Abstract:
A fluid circuit for cell washing is provided that comprises a spinning membrane separator and a fluid management system comprising a cassette that defines the fluid pathways, and including internally mechanical valving, pressure sensing and air sensing for controlling flow through the fluid pathways, thus minimizing the volume of the fluid circuit. Additionally, the fluid circuit comprises syringes that are acted on by syringe pumps associated with the hardware component of the system to provide pressure for moving fluid through the circuit. Preferably, the syringes are connected directly to the cassette, or formed integrally within the cassette housing, thus further minimizing the volume of the fluid circuit.

Description:
TECHNICAL FIELD 
       [0001]    The present disclosure is directed to systems and methods for washing suspensions of biological cells. More particularly, the present disclosure is directed to systems and methods for washing small volumes of biological cells. 
       BACKGROUND 
       [0002]    A number of well-known therapies are currently practiced in which a targeted cellular blood component (e.g., red blood cells, white blood cells, and platelets) is separated from whole blood and stored for later infusion to a patient. The targeted cellular product (e.g., red blood cells or platelets) may be in a suspension that includes plasma and/or some other supernatant. As such, it is sometimes desirable to “wash” the cellular suspension (typically with saline) to remove the plasma/supernatant, as well as any non-target cellular material, prior to reinfusion. 
         [0003]    Systems and methods for cell washing are exemplified by US 2013/0341291, US 2013/0092630, and US 2014/0199680, each of which is incorporated herein by reference. Each of these published applications discloses cell washing methods utilizing systems and fluid circuits including a spinning membrane separator. Such systems include peristaltic pumps and pinch valves that act on tubing to direct flow within the fluid circuit. 
         [0004]    The fluid circuits in the cited published applications have a relatively large internal volume, and thus require relatively large volumes of wash or flush media to clear processed fluid through the fluid circuit. While such systems and fluid circuits are capable of washing and reducing the volume of the targeted cellular component into final volumes of ranging from approximately 50 mL to 5,000 mL, there are instances in which smaller final volumes (e.g., 10 mL) are desired, such as when processing single-dose quantities of mononuclear cell products. Thus, it would be desirable to provide systems and methods for washing small volumes of cellular suspensions. 
       SUMMARY 
       [0005]    In a first aspect of the disclosure, a fluid circuit for cell washing is provided that comprises a spinning membrane separator and a fluid management system comprising a cassette that defines the fluid pathways, and including internal mechanical valving and sensors (for sensing, e.g., pressure, air, fluid interfaces, etc.) for controlling flow through the fluid pathways, thus minimizing the volume of the fluid circuit by minimizing the tubing required. Additionally, the fluid circuit comprises syringes that are acted on by syringe pumps associated with the hardware component of the system to provide pressure for moving fluid through the circuit. Preferably, the syringes are connected directly to the cassette, or the barrels of the syringes may be integrally formed with the cassette, thus further minimizing the volume of the fluid circuit. 
         [0006]    In a second aspect, a disposable kit for washing a suspension of cellular material is provided comprising a spinning membrane separator having an inlet for flowing the suspension of cellular material to be washed and a wash medium into the spinning membrane separator, a first outlet for flowing retentate comprising target components from the spinning membrane separator, and a second outlet for flowing filtrate comprising non-target components of the cellular suspension (including supernatant) and wash medium from the spinning membrane separator. The kit further includes containers for receiving the retentate and the filtrate, and also either includes a container of wash medium integrally connected to the kit or is configured to be connected to a container of wash medium. Alternatively, a sterile vent can replace each of the containers for receiving the retentate and the filtrate. Optionally, the kit may also include either a container of diluent integrally connected to the kit or is configured to be connected to a container of diluent. 
         [0007]    Fluid management of the kit is controlled by a flow control cassette comprising a housing and having a first fluid pathway with a first inlet configured to be in fluid communication with a source of the suspension of cellular material to be washed, a second inlet configured to be in fluid communication with the container of wash medium, and an outlet in fluid communication with the inlet of the spinning membrane separator; a second fluid pathway with an inlet in fluid communication with the first outlet of the spinning membrane separator for flowing retentate, a first outlet in fluid communication with the container for receiving the retentate, and a second outlet in fluid communication with a first syringe; a third fluid pathway with an inlet in fluid communication with the second outlet of the spinning membrane separator for flowing filtrate, a first outlet in fluid communication with the container for receiving the filtrate, and a second outlet in fluid communication with a second syringe; at least one device for selectively occluding the fluid pathways associated with each of the first, second and third fluid pathways; and at least one fluid interface detector associated with each of the first, second and third fluid pathways. Preferably, a device for selectively occluding is associated with each of the first inlet and second inlet of the first fluid pathway, the inlet and first outlet of the second fluid flow pathway, and the inlet and first outlet of the third fluid pathway. Optionally, the second fluid pathway may include a second inlet configured to be in fluid communication with a source of diluent, and a device for selectively occluding is associated with the second inlet. 
         [0008]    In a third aspect, each of the first and second syringe comprises a plunger and a body or barrel having a discharge port, each syringe being removably secured directly to the housing of the cassette by the discharge port. 
         [0009]    In a fourth aspect, a method for washing a suspension of cellular material is provided. The method includes priming various portions of the disposable kit with wash media, loading the spinning membrane separator with a volume of the suspension of cells to be washed, removing the supernatant and non-target materials from the separator, washing the components remaining in the separator, and removing or clearing the washed components from the separator. 
         [0010]    More particularly, the disposable kit may be primed with wash media by withdrawing the plunger of the first syringe while occluding the first fluid pathway adjacent its first inlet, the second fluid pathway adjacent its first outlet, and the third fluid pathway adjacent its inlet to draw wash media into the first fluid pathway; at least partially depressing the plunger of the first syringe while opening the first fluid pathway adjacent its first outlet and occluding the first fluid pathway adjacent its second inlet to prime the first fluid pathway up to the source of the suspension of cellular material to be washed; and further depressing the plunger of the first syringe while opening the second fluid pathway adjacent its first outlet and occluding the first fluid pathway adjacent its inlet to vent air to the container for receiving retentate. 
         [0011]    Alternatively, to further reduce the volume of wash media, the disposable kit may be primed with wash media by drawing wash media from its source only up to the inlet to the first fluid pathway. 
         [0012]    The spinning membrane separator is then loaded with a volume of the suspension of cellular material to be washed by withdrawing the plunger of the first syringe while opening the first fluid pathway adjacent its first inlet and occluding the first fluid pathway adjacent its second inlet, opening the second fluid pathway adjacent its inlet and occluding the second fluid flow path adjacent its first outlet; and occluding the third fluid pathway adjacent its inlet to draw the volume of suspension into the separator; and depressing the plunger of the first syringe while opening the second fluid pathway adjacent its first outlet and occluding the first fluid pathway adjacent its inlet to vent air either to the container for receiving retentate or to the vent filter. 
         [0013]    The volume of the suspension of cells in the separator is then washed by withdrawing the plunger of the second syringe while opening the first fluid pathway adjacent its first inlet and occluding the first fluid pathway adjacent its second inlet, occluding the second fluid pathway adjacent its inlet, and opening the third fluid path way adjacent its inlet and occluding the third fluid flow path adjacent its first outlet to simultaneously draw additional suspension into the separator and supernatant into the second syringe; further withdrawing the plunger of the second syringe while occluding the first fluid pathway adjacent its first inlet and opening the first fluid pathway adjacent its second inlet, occluding the second fluid pathway adjacent its inlet, and occluding the third fluid pathway adjacent its first outlet to draw wash media into and through the spinning membrane separator and into the second syringe. 
         [0014]    The spinning membrane separator is then cleared of washed cells by occluding the third fluid pathway adjacent its inlet and opening the third fluid pathway adjacent its first outlet while depressing the plunger of the second syringe to flow supernatant and wash media into the container for filtrate, and opening the second fluid pathway adjacent its inlet and occluding the second fluid pathway adjacent its first outlet, occluding the first fluid pathway adjacent its first inlet and opening the first fluid pathway adjacent its second inlet while withdrawing the plunger of the first syringe to draw washed cellular matter into the first syringe. 
         [0015]    Washed cellular material may then be flowed from the first syringe to the container for receiving retentate by depressing the plunger of the first syringe while occluding the second fluid pathway adjacent its inlet and opening the second fluid pathway adjacent its first outlet. The steps of loading the spinning membrane separator, washing the volume of cells in the separator, and clearing the spinning membrane of washed cells are repeated until the source of the suspension of cellular material to be washed is emptied. 
         [0016]    Optionally, after the washed cellular material is flowed into the container for receiving retentate, a diluent, such as a cryoprotectant, may be introduced into the collection container for the washed cellular material. 
     
    
     
       DESCRIPTION OF THE DRAWINGS 
         [0017]      FIG. 1  is a perspective view of a system for washing small volumes of cellular suspensions in accordance with the present invention. 
           [0018]      FIG. 2  is a schematic view of a disposable kit for use in the system of  FIG. 1 . 
           [0019]      FIG. 3  is a schematic view of an alternate configuration of the disposable kit of  FIG. 2 . 
           [0020]      FIGS. 4-15  are schematic views of the disposable kit of  FIG. 2  showing the configuration of the kit during the various stages of a cell washing procedure, with  FIGS. 4-6  illustrating the prime phase of the procedure,  FIGS. 7-11  illustrating the steps of the first wash phase, and  FIGS. 12-15  illustrating the steps of a subsequent wash phase. 
           [0021]      FIG. 16  is a schematic view of a second embodiment of a disposable kit for use in the system of  FIG. 1  that permits the addition of a diluent to the washed cells. 
           [0022]      FIGS. 17 and 18  are schematic views of the disposable kit of  FIG. 16  illustrating the steps of adding a diluent to the washed cells. 
       
    
    
     DETAILED DESCRIPTION 
       [0023]    A more detailed description of the systems and methods in accordance with the present disclosure is set forth below. It should be understood that the description below of specific devices and methods is intended to be exemplary, and not exhaustive of all possible variations or applications. Thus, the scope of the disclosure is not intended to be limiting, and should be understood to encompass variations or embodiments that would occur to persons of ordinary skill. 
         [0024]    Turning to  FIG. 1 , there is seen a system  10  for cell washing in accordance with the present disclosure including a reusable hardware component  12  and a disposable kit component  14 , best seen in  FIG. 2 . 
         [0025]    The disposable kit  14  includes a spinning membrane separator  16 , such as is well known in the art, a cassette  18 , for providing fluid management through the kit, and various containers  20 ,  22 ,  24  and  26 , and syringes  28  and  30 , each comprising a body or barrel portion and a plunger, in fluid communication with the cassette, which are described in greater detail below. Tubings interconnect each of the various containers, as well as the inlet and outlets of the spinning membrane separator, to the cassette. Preferably the length of each of the interconnecting tubings is kept as short as possible to further minimize the internal volume of the kit. Also, it is preferable that discharge ports of the syringes be configured to be removably connected directly to the cassette, again to minimize the internal volume of the kit. Alternatively, the syringes and/or the spinning membrane separator may be integrally formed as part of the cassette, so as to be internal to the cassette housing, to further reduce the tubing volume associated with the kit. 
         [0026]    The reusable hardware component  12  includes a drive system/support  32  for the spinning membrane separator  16 , supports  34  for the various containers of the disposable kit, a syringe pump  36 ,  38  for each syringe  28 ,  30 , and a programmable controller  40  for automatically controlling operation of the system. 
         [0027]    Specifically, the disposable kit  14  comprises a spinning membrane separator  16  having an inlet  42  for flowing the suspension of cellular material to be washed and a wash medium into the spinning membrane separator, a first outlet  44  for flowing retentate comprising washed cells from the spinning membrane separator, and a second outlet  46  for flowing filtrate comprising a non-cellular component of the cellular suspension and wash medium from the spinning membrane separator. 
         [0028]    The kit further includes containers  24 ,  26  for receiving the retentate and the filtrate, respectively, and also either includes a container  22  of wash medium integrally connected to the kit at the time of manufacture or is configured to be connected to a container of wash medium at the point of use. Alternatively, with reference to  FIG. 3 , a sterile vent  48 ,  50  can replace each of the containers  24 ,  26  for receiving the retentate and the filtrate. 
         [0029]    Fluid management of the kit is controlled by the cassette  18 . The cassette  18  comprises a housing  52  having a series of fluid pathways therein interconnecting the various other components of the disposable kit, each of the fluid pathways having flow control mechanisms, such as valves/clamps and air detectors/pressure sensors associated therewith that are automatically operated by the controller  40 . By having the valves/clamps, detectors and sensors integral with the cassette, the lengths of the tubings interconnecting the various containers of the system to the cassette can be minimized, thus reducing the internal volume of the kit. 
         [0030]    Specifically, the cassette  18  includes a first fluid pathway  54  with a first inlet  56  configured to be in fluid communication with container  20  of the suspension of cellular material to be washed. The first fluid pathway  54  further includes a second inlet  58  is in fluid communication with the container of wash media  20 , and an outlet  60  in fluid communication with the inlet  42  of the spinning membrane separator  16 . 
         [0031]    The cassette  18  includes a second fluid pathway  62  having an inlet  64  in fluid communication with the first outlet  44  of the spinning membrane separator  16  for flowing the retentate. The second fluid pathway further includes a first outlet  66  in fluid communication with the container  24  for receiving the retentate, and a second outlet  68  in fluid communication with the first syringe  28 . 
         [0032]    A third fluid pathway  70  is provided that includes an inlet  72  in fluid communication with the second outlet  46  of the spinning membrane separator  16  for flowing filtrate. The third fluid pathway  70  further includes a first outlet  74  in fluid communication with the container  26  for receiving the filtrate, and a second outlet  76  in fluid communication with the second syringe  30 . 
         [0033]    Devices for selectively occluding the fluid pathways are associated with each of the first, second and third fluid pathways. Such occluding devices may take the form of valves or clamps. Preferably, a first such valve/clamp  78  is associated with the  56  first inlet of the first fluid pathway  54 , a second valve/clamp  80  is associated with the second inlet  58  of the first fluid pathway  54 , a third valve/clamp  82  is associated with the inlet  64  of the second fluid pathway  62 , a fourth valve/clamp  84  is associated with the first outlet  66  of the second fluid flow pathway  62 , a fifth valve/clamp  86  is associated with the inlet  72  of the third fluid pathway  70 , and a sixth valve/clamp  88  is associated with the first outlet  74  of the third fluid pathway  70 . 
         [0034]    Each of the first, second and third fluid pathways is also provided with a sensor  90 ,  92 ,  94 , respectively, that is able to detect differences in the fluid passing by. Specifically, the sensors  90 ,  92  and  94  are able to detect interfaces between different types of fluids, such as an air-liquid interface, a wash media-retentate interface, and a wash media-filtrate interface. Upon the detection of such interfaces, a signal is sent to the controller that will act to control the configuration of the valves/clamps (open or closed) and actuate the syringe pumps  36 ,  38  to move fluid through the kit in accordance with a cell washing procedure. The cassette  14  may also include a pressure sensor  96  for monitoring purposes. 
         [0035]    A cell washing procedure utilizing the system set forth above will now be described. The procedure includes three relatively distinct phases: a priming phase, as illustrated in  FIGS. 4-6 , during which the kit is primed with wash media, a loading phase, as illustrated in  FIGS. 7 and 8 , in which the annulus of the spinning membrane separator is filled with the cellular suspension that is to be washed, and a wash phase, as illustrated in  FIGS. 9-11 , in which filtrate (supernatant and wash media) and retentate (the washed cells) are drawn through the cassette and flowed to their respective containers. 
         [0036]    Once the disposable kit  14  is loaded onto the hardware component  12 , with a container  20  of the cell suspension to be washed connected to the cassette  18 , the cell washing procedure may commence. As is appreciated, the procedure is automatically controlled by means of the programmable controller  40 , which sequentially operates the valves/clamps and the syringe pumps, in accordance with signals received from the sensors. 
         [0037]    The priming sequence, as illustrated, comprises three steps. In a first step, shown in  FIG. 4 , the first fluid flow path  54  is primed with wash media from the second inlet  58  to the valve/clamp  78  adjacent the first inlet  56  for the source container  20  to the outlet  60  connecting with the inlet  42  of the separator  16 . In this step, the plunger of the first syringe  28  is withdrawn after closing valves/clamps  78 ,  84  and  86  and opening valves/clamps  80  and  82 , thus drawing wash media out of the container  22  into the first fluid pathway  54 . Wash media is drawn through the spinning membrane separator  16  and out the first outlet  44  into the second fluid pathway  62  until the sensor  92  detects an air-fluid interface, at which time the syringe pump is stopped and the plunger of the first syringe  28  no longer withdrawn. Alternatively, withdrawal (and depression) of the plunger can be controlled based on changes in volume within the barrel of the syringe that is correlated to volumes of fluid drawn through the kit. As previously noted, the disposable kit may be primed with wash media by drawing wash media from its source  22  only up to the inlet  58  to the first fluid pathway  54 , to further educe the volume of wash media. 
         [0038]    In a second step of the priming sequence, shown in  FIG. 5 , the plunger of the first syringe  28  is at least partially depressed, after opening valve/clamp  78  and closing valve/clamp  80 , to prime the first fluid pathway  54  to the source container  20 , thus completing the priming of the first fluid pathway. 
         [0039]    In a third step of the priming sequence, shown in  FIG. 6 , the plunger of the first syringe  28  is completely depressed, so that no air remains in the syringe, after closing valves/clamps  78  and  82  and opening valve/clamp  84 , to vent air to the retentate container  24 . While not shown in the drawings, the third fluid flow path  70  may also be primed with wash media by withdrawing the plunger of the second syringe  30  after valves/clamps  78 ,  82  and  88  are closed and valves/clamps  80  and  86  opened, to draw wash media into the third fluid pathway. The air drawn into the second syringe  30  would then be vented into the filtrate container  26  by closing valve/clamp  86  and opening valve/clamp  88  and completely depressing the plunger. 
         [0040]    The system is now ready for loading the annulus of the spinning membrane separator  16  with the suspension of cells to be washed. With reference to  FIG. 7 , this is accomplished by withdrawing the plunger of the first syringe  28  after opening valves/clamps  78  and  82  and closing valve/clamp  84 . This draws cell suspension out of the source container  20  into the first fluid pathway  54  and into the spinning membrane separator  16 . The wash media in the first fluid pathway  54  that resulted from priming is drawn into the second fluid pathway. The withdrawal of the plunger of the first syringe  28  is stopped when the annulus of the separator  16  is filled with cell suspension, and prior to the cell suspension reaching the second fluid pathway, as determined by, e.g., detection of an air-fluid interface by sensor  92 , or upon a change in volume of the barrel of the syringe. The air drawn into the syringe  28  due to loading the separator  16  is then vented to the retentate container  24  by completely depressing the plunger of the first syringe  28  after closing the valve/clamp  82  and opening the valve/clamp  84 , as shown in  FIG. 8 . 
         [0041]    The supernatant is then separated from the cell suspension by the separator  16  and removed. With reference to  FIG. 9 , this is accomplished by withdrawing the plunger of the second syringe  30  after opening valves/clamps  78  and  86 , while valves/clamps  80 ,  82  and  88  remain closed. As such, additional cell suspension is drawn into the separator as the supernatant flows out of the separator through outlet  46 , into the third fluid flow path  70  and into the barrel of the second syringe  30 , while cellular content accumulates in the annulus of the separator. 
         [0042]    Withdrawal of the plunger of the second syringe  30  continues drawing supernatant into the barrel until the cellular content of the annulus of the separator  16  is exceeds the configured volume (based on an empirical determination of the internal volume of the spinner annulus, the rotational velocity of the spinner, the filtrate flow rate). Alternatively, the plunger of the second syringe  30  continues to draw supernatant into the barrel of the second syringe  30  until it is filled with supernatant, or the sensor  90  detects an air fluid interface, indicating that the source container  20  is empty. 
         [0043]    The cells accumulated in the annulus of the separator  16  are then washed. With reference to  FIG. 10 , this is accomplished by further withdrawing the plunger of the second syringe  30  after closing valve/clamp  78  and closing valve/clamp  80 , while valves/clamps  82 ,  84  and  88  remain closed. As such, wash media is drawn into and through the separator  16  into the second syringe  30 . The plunger of the syringe continues to be withdrawn until it is either filled or container  22  is emptied of wash media. 
         [0044]    The cells accumulated in the annulus of the separator  16  are then withdrawn to clear the annulus. With reference to  FIG. 11 , to this end, the plunger of the first syringe  28  is withdrawn after opening valve/clamp  82  and closing valve/clamp  86 , while valves/clamps  78  and  84  are closed, thus drawing the washed cells into the barrel of the first syringe. 
         [0045]    If the source container  20  contains additional cell suspension that is to be washed, the supernatant/wash media contained in the second syringe can be flowed into the filtrate container  26  by depressing the plunger of the second syringe after the valve/clamp  86  is closed and the valve/clamp  88  opened. 
         [0046]    If additional cell suspension is contained in the source container  20 , it can be washed by repeating the steps illustrated in  FIGS. 9-11 , as described above, until the container  20  is depleted. At the completion of each wash cycle, the washed cells contained in the first syringe  28  may be flowed to the retentate container  24  by fully depressing the plunger of the first syringe  28  after opening valve/clamp  84  and closing valve/clamp  82 . 
         [0047]    Alternatively, subsequent wash cycles may be performed as illustrated in  FIGS. 12-15 . Specifically, a second or subsequent volume of cell suspension is pulled from the source container  20  into the annulus of the separator  16  by closing the valves/clamps  80 ,  82  and  88 , opening the valves/clamps  78  and  86 , and withdrawing the plunger of second syringe  30  ( FIG. 12 ). At the same time, the previous cycle&#39;s washed retentate is dumped into the retentate container  24  by opening the valve/clamp  84  and depressing the plunger of the first syringe  28 . 
         [0048]    Then, with reference to  FIG. 13 , the supernatent in the cell suspension is removed by closing the valve/clamp  78  and opening the valve/clamp  80 , so that additional wash media is drawn from the container  22  into the annulus of the spinner  16  by further withdrawing the plunger of the second syringe  30 . 
         [0049]    Then, the annulus of the spinner  16  is cleared by opening the valve/clamp  82  and withdrawing the plunger of the first syringe  28 , thus drawing the retentate into the syringe  28  ( FIG. 14 ). Simultaneously, the filtrate in the second syringe  30  is flowed into the filtrate container  28  by closing the valve/clamp  86  and depressing the plunger of the second syringe  30 . 
         [0050]    The retentate in the first syringe  28  is then flowed to the retentate container  24  by closing the valves/clamps  80 ,  82 , opening the valve/clamp  84 , and depressing the plunger of the first syringe  28 , as shown in  FIG. 15 . The steps illustrated in  FIGS. 12-15  may be repeated until the source container  20  is emptied of cell suspension. 
         [0051]    Under certain circumstances, it may be desirable to dilute the washed cells comprising the retentate, for example if the retentate is to be frozen, in which case a cryoprotective agent would be used to dilute the retentate, To this end, and as illustrated in  FIG. 16 , the cassette  18  may be provided with a further, fourth fluid pathway  100  that provides fluid communication between the first syringe  28  and a container  102  for the diluent. The fluid pathway  100  includes an inlet  104  and a valve/clamp  106  adjacent the inlet  104  for controlling fluid flow through the pathway  100 . 
         [0052]    To add a diluent to the retentate in the container  24 , the valves/clamps  82  and  84  are closed, while the valve/clamp  106  is opened. The plunger of the first syringe  28  is withdrawn to flow diluent out of the container  102  and into the syringe  28  (as shown in  FIG. 17 . Then, the valve/clamp  106  is closed and the valve/clamp  84  opened. The plunger of the first syringe  28  is then depressed to flow diluent into container  24  (as shown in  FIG. 18 ). 
         [0053]    Thus, an improved method and system for washing small volumes of biological cells has been disclosed. The description provided above is intended for illustrative purposes, and is not intended to limit the scope of the disclosure to any particular method, system, apparatus or device described herein.