing. For example, the separated second chamber  26  with the desired cells may be placed in fluid communication with a further fluid destination or source, such as an additional chamber, for further treatment and concentration, as described below in reference to another example. 
     The example sedimentation assembly  10  may be used to reconstitute, wash, treat or concentrate a diverse set of cell preparations. For example, the biological material received by the first chamber  12  may be a relatively crude suspension of cells and may include individual cells, multi-cellular aggregates and/or cells associated with non-cellular material. The suspension of cells may include one or more cell types. The suspension of cells also may include stem cells alone or in combination with other cell types, including other types of stem cells. 
     The sedimentation assembly  10  also may be used with cell preparations that have been subjected to purification procedures. For example, the sedimentation assembly  10  may be linked, connected to or otherwise incorporated into a system for purifying cells. In such an arrangement, the first chamber  12  of the sedimentation assembly  10  may receive a suspension of cells from the cell purification system. For instance, the suspension of cells received by the first chamber may be stem cells that have been isolated according to the presence or absence of a selected cell marker using affinity techniques. The suspension of cells may have been, for example, isolated as being CD34 positive. 
     As indicated, centrifugation may be used to produce a sedimentation force field to flow a suspension of cells from the first chamber  12  to the second chamber  26 . When centrifugation is used, the sedimentation assembly  10  may be placed in a holder, for convenient further placement of the assembly in a centrifuge. The holder also may assist in stabilizing the assembly during centrifugation. The size and shape of the holder may be adapted to a given sedimentation assembly and centrifuge bucket. Such a holder also may be used to hold a sedimentation assembly for sedimentation at normal gravity force. 
       FIGS. 4-6  show an example of a holder  44  that may be used with a further example of a sedimentation assembly  48 .  FIG. 4  shows the example of a holder  44  that may be used to hold a sedimentation assembly  48  in a centrifuge bucket during centrifugation. The holder includes an opening  46 , best seen in  FIG. 5 , for placement of the sedimentation assembly into the holder  44 . In this example, the overall shape of the holder generally is cylindrical, to fit the most common shape of centrifuge buckets. 
       FIG. 5  shows the placement of the sedimentation assembly  48  into the holder  44  of  FIG. 4 . As shown, the sedimentation assembly includes a first chamber  50  with a concentration zone,  52  a second chamber  54 , and a coupling  56 . In this example, the first chamber  50  includes an inlet  58  for receiving a suspension of cells. The inlet  58  may be covered, for example, with a screw cap  60 . 
     In  FIG. 6 , the sedimentation assembly  48  is shown placed within the holder  44 , shown in cross-section, for use in a sedimenting procedure, as would occur during centrifugation. During the sedimenting procedure, the desired cells, initially in the first chamber  50 , will become concentrated within the concentration zone  52 , and will tend to flow into the second chamber  54 , via the coupling  56 . 
       FIGS. 7 a -7 g    exemplify a use of a sedimentation assembly  61  according to the disclosure for performing multiple washing and/or treating steps of a cell population. The sedimentation assembly  61  includes a first chamber  64  and a second chamber  26 . In  FIG. 7 a   , the second chamber  26  contains a suspension of cells  62  that may require further processing. The suspension of cells in the second chamber  26  may result from processing according to previously described examples for obtaining a concentrated cell population such as is discussed, for example, with respect to use of the first chamber  12  in  FIGS. 3 a   - 3   f.    
     As shown in  FIG. 7 b   , the second chamber  26  with the suspension of cells  62  may be placed in fluid communication with another fluid destination or source, such as an additional first chamber  64  which may contain a washing or treatment solution. The connection of the two chambers may be facilitated by the presence of a coupling, such as previously discussed coupling  32  that allows for repeated coupling (in fluid communication) and uncoupling (not in fluid communication) of the chambers. The cells  62  then may flow into the additional first chamber  64 , with the flow being enhanced simply by applying manual force to a wall of the second chamber  26 , such as by squeezing the second chamber  26  while the sedimentation assembly  61  is in an inverted position. It will be appreciated that a sedimentation force field, such as a centrifugal force field, also may be applied to the inverted sedimentation assembly so as to facilitate the flow of cells from the second chamber  26  to the first chamber  64 . 
     In examples where the cells are to be washed, the suspension of cells may be flowed from the second chamber  26  to an additional first chamber  64  that contains a large volume of a wash solution. In other examples, the cells may be flowed into an additional first chamber containing a relatively small volume of fluid, as might occur when the cells are to be treated with an expensive reagent. After flowing the cells from the second chamber  26  to the additional first chamber  64 , to limit cell loss the second chamber  26  may remain connected with the first chamber  64 , or alternatively may be disconnected from the first chamber  64 . 
     After washing or treatment of the cells within the additional first chamber  64 , the cells may be flowed back into the second chamber  26 , which remains attached to the additional first chamber thereby allowing complete recovery of all the cells or at least reducing cell loss. This may be accomplished using a sedimentation force field, such as shown in  FIG. 7 c   . Alternatively, the additional first chamber  64  may be connected to and placed in fluid communication with a new second chamber. The second chamber  26  then may be separated from the additional first chamber  64 , resulting in a suspension of cells in the second chamber  26  that has been washed and re-concentrated, as seen in  FIG. 7   d.    
     If desired, the washed suspension of cells in the further second chamber  26  then may be flowed to yet another first chamber  68  for further processing, such as by additional washing or treatment. The connection and flowing of the suspension of cells from the second chamber  26  to the additional first chamber  68  is represented in  FIG. 7 e    and is accomplished in a similar manner as with respect to the above description of  FIG. 7 b   . As shown in  FIG. 7 f   , the cells then may be flowed back to the original second chamber  26  or a new second chamber, such as by use of a sedimentation force field. The first and second chambers may remain attached and the use of the same second chamber may reduce cell loss. In this way, a suspension of cells may be repeatedly moved between “first” and “second” chambers that are placed in fluid communication, providing for repeated washing, treatment and/or re-concentration of the cells, shown deposited in the second chamber  26  in  FIG. 7   g.    
       FIGS. 8 a -8 h    shows a further example of a sedimentation assembly  70  and a method of use thereof in accordance with the disclosure. The sedimentation assembly  70  includes a first chamber  72  for receiving a cell suspension and a second chamber  76 , which can be in the form of a syringe. A coupling  78  can be used to place the chambers  72 ,  76  in fluid communication. As described with respect to the other examples, the second chamber  76  may be placed in fluid communication with a first chamber  72 . The sedimentation assembly  70  with the first chamber  72  connected to the second chamber  76  may be placed in a sedimentation force field, such as shown in  FIG. 8 b   , to flow a cell population  74  into the second chamber  76 . 
     The flow of the cell population  74  to the second chamber  76 , in the form of a syringe, also may be facilitated or accomplished by moving a piston  80  of the syringe  76 , so as to create a vacuum in the second chamber  76 , as shown by the displacement of the piston  80  in  FIGS. 8 c  and 8 d   . This movement of the piston  80  causes fluid to be drawn into the second chamber  76  from the first chamber  72  to relieve the vacuum. The volume of the syringe chamber may be configured as fixed or variable, depending on anticipated fluid volume. In one example, retraction of the piston  80  will draw fluid into the second chamber thereby helping to recover cells that remain in the first chamber  72  or in the area of the coupling  78  even after the application of a sedimentation force field. In addition, retraction of the piston may be used to increase the amount of fluid in the second chamber, if desired. The piston  80  of the syringe  76  also may be pushed after the cell population has been flowed into the syringe  76 , thereby removing excess supernatant from the second chamber and adjusting the volume in which the cells are suspended in the second chamber  76 . 
     The second chamber  76  then may be removed from fluid communication with the first chamber  72 , as illustrated in  FIG. 8 e   . Given that the second chamber  76  is in the form of a syringe, the second chamber  76  may be used to administer the cells to a patient or used for other purposes. As indicated in  FIG. 8 f   , the syringe also may be placed in fluid communication with a further fluid destination or source, such as a further first chamber  82 , for further washing or treatment. The cells  74  may be flowed into the further first chamber  82  by movement of the piston  80  of the second chamber syringe  76 , as shown in  FIGS. 8 f  and 8 g   , or by application of a sedimentation force field, such as described above in reference to  FIG. 8 b   . The cells also may be flowed back into the second chamber  76  (or into a further “second” chamber) to result in a concentrated cell population in the second chamber  76 , as shown in  FIG. 8   h.    
     A further example of a sedimentation assembly according to the disclosure is shown in  FIG. 9 . According to this example, one or both chambers of the sedimentation assembly is adapted by the provision of one or more air pockets to more easily allow the trapping of air in the chamber. This feature is beneficial when it is necessary to easily compress the contents of a chamber, such as occurs, for example, when a a structure such as needle or cannula must be introduced into a chamber filled with liquid. 
     The sedimentation assembly  84  shown in  FIG. 9  is substantially similar to the example shown in  FIGS. 1 and 2 . That is, the sedimentation assembly  84  includes a first chamber  86 , a second chamber  88 , and a coupling  90 . The coupling  90  shown in  FIG. 9  is identical to that shown in  FIG. 1 a   . In  FIG. 9 , the wall of the second chamber  88  curves upwards on both sides of inlet port  92 , forming air-trapping pockets or regions  94  within the second chamber  88 . 
     According to the example of  FIG. 9 , air is trapped in the air-trapping regions  94  when the chamber is placed upright and filled with liquid. When a syringe needle or similar device is inserted into the second chamber  88  through, for example, the septum  96 , liquid is forced into the air-trapping regions because the trapped air is compressible, allowing a structure such as needle or cannula to more easily penetrate the chamber. 
     Further, other types of valves and couplings may be used with the sedimentation assembly of the disclosure. Resealable valves are preferred (and particularly preferably automatically resealable) to regulate the flow of fluid between the chambers, either alone or in combination with other valves. For example, stopcock valves as well as clamps are examples of manually resealable elements that may be used. In one example, a syringe-type needle may be used with a rubber plug forming a valve. 
     Other valves and couplings that may be used are disclosed, for example, in U.S. Pat. Nos. 4,683,916, 5,188,620, 5,957,898, 6,039,302 6,261,282 and 6,605,076 which are herein incorporated by reference in their entirety. These valves and others may employ a variety of septums and septum opening mechanisms, and may be employed with various types and shapes of coupling members such as needles, Luer members, cannulas, nozzles and hybrid structures. 
       FIG. 10 a - d    shows examples of such valves and connectors. In  FIG. 10 a   , valve  100  has a resealable pre-slit septum  102  mounted on the first end  104  of a housing  106 . The septum is mounted between annular, U-shaped, swaged end members  108  and an internal septum supporting ridge  110 . As described more fully in U.S. Pat. Nos. 5,188,620 and 6,605,076, this septum is co-operative with a blunt cannula that may be inserted through septum slit  102  for introducing fluid into and through the valve. 
     A further example of a valve connector  200  is shown in  FIG. 10 b   . In this example, a nozzle  202  in the form of a male Luer fitting is shown partially inserted into the valve  200  to establish a fluid flow path. Briefly, the insertion of the nozzle  202  depresses a gland or elastomeric member  204  and axially displaces a hollow internal post  206  to open a fluid flow path through the gland and the hollow post to valve outlet  208 . 
       FIG. 10 c    shows a further example of a valve connector that may be used with an apparatus according to the disclosure. The valve connector  300  includes a resealable valve member  302  having an upper portion  304 , middle portion  306  and annular skirt (not shown). One valve slit  308 , extends downwardly through the upper portion  304  and middle portion  306  into a chamber  310 . Engagement of a cannula against the face of the valve  302  causes the slit  314  to open and provides a fluid flow path through the slit and chamber  310  to the valve outlet. 
       FIG. 10 d    shows one further valve that may be used with the present apparatus. Specifically, the valve body  400  of  FIG. 10 d    includes a male Luer portion  402  and a female Luer portion  404 . A valve disc  406  is located within the valve body and rests on a triangular projection  408 . The inherent resiliency of the valve disc normally biases it in a closed position as shown in solid lines. A valve actuator  410  is located in the female Luer bore, so that the insertion of a connecting male Luer forces the actuator  410  axially to engage and bend the edges of the valve disc  406  downwardly to an open position. The disc reseals upon removal of the connecting male Luer. 
     It will be understood that the examples provided in the present disclosure are illustrative of some of the applications of the principles of the present disclosure. Numerous modifications may be made by those skilled in the art without departing from the true spirit and scope of the disclosure. Various features which are described herein can be used in any combination and are not limited to particular combinations that are specifically described herein.