Patent Description:
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'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.

Methods and devices for harvesting cells are described in patent application <CIT>, 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 <CIT> 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 <CIT>.

In addition, embodiments of the cell harvesting devices are shown <CIT> 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. <CIT> discloses an automated tissue engineering system comprising - a housing with a tissue engineering module removably accommodated within said housing, said tissue engineering module comprising a support structure that holds at least one bioreactor. The system also comprises a fluid containment system in fluid communication with said bioreactor. Filters are used to selectively control the movement of cell suspensions within the system and to limit the passage of cell aggregates during washing and transition stages of the tissue engineering process.

The inventors have devised improvements to the methods and devices disclosed in <CIT>, 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.

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:.

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> there is shown a cell harvesting instrument <NUM>, 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 <CIT>.

The instrument <NUM> comprises a housing <NUM> which has a touch screen <NUM> and a door <NUM>, shown closed and, in chain dotted lines, shown in an open position <NUM>'. The door <NUM> allows the insertion and removal of a disposable processing kit <NUM>. The kit <NUM> is generally flat with a peripheral support frame <NUM> of thickness x' in the x direction of around <NUM>- <NUM>. In other words, fluid paths <NUM> within the frame, and additional components of the kit described below, lie substantially in a generally flat, single, plane. The liquid paths <NUM>, shown in chain dotted lines have, in this case, four inlets/outlets <NUM>, <NUM>, <NUM>, and <NUM>. The fluid paths <NUM> are mostly constructed from medical grade tubing, for example PVC tubing. Other than those inlets/outlets <NUM>-<NUM>, the fluid paths <NUM> 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 <NUM>, do not contact any fluids in the paths, thereby maintaining sterility of the paths in use. The frame <NUM> also includes through-apertures <NUM> and <NUM> which run from one side of the frame <NUM> 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 <NUM> further includes a tangential flow filter <NUM>, and a detachable process reservoir <NUM>, in this case in the form of a moulded plastics container. The processing kit <NUM> is inserted into and removed from the housing <NUM> in the direction of arrow y.

<FIG> is a side view of the processing kit <NUM> and shows the layout of the fluid paths <NUM> within the support frame <NUM>, and its external connections which in practice are made externally of the housing <NUM> when the kit <NUM> is inserted into the housing <NUM> in use. The kit <NUM> once inserted, is connected to a buffer/wash liquid supply <NUM>, to a source of suspended cells or similar biological material <NUM>, to a waste collection <NUM> and to a harvesting collection chamber <NUM>, each by means of a respective sterile connector <NUM>, <NUM>, <NUM> and <NUM>. In the alternative, any of the buffer supply <NUM>, source <NUM>, waste collection <NUM>, and harvesting collection chamber <NUM> can be pre-connected to the fluid paths <NUM>. In practice, extended respective fluid connection tubing is coiled close to the frame <NUM> initially, terminating in said buffer supply <NUM>, source <NUM>, waste collection <NUM>, and/or harvesting collection chamber <NUM>, and the extended tubing is uncoiled to be fed outside of the housing <NUM> once the kit <NUM> is inserted into the housing. The through aperture <NUM> allows a pumping action to be exerted on fluids within the flexible tubular paths 120a, 120b, 120c and 120d which cross the aperture. Likewise, the through aperture <NUM> allows the tubular paths 130a and 130b that cross that aperture to be pinched to provide a valve action. The processing reservoir <NUM> 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 <NUM>. It is important to determine the total volume/mass of fluids in the whole processing loop, which includes the fluid paths110, the filter <NUM> and the processing reservoir <NUM>. 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 <NUM> have a fixed working volume, the variable mass in the processing reservoir <NUM> is all that needs to be measured to determine the total processing loop volume/mass. Thus, the reservoir <NUM> includes a hanger <NUM> which allows its weight to be measured and thereby the total fluid volume/mass can be determined.

<FIG> shows a part of the frame <NUM> inserted into the housing <NUM>. In this instance the frame includes guiding formations for example I the ofrm of pegs , or ribs <NUM> top and bottom which locate slideably in an open ended groove <NUM> formed in a top guide rail <NUM> supported by a rigid device frame <NUM> within the housing <NUM>, to slideably support and locate the kit <NUM>.

In <FIG> a bottom guide rail <NUM> is shown which also includes a groove <NUM> to accept pegs or a rib (not shown) on the bottom of the frame <NUM>. The processing kit <NUM> is loaded into the housing <NUM>. The bottom guide rail <NUM> and a top rail (<NUM> <FIG>), 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 <NUM>, 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 <NUM> 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> shows the device frame <NUM> in more detail, with the housing <NUM> removed for clarity. The direction of insertion of the kit <NUM> is shown by arrow, so the device frame <NUM> is viewed in this illustration from the rear of the housing <NUM> shown in <FIG>. The device frame <NUM> comprises two plates <NUM> held in spaced relation by spacer fixings <NUM>. The top and bottom guide rails <NUM> and <NUM> run in parallel each mounted to both of the two spaced plates <NUM>. Also mounted to the plates are a shoe <NUM> 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 <NUM> and anvil <NUM>, in use align with the through apertures <NUM> and <NUM> respectively.

<FIG> shows the device frame <NUM>, and pivotably mounted on the frame via a pivot <NUM>, a pump assembly <NUM>. The pump assembly in use, with the processing kit inserted into the housing <NUM> between guide rails <NUM> and <NUM>, is pivoted in the direction of arrow R about pump pivot <NUM>, relative to the stationary frame <NUM>, to interact with the flexible tubes 120a,b,c and d as well as the flexible tubes 130a and b, using the shoe <NUM> and anvil <NUM> as reaction faces. Additional alignment is effected by guide pins <NUM> rigidly mounted to the assembly <NUM>. The pump assembly <NUM> interfaces with a processing kit <NUM> to selectively pump fluid through the fluid paths <NUM> with, in this instance, a peristaltic action. The assembly <NUM> includes a <NUM> 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 <NUM> 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 <NUM>.

<FIG> shows the pump assembly in more detail, removed from the frame <NUM>, and viewed in the direction of arrow A in <FIG>. In this view, four pump heads 44a, 44b, 44c and 44d are visible, which interact with the flexible tubes 120a,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 <NUM> acting on drive belts to provide controlled and reversible fluid pressure differentials in the fluid paths 120a to d. The pivoting of the whole pump assembly <NUM> into a pumping position is effected by an electrical actuator <NUM> mounted to the assembly <NUM> and reacting against the frame <NUM>. During the movement of the pump assembly into an operative position, guide pins <NUM> cooperate with complementary formations on the processing kit support frame <NUM>, so that the kit and pump heads are aligned more accurately than relying only on the guide rails <NUM> and <NUM>. The pump heads have six generally evenly spaced rotors, which when engaged against a shoe <NUM> of approximately <NUM>° 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>, where each of the four pump drive motors <NUM> are visible along with one of the toothed drive belts <NUM> and tension screws <NUM>, used to impart tension in the drive belts <NUM>. The drive belts' pulleys are sized to provide approximately a <NUM>:<NUM> reduction in speed of the motor at the pump head.

<FIG> shows another view of the pump assembly. In this view the pump head 44d 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 44d 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 <NUM>/min at around <NUM> rpm motor speed.

<FIG> shows the pumps heads 44a,b,c and d. As labelled, it can be seen that the four heads function to circulate fluid from the processing reservoir <NUM>, to the filter <NUM>, and back to the reservoir or to a collection point <NUM> (head 44d acting on tube 120d), to bring in cells in suspension from the source <NUM> (head 44c acting on tube 120c), to bring in buffer/wash solution <NUM> (head 44b acting on tube 120b) and to remove waste permeate <NUM> from the filter <NUM> (head 44a acting on tube 120a). As mentioned above, from speedier processing more than one tube <NUM> may be provide for each pump head, thus wider head 44d may in other arrangements act on more than one tube <NUM>.

<FIG> shows a pinch valve assembly <NUM> which is mounted underneath the pump motor <NUM> and pump head 44and pivots into position ready for operation together with the pump assembly <NUM>. The pinch valve assembly <NUM> closes and opens process and collection fluid paths by pinching the tubes 130a and 130b against the anvil surface <NUM>. The assembly includes a single linear actuator <NUM> which includes an electric stepper motor <NUM>, for rotatably driving a lead screw <NUM> both clockwise and counterclockwise, which in turn moves a carriage <NUM> linearly back and forth in the direction of arrow C on a rail <NUM>. The carriage <NUM> includes two rollers 54a and 54b, which act on cam profiles 51a and 51b formed on the back of two spring loaded valve arms 53a and 53b. The arms 53a and 53b are urged against the respective rollers 54a and 54b. The arms have fingers 52a and 52b, the tips of which press against the tubes 130a and 130b aligned in the valve's operative position with the anvil <NUM>. The cam profiles 51a and 51b have 'open' portions (58a and 58b) which allow fluid flow and 'closed' portions (59a and 59b) which prevent substantial flow. Since the fingers are arranged in opposite orientations, the sequence of open and closed positions for the two fingers is: 130a closed, 130b open (the position shown in <FIG>); 130a closed, 130b closed (at the mid-position of carriage <NUM>); and 130a open, 130b closed (at the rightmost position of the carriage <NUM> when viewed in the same direction of view as illustrated in <FIG>). 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> shows a horizontal cross section through the anvil <NUM>, through the valve arms 53a and 53b and through carriage rollers 54a and 54b, which in this view are in their mid-position, such that both fingers 52a and 52b are acting to compress and thereby close flexible tubes 130a and 130b (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 <NUM> can be accommodated, the fingers 52a and 52b 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 <NUM> once the processing kit <NUM> is in place. Then the fingers operate by opening or closing the tubes according to an operation protocol. The valve assembly <NUM> can be adjusted initially independently of the position of the pump assembly <NUM>, so that the correct pinch load can be obtained.

<FIG> shows a transfer mechanism <NUM> housed within the housing <NUM> for transferring the processing reservoir <NUM> of the processing kit <NUM> onto a weighing hook <NUM> so that the volume of liquids in the reservoir can be estimated in use. In practice the mechanism <NUM> removes the reservoir <NUM> from the processing kit support frame <NUM>, transfers it to hook <NUM>, which is supported by a load cell <NUM> where it will stay for the duration of a processing run, and then returns the reservoir <NUM> to the support frame <NUM>. The processing reservoir <NUM> is mounted on the support frame <NUM> 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 <NUM> is controlled by one stepper motor <NUM> and a lead screw <NUM> which directly controls X direction movement of a rear carriage <NUM>, travelling on a linear rail <NUM> as the lead screw <NUM> is rotated by the motor <NUM>. The rear carriage <NUM> supports an extension shaft <NUM> that moves with the carriage <NUM>. The shaft <NUM> has a distal end <NUM> which includes a profiled head <NUM> (<FIG>). A front carriage <NUM> is moveable on the rail <NUM> also, but is not driven by the lead screw. Rather its movement is controlled by movement of the profiled head <NUM> and explained in more detail below.

The mechanism <NUM> starts in the position shown in <FIG>, which is a side view in the direction of arrow y in <FIG>. That position allows for insertion of the processing kit <NUM> into the housing <NUM>, and brings the hanger <NUM> of the processing reservoir into an alignment with the mechanism <NUM>. The hanger <NUM> includes two resilient arms <NUM> which sit in supporting apertures in the processing kit frame <NUM>. In this initial position the hanger arms support the processing reservoir and keep it resiliently in place on the frame <NUM>. On the hanger <NUM>, above the arms is a further aperture <NUM> which accepts the hook <NUM>.

The rear carriage <NUM> is then driven in the positive X-direction as shown in <FIG>. This movement ultimately pushes the profiled head <NUM> into a latch arrangement which has a pair of sprung expansion arms <NUM>. The spring force required to open the expansion arms <NUM> is such that the expansion arms remain closed and the front carriage <NUM> is driven forward in the positive X direction also as shown in <FIG>. The front carriage <NUM> 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>. The frame <NUM> cannot move because it is being held in place by the upper and lower guides of the guide rails <NUM> and <NUM>. Thus, the rear carriage continues to move forward while the front carriage is stopped, causing the profiled head <NUM> to force apart the expansion arms <NUM> apart and into latching cooperating engagement with the resilient arms <NUM> of the hanger <NUM>. In this position the expansion arms distort the resilient arms to release their grip on the hanger <NUM>, and the hook <NUM> enters the aperture <NUM>.

Next, as shown in <FIG>, the rear carriage is driven by the motor <NUM> and leadscrew <NUM> in the negative X direction, thereby detaching the hanger <NUM> from the frame <NUM>, and moving the hanger <NUM>, with the reservoir <NUM> away from the frame <NUM>.

The front carriage <NUM> is dragged backwards until it hits a stop. In this position the hanger <NUM> drops onto the load cell hook <NUM>. The rear carriage <NUM> continues moving and the profiled head <NUM> is pulled out from between the expansion arms <NUM>, thus returning them to their neutral position shown. At this point the hanger <NUM> is no longer held in place by the expansion arms and therefore slides down the load cell hook <NUM>, finally bringing weight to bear on the load cell <NUM>. The rear carriage <NUM> is now back to its initial, home position, and no parts of the mechanism, apart from the hook <NUM> touch the reservoir <NUM>, or its hanger <NUM>.

Returning the reservoir <NUM> to the frame <NUM> is carried out by reversing the steps described above. The front carriage <NUM> reaches a stop when the hanger <NUM> is flush against the support frame <NUM>, with the support frame <NUM> held in place by the upper and lower guides <NUM> and <NUM>. The rear carriage <NUM> continues to drive forward and pushes the expansion arms apart. This step ensures that the hanger <NUM> is properly located in the Z-dimension and that the resilient arms <NUM> are met with no resistance passing through their apertures on the frame <NUM>. This action is different from the reservoir retrieval described above; the profiled head <NUM> is driven past the ends of the expansion arms <NUM>, as shown in <FIG>.

In this position, the hanger <NUM> will be securely reattached to the support frame <NUM> and the expansion arms <NUM>, profiled head <NUM>, and load cell hook <NUM> can be extracted. The rear carriage <NUM> drives backwards, dragging the front carriage <NUM> with it. The front carriage <NUM> reaches a stop while the rear carriage <NUM> continues moving backward. This allows the profiled head to be pulled through the expansion arms <NUM> once again and reset for a new process kit and new processing reservoir, as shown in <FIG>.

Claim 1:
A cell harvesting processing kit for separating cells from fluids, and/or for washing cells, the kit comprising sealed fluid paths at least a portion of which are formed from flexible tubing suitable for being externally manipulated by mechanical elements, the kit comprising also a fluid processing reservoir and a filter suitable for retaining cells whilst permeating fluids in said paths, the kit being characterized in that the fluid paths are supported by a generally flat supporting frame such that said paths, reservoir and filter are arranged in a generally flat plane, wherein said fluid processing reservoir is in the form of a container which lies generally flat in the frame and is demountable from the frame and re-attachable to the frame in use,
the kit being characterized in that said supporting frame includes through-apertures, and said flexible tubing is arranged to extend across said apertures such that the tubing is accessible from both sides of the flat supporting frame to allow said external manipulation from both sides of the frame.