Patent Publication Number: US-11649423-B2

Title: Cell harvesting apparatus

Description:
This invention relates to cell harvesting apparatus, to the improved functioning of such apparatus, and to improvements in the components thereof. 
     BACKGROUND 
     Effective harvesting of cells from various sources is required for different therapeutic applications, such as cell therapy, or tissue engineering. The examples of therapeutic applications include but are not limited to autologous or allogeneic transplantation of stem cells, transplantation of matured functional cells, T cells, modified human cells including T cells, or xenotransplantation of non-human cells. The applications facilitate healing of the damaged tissue or an organ, by regenerating cells to improve the condition of a diseased state. 
     For translational research, which facilitates the development and implementation of scientific discoveries to prevent, diagnose, and treat disease using state-of-the-art technologies, a range of potential cell types require isolation prior to modification, activation, and/or expansion. To meet this translational market need, the cells are first required to be concentrated and washed to remove any impurities. For preserved cell applications, where previously separated mononucleated cells (MNC) are stored in cryogenic temperatures after suspension in media containing preservatives such as dimethylsulfoxide (DMSO), the cells need to be washed, typically through a dilution process, several times to minimize the preservative&#39;s concentration before re-concentrating and re-suspending the cells for use. Therefore, the processing of cryo-preserved cells is necessary before use in any application, specifically for therapeutic application or research purposes. 
     For both of the examples, a suspension of such cells should be processed to concentrate and should be washed extensively to ensure high quality-herein, such concentration optionally including one or more wash cycles is referred to as cell harvesting. Although various methods and systems for harvesting cells are known in the art, the quality and quantity output of these systems are insufficient for therapeutic application. Therefore, systems and methods for harvesting cells under aseptic conditions not necessarily in large scale processing facilities, but with reduced infrastructure requirements and robust operational efficiency, are highly desirable. In additional, equipment which is simple to operate and to maintain is desirable also. 
     BRIEF DESCRIPTION 
     Methods and devices for harvesting cells are described in patent application US2013/0029411, the contents of which are incorporated herein by reference, and result in high quality cell samples, which are devoid of significant residual impurities or preservatives. These methods and devices resolve some of the problems associated with the cells used for translational applications or cells recovered from cryogenic preserved cells. 
     An example of method of harvesting cells from a fluidic material in a processing loop as shown in US2013/0029411 comprises, a processing chamber and a filtering device wherein the fluidic material has a volume and the processing chamber has an overall capacity, comprises circulating the fluidic material through the processing loop and balancing an influx of the fluidic material into the processing chamber with a permeate flux of the filtering device to maintain the volume of the fluidic material in the processing chamber at a constant value, concentrating the cells by increasing the permeate flux of the filtering device relative to the influx of the fluidic material into the processing chamber; and collecting the concentrated cells in a collection chamber. Other examples of the method of harvesting cells from a fluidic material in a processing loop are shown in US2013/0029411. 
     In addition, embodiments of the cell harvesting devices are shown US2013/0029411 comprising, for example, a processing loop comprising a processing chamber and a filtering device; a network of input and output lines operatively coupled to one or more of a source chamber, buffer chamber, waste chamber and collection chamber, and a controller that controls a mass of the processing chamber at a desired value based on an influx and a permeate flux of the processing loop. 
     The inventors have devised improvements to the methods and devices disclosed in US2013/0029411, which have resulted in improved performance and reliability, as well as reduced costs in the consumable parts of the improvements. Embodiments of the invention address the shortcomings of known cell harvesting equipment. The invention is set out in the independent claims herein, with preferred features defined in dependent claims. 
    
    
     
       DRAWINGS 
       These and other features, aspects, and advantages will become better understood when the following detailed description is read with reference to the accompanying drawings in which like characters represent like parts throughout the drawings, wherein: 
         FIG.  1    shows a cell harvesting instrument together with its disposable processing kit; 
         FIG.  2    shows a schematic representation of the disposable processing kit shown in  FIG.  1   ; 
         FIG.  3    shows the processing kit in place in the instrument; 
         FIG.  4    shows a receiving rail mountable in the housing of the instrument of  FIG.  1    for guiding the processing kit into place in the housing; 
         FIG.  5    shows a processing kit receiving frame which is housed within the housing of the instrument shown in  FIG.  1   ; 
         FIGS.  6  to  10    show details of a peristaltic pump mountable in the frame shown in  FIG.  5     
         FIGS.  11  and  12    show details of a pinch valve again mountable to the frame shown in  FIG.  5   ; 
         FIG.  13    shows a pictorial view of a processing reservoir transfer mechanism; and 
         FIGS.  14  to  19    show side views of the transfer mechanism of  FIG.  13   , in different functional positions. 
     
    
    
     DETAILED DESCRIPTION 
     To more clearly and concisely describe and point out the subject matter of the claimed invention, the following definitions are provided for specific terms, which are used in the following description and the appended claims. Throughout the specification, use of specific terms should be considered as non-limiting examples. 
     Referring to  FIG.  1    there is shown a cell harvesting instrument  10 , which in use functions to take in liquids which include suspended cells or similar microbiological material, for the purpose of largely separating the cells from the liquid or reducing the liquid content of the suspension. The instrument can function to wash the cells etc. one or more times to rid the separated cells of unwanted material. A preferred functioning regime can be found in US2013/0029411. 
     The instrument  10  comprises a housing  12  which has a touch screen  14  and a door  16 , shown closed and, in chain dotted lines, shown in an open position  16 ′. The door  16  allows the insertion and removal of a disposable processing kit  100 . The kit  100  is generally flat with a peripheral support frame  105  of thickness x′ in the x direction of around 30-40 mm. In other words, fluid paths  110  within the frame, and additional components of the kit described below, lie substantially in a generally flat, single, plane. The liquid paths  110 , shown in chain dotted lines have, in this case, four inlets/outlets  122 ,  124 ,  126 , and  128 . The fluid paths  110  are mostly constructed from medical grade tubing, for example PVC tubing. Other than those inlets/outlets  122 - 128 , the fluid paths  110  are functional closed circuits, which are sealed, other than at vents which have filters containing sub-micron pore size filters to allow gases to escape, but to prevent ingress of contamination. In particular, mechanical parts contained within the housing  12 , do not contact any fluids in the paths, thereby maintaining sterility of the paths in use. The frame  105  also includes through-apertures  120  and  130  which run from one side of the frame  105  to the other, providing regions where the tubes of the fluid paths which pass across the apertures can be manipulated from both sides of the frame by said mechanical parts. Where the fluid paths cross the apertures, these tubes need to be flexible, and so these tubes are preferably formed from silicon tubing. 
     The kit  100  further includes a tangential flow filter  140 , and a detachable process reservoir  150 , in this case in the form of a moulded plastics container. The processing kit  100  is inserted into and removed from the housing  12  in the direction of arrow y. 
       FIG.  2    is a side view of the processing kit  100  and shows the layout of the fluid paths  110  within the support frame  105 , and its external connections which in practice are made externally of the housing  12  when the kit  100  is inserted into the housing  12  in use. The kit  100  once inserted, is connected to a buffer/wash liquid supply  123 , to a source of suspended cells or similar biological material  125 , to a waste collection  129  and to a harvesting collection chamber  127 , each by means of a respective sterile connector  122 ,  124 ,  128  and  126 . In the alternative, any of the buffer supply  123 , source  125 , waste collection  129 , and harvesting collection chamber  127  can be pre-connected to the fluid paths  110 . In practice, extended respective fluid connection tubing is coiled close to the frame  105  initially, terminating in said buffer supply  123 , source  125 , waste collection  129 , and/or harvesting collection chamber  127 , and the extended tubing is uncoiled to be fed outside of the housing  12  once the kit  100  is inserted into the housing. The through aperture  120  allows a pumping action to be exerted on fluids within the flexible tubular paths  120   a ,  120   b ,  120   c  and  120   d  which cross the aperture. Likewise, the through aperture  130  allows the tubular paths  130   a  and  130   b  that cross that aperture to be pinched to provide a valve action. The processing reservoir  150  acts as fluid holding chamber and is part of the recirculating loop, through which the cell-containing fluid actively recirculates during most of the concentration and washing process performed by the instrument  10 . It is important to determine the total volume/mass of fluids in the whole processing loop, which includes the fluid paths  110 , the filter  140  and the processing reservoir  150 . That total will vary in use because, for example, the amount of waste fluid taken away and the amount of buffer added will alter the total volume. However, since all components except the processing reservoir  150  have a fixed working volume, the variable mass in the processing reservoir  150  is all that needs to be measured to determine the total processing loop volume/mass. Thus, the reservoir  150  includes a hanger  152  which allows its weight to be measured and thereby the total fluid volume/mass can be determined. 
       FIG.  3    shows a part of the frame  105  inserted into the housing  12 . In this instance the frame includes guiding formations for example I the ofrm of pegs, or ribs  112  top and bottom which locate slideably in an open ended groove  23  formed in a top guide rail  22  supported by a rigid device frame  20  within the housing  12 , to slideably support and locate the kit  100 . 
     In  FIG.  4    a bottom guide rail  24  is shown which also includes a groove  25  to accept pegs or a rib (not shown) on the bottom of the frame  105 . The processing kit  100  is loaded into the housing  12 . The bottom guide rail  24  and a top rail ( 22   FIG.  5   ), both have grooves that interface with respective pegs or ribs on the processing kit. The lower peg or rib and groove are wider than the top for two reasons: a) to make it obvious to the user which end is the top and to prevent incorrect insertion of frame  105 , and b) to make it easier to clean the lower rail in the event of a processing kit leak. To aid cleanup, the bottom guide rail  24  has large radii and is dish shaped to catch any leakage. An adjustable roller detent feature (not shown) provides user tactile feedback to alert the user to stop pushing the processing kit into the housing. 
       FIG.  5    shows the device frame  20  in more detail, with the housing  12  removed for clarity. The direction of insertion of the kit  100  is shown by arrow, so the device frame  20  is viewed in this illustration from the rear of the housing  12  shown in  FIG.  1   . The device frame  20  comprises two plates  26  held in spaced relation by spacer fixings  28 . The top and bottom guide rails  22  and  24  run in parallel each mounted to both of the two spaced plates  26 . Also mounted to the plates are a shoe  30  for reacting the forces of a peristaltic pump rotor (described in more detail below) and an anvil to react forces exerted by a pinch valve (described in more detail below). The shoe  30  and anvil  32 , in use align with the through apertures  120  and  130  respectively. 
       FIG.  6    shows the device frame  20 , and pivotably mounted on the frame via a pivot  42 , a pump assembly  40 . The pump assembly in use, with the processing kit inserted into the housing  12  between guide rails  22  and  24 , is pivoted in the direction of arrow R about pump pivot  42 , relative to the stationary frame  20 , to interact with the flexible tubes  120   a,b,c  and d as well as the flexible tubes  130   a  and  b , using the shoe  30  and anvil  32  as reaction faces. Additional alignment is effected by guide pins  48  rigidly mounted to the assembly  40 . The pump assembly  40  interfaces with a processing kit  100  to selectively pump fluid through the fluid paths  110  with, in this instance, a peristaltic action. The assembly  40  includes a 3 state pinch valve to direct the flow appropriately by the use of cams which compress and close the cooperating flexible tubes. The pump and valve, each described in more detail below, are supported on the frame  20  such that operational forces are isolated from the surrounding housing. Disengagement of the pump and valve is effected by pivoting in a direction opposite to arrow R, prior to removal of a used processing kit  100 . 
       FIG.  7    shows the pump assembly in more detail, removed from the frame  20 , and viewed in the direction of arrow A in  FIG.  6   . In this view, four pump heads  44   a ,  44   b ,  44   c  and  44   d  are visible, which interact with the flexible tubes  120   a,b,c  and  d  respectively. The heads are each formed from sets of rollers each mounted for rotation about a roller pin, and each pin mounted for rotation about a pump axis P, thereby forming the head of a peristaltic pump. The four heads share the same pump axis P but can be rotated independently by four different servo type motors  46  acting on drive belts to provide controlled and reversible fluid pressure differentials in the fluid paths  120   a  to  d . The pivoting of the whole pump assembly  40  into a pumping position is effected by an electrical actuator  44  mounted to the assembly  40  and reacting against the frame  20 . During the movement of the pump assembly into an operative position, guide pins  48  cooperate with complementary formations on the processing kit support frame  105 , so that the kit and pump heads are aligned more accurately than relying only on the guide rails  22  and  24 . The pump heads have six generally evenly spaced rotors, which when engaged against a shoe  30  of approximately 70° arc provides at least one roller always in contact with the shoe, thereby preventing reverse fluid flow and fluid flow if the pump is not turning. 
     The pump assembly is shown in yet more detail in  FIG.  8   , where each of the four pump drive motors  46  are visible along with one of the toothed drive belts  47  and tension screws  49 , used to impart tension in the drive belts  47 . The drive belts&#39; pulleys are sized to provide approximately a 2:1 reduction in speed of the motor at the pump head. 
       FIG.  9    shows another view of the pump assembly. In this view the pump head  44   d  is shown. It will be observed that this pump head is wider than the other pump heads in the pump axis direction P. This wider pump head  44   d  allows two or more flexible tubes to be engaged simultaneously, thereby providing increased fluid flow if required. This wider head arrangement allows a processing pump flow rate of up to 3000 mL/min at around 280 rpm motor speed. 
       FIG.  10    shows the pumps heads  44   a,b,c  and  d . As labelled, it can be seen that the four heads function to circulate fluid from the processing reservoir  150 , to the filter  140 , and back to the reservoir or to a collection point  127  (head  44   d  acting on tube  120   d ), to bring in cells in suspension from the source  125  (head  44   c  acting on tube  120   c ), to bring in buffer/wash solution  123  (head  44   b  acting on tube  120   b ) and to remove waste permeate  129  from the filter  130  (head  44   a  acting on tube  120   a ). As mentioned above, from speedier processing more than one tube  120  may be provide for each pump head, thus wider head  44   d  may in other arrangements act on more than one tube  120 . 
       FIG.  11    shows a pinch valve assembly  50  which is mounted underneath the pump motor  464  and pump head  44  and pivots into position ready for operation together with the pump assembly  40 . The pinch valve assembly  50  closes and opens process and collection fluid paths by pinching the tubes  130   a  and  130   b  against the anvil surface  32 . The assembly includes a single linear actuator  55  which includes an electric stepper motor  56 , for rotatably driving a lead screw  58  both clockwise and counterclockwise, which in turn moves a carriage  57  linearly back and forth in the direction of arrow C on a rail  53 . The carriage  57  includes two rollers  54   a  and  54   b , which act on cam profiles  51   a  and  51   b  formed on the back of two spring loaded valve arms  53   a  and  53   b . The arms  53   a  and  53   b  are urged against the respective rollers  54   a  and  54   b . The arms have fingers  52   a  and  52   b , the tips of which press against the tubes  130   a  and  130   b  aligned in the valve&#39;s operative position with the anvil  32 . The cam profiles  51   a  and  51   b  have ‘open’ portions ( 58   a  and  58   b ) which allow fluid flow and ‘closed’ portions ( 59   a  and  59   b ) which prevent substantial flow. Since the fingers are arranged in opposite orientations, the sequence of open and closed positions for the two fingers is:  130   a  closed,  130   b  open (the position shown in  FIG.  11   );  130   a  closed,  130   b  closed (at the mid-position of carriage  57 ); and  130   a  open,  130   b  closed (at the rightmost position of the carriage  57  when viewed in the same direction of view as illustrated in  FIG.  11   ). It will be noted that no power is needed to hold the arms in the open or closed positions, because such positions may need to be maintained for long periods of time during possessing. It should also be noted that an open/open position is deliberately not possible to prevent unwanted fluid flows. 
       FIG.  12    shows a horizontal cross section through the anvil  32 , through the valve arms  53   a  and  53   b  and through carriage rollers  54   a  and  54   b , which in this view are in their mid-position, such that both fingers  52   a  and  52   b  are acting to compress and thereby close flexible tubes  130   a  and  130   b  (shown schematically in this illustration). It will be noted that the starting positions of the tubes is also illustrated. In order that the thickness of the processing kit frame  105  can be accommodated, the fingers  52   a  and  52   b  are initially retracted (along with the pump heads), and are only brought into a position ready to operate by pivoting forward of the pump assembly  40  once the processing kit  100  is in place. Then the fingers operate by opening or closing the tubes according to an operation protocol. The valve assembly  50  can be adjusted initially independently of the position of the pump assembly  40 , so that the correct pinch load can be obtained. 
       FIG.  13    shows a transfer mechanism  60  housed within the housing  12  for transferring the processing reservoir  150  of the processing kit  100  onto a weighing hook  62  so that the volume of liquids in the reservoir can be estimated in use. In practice the mechanism  60  removes the reservoir  150  from the processing kit support frame  105 , transfers it to hook  62 , which is supported by a load cell  61  where it will stay for the duration of a processing run, and then returns the reservoir  150  to the support frame  105 . The processing reservoir  150  is mounted on the support frame  105  as supplied to the user and inserted into the housing in that state. It is reattached to the support frame before the user removes the processing kit from the housing. During a run, the process reservoir and connected tubing will hang freely on the load cell hook to enable mass measurement. 
     The motion of the mechanism  60  is controlled by one stepper motor  64  and a lead screw  66  which directly controls X direction movement of a rear carriage  65 , travelling on a linear rail  68  as the lead screw  66  is rotated by the motor  64 . The rear carriage  66  supports an extension shaft  73  that moves with the carriage  66 . The shaft  73  has a distal end  71  which includes a profiled head  72  ( FIG.  14   ). A front carriage  70  is moveable on the rail  68  also, but is not driven by the lead screw. Rather its movement is controlled by movement of the profiled head  74  and explained in more detail below. 
     The mechanism  60  starts in the position shown in  FIG.  14   , which is a side view in the direction of arrow y in  FIG.  5   . That position allows for insertion of the processing kit  100  into the housing  12 , and brings the hanger  152  of the processing reservoir into an alignment with the mechanism  60 . The hanger  152  includes two resilient arms  154  which sit in supporting apertures in the processing kit frame  105 . In this initial position the hanger arms support the processing reservoir and keep it resiliently in place on the frame  105 . On the hanger  152 , above the arms is a further aperture  156  which accepts the hook  62 . 
     The rear carriage  65  is then driven in the positive X-direction as shown in  FIG.  15   . This movement ultimately pushes the profiled head  72  into a latch arrangement which has a pair of sprung expansion arms  75 . The spring force required to open the expansion arms  75  is such that the expansion arms remain closed and the front carriage  70  is driven forward in the positive X direction also as shown in  FIG.  15   . The front carriage  70  is driven forward in this way until it reaches a hard stop formed by the reservoir clip on the support frame, as shown in  FIG.  16   . The frame  105  cannot move because it is being held in place by the upper and lower guides of the guide rails  22  and  24 . Thus, the rear carriage continues to move forward while the front carriage is stopped, causing the profiled head  72  to force apart the expansion arms  75  apart and into latching cooperating engagement with the resilient arms  154  of the hanger  152 . In this position the expansion arms distort the resilient arms to release their grip on the hanger  152 , and the hook  62  enters the aperture  156 . 
     Next, as shown in  FIG.  17   , the rear carriage is driven by the motor  64  and leadscrew  68  in the negative X direction, thereby detaching the hanger  152  from the frame  105 , and moving the hanger  152 , with the reservoir  150  away from the frame  105 . 
     The front carriage  70  is dragged backwards until it hits a stop. In this position the hanger  152  drops onto the load cell hook  62 . The rear carriage  65  continues moving and the profiled head  72  is pulled out from between the expansion arms  75 , thus returning them to their neutral position shown. At this point the hanger  152  is no longer held in place by the expansion arms and therefore slides down the load cell hook  62 , finally bringing weight to bear on the load cell  61 . The rear carriage  65  is now back to its initial, home position, and no parts of the mechanism, apart from the hook  61  touch the reservoir  150 , or its hanger  152 . 
     Returning the reservoir  150  to the frame  105  is carried out by reversing the steps described above. The front carriage  70  reaches a stop when the hanger  152  is flush against the support frame  105 , with the support frame  105  held in place by the upper and lower guides  22  and  24 . The rear carriage  65  continues to drive forward and pushes the expansion arms apart. This step ensures that the hanger  152  is properly located in the Z-dimension and that the resilient arms  154  are met with no resistance passing through their apertures on the frame  105 . This action is different from the reservoir retrieval described above; the profiled head  72  is driven past the ends of the expansion arms  75 , as shown in  FIG.  18   . 
     In this position, the hanger  152  will be securely reattached to the support frame  105  and the expansion arms  75 , profiled head  72 , and load cell hook  62  can be extracted. The rear carriage  65  drives backwards, dragging the front carriage  70  with it. The front carriage  70  reaches a stop while the rear carriage  65  continues moving backward. This allows the profiled head to be pulled through the expansion arms  75  once again and reset for a new process kit and new processing reservoir, as shown in  FIG.  19   . 
     In operation, the instrument  10  includes mechanical elements including the pump, pinch valve and weighing mechanisms described above, which are reusable, together with a removeable and disposable low cost processing kit  100  which comprises all the fluid elements (e.g. paths  110 , filter  140  and fluid processing reservoir  150 ) necessary for cell harvesting. The combination of these features results in a cell harvesting instrument which is easy to use and can be readied for the next harvesting batch quickly. No mechanical parts come into contact with fluids, which means that cleaning of the mechanical parts between harvesting is not required. The instrument  10  is particularly suitable for concentrating and/or washing human cells, for example for subsequent use in cellular therapeutic applications where the readily achievable aseptic operating conditions of the instrument provide a much improved chance of therapeutic success, as well as reduced costs and turn-around times. 
     While only certain features of the invention have been illustrated and described herein, many modifications and changes will occur to those skilled in the art. It is, therefore, to be understood that the appended claims are intended to cover all such modifications and changes as fall within the scope of the invention. Further, it is intended that combinations of features contained in dependent claims are so combined for convenience, and any one or more of those combined features may be removed, replaced or moved into other claims without introducing new matter.