Improvements in and relating to cell harvesting devices

Abstract

Disclosed herein is a cell harvesting instrument suitable for concentrating and / or washing cells from a source suspension of cells, the instrument comprising: a housing for accommodating mechanical elements comprising at least one fluid pump, at least one valve; and a treatment kit removably inserted into the housing, the kit comprising a substantially flat frame having or supporting a plurality of sealed fluid paths, the fluid paths being arranged in a substantially plane and such that fluid in the paths does not contact the mechanical element, where at least a portion of the fluid paths comprises a flexible tube, the flexible tube being configured to receive fluid from the fluid paths. The outer surface of the flexible tube may be manipulated by the or each fluid pump to provide fluid flow in one or more of the paths, and / or by the or each valve to constrain fluid flow in one or more of the paths. In an embodiment, the kit also includes a fluid handling reservoir and a filter adapted to separate cells from the fluid in the pathway. Transfer mechanisms for moving and weighing fluid processing reservoirs are also disclosed.

Description

[0001] This application is a divisional application of the PCT patent application "In a cell harvesting device and improvements thereof" (application number: 201680082332.6, applicant: Global Life Science Consulting, USA LLC), which entered the Chinese national phase on August 21, 2018. Technical Field

[0002] This invention relates to a cell harvesting device, improved functionality of the device, and improvements in its components. Background Technology

[0003] For various therapeutic applications (such as cell therapy or tissue engineering), it is necessary to efficiently harvest cells from a variety of sources. Examples of therapeutic applications include, but are not limited to, autologous or allogeneic transplantation of stem cells, transplantation of mature functional cells, T cells, human cells modified to contain T cells, or xenograft transplantation of non-human cells. This application promotes the healing of damaged tissues or organs by regenerating cells to improve the condition of the disease.

[0004] For translational research that utilizes state-of-the-art technologies to advance the development and implementation of scientific discoveries for the prevention, diagnosis, and treatment of diseases, a range of potential cell types need to be isolated before modification, activation, and / or expansion. To meet this translational market demand, cells must first be concentrated and washed to remove any impurities. For cell preservation applications, where previously isolated mononuclear cells (MNCs) are suspended in a medium containing preservatives such as dimethyl sulfoxide (DMSO) and stored at low temperatures, the cells typically require several washes through a dilution process to minimize the concentration of preservatives before reconcentrating and resuspending the cells for use. Therefore, cryopreservation of cells is essential before use in any application, particularly for therapeutic applications or research purposes.

[0005] For both implementations, the suspension of such cells should be treated to concentrate and should be extensively washed to ensure high quality; herein, such concentration, optionally including one or more washing 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 applications. 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. Furthermore, equipment that is easy to operate and maintain is also desirable. Summary of the Invention

[0006] Methods and apparatus 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 free of significant residual impurities or preservatives. These methods and apparatus address several problems associated with cells used for transformation applications or cells recovered from cryopreserved cells.

[0007] An example of a method for harvesting cells from a fluid material in a processing loop (as shown in US2013 / 0029411) includes a processing chamber and a filtration device, wherein the fluid material has a volume and the processing chamber has a total capacity. The method includes circulating the fluid material through the processing loop, and using the permeation flux of the filtration device to balance the inflow flux of the fluid material into the processing chamber to maintain the volume of the fluid material in the processing chamber at a constant value, causing cells to concentrate by increasing the permeation flux of the filtration device relative to the inflow flux of the fluid material into the processing chamber; and collecting the concentrated cells in a collection chamber. Other examples of methods for harvesting cells from a fluid material in a processing loop are shown in US2013 / 0029411.

[0008] Furthermore, US2013 / 0029411 discloses an embodiment of a cell harvesting apparatus, which includes, for example, a processing loop comprising a processing chamber and a filtration device; a network of input and output lines operatively coupled to one or more of a source chamber, a buffer chamber, a waste chamber, and a collection chamber; and a controller that controls the quality of the processing chamber to a desired value based on the inflow flux and permeation flux of the processing loop.

[0009] The inventors have designed improvements to the method and apparatus disclosed in US2013 / 0029411, resulting in improved performance and reliability, as well as reduced costs in terms of improved consumable components. Embodiments of the present invention address the shortcomings of known cell harvesting equipment. The invention is set forth in the independent claims herein, wherein preferred features are defined in the dependent claims. Attached Figure Description

[0010] These and other features, aspects, and advantages of the invention will be better understood when the following detailed description is read with reference to the accompanying drawings, in which similar reference numerals denote similar parts throughout the drawings, wherein: Figure 1 The cell harvesting device along with its disposable processing kit is shown; Figure 2 It shows Figure 1 A schematic diagram of the disposable treatment kit shown; Figure 3 The treatment kit is shown in place within the instrument; Figure 4 It shows that it can be installed Figure 1The receiving track in the housing of the instrument is used to guide the processing kit to the appropriate position in the housing; Figure 5 The processing kit receiving frame is shown, which is housed in Figure 1 Inside the housing of the device shown; Figures 6 to 10 It shows that it can be installed Figure 5 Details of the peristaltic pump in the frame shown; Figure 11 and Figure 12 It also shows that it can be installed to Figure 5 Details of the clamping valve of the frame shown; Figure 13 A graphical view of the storage transfer mechanism is shown; and Figures 14 to 19 It shows the different functional positions. Figure 13 Side view of the transfer mechanism. Detailed Implementation

[0011] To more clearly and concisely describe and indicate the subject matter of the claimed invention, the following definitions are provided for use with particular terms, which are used in the following description and the appended claims. Throughout the specification, the use of particular terms should be considered as non-limiting instances.

[0012] Reference Figure 1 The image shows a cell harvesting device 10 that functions in use to absorb a liquid containing suspended cells or similar microbial material, with the aim of largely separating the cells from the liquid or reducing the liquid content of the suspension. The device can function to wash the cells, etc., once or multiple times to remove unwanted material from the separated cells. Preferred functional mechanisms can be found in US2013 / 0029411.

[0013] The device 10 includes a housing 12 having a touchscreen 14 and a door 16, shown closed and indicated by a dashed line as being in the open position 16'. Door 16 allows for the insertion and removal of the disposable treatment kit 100. The kit 100 is generally flat, having a peripheral support frame 105 with a thickness x' (approximately 30-40 mm) in the x-direction. In other words, the fluid path 110 within the frame and the additional components of the kit described below are generally located in a generally flat single plane. The fluid path 110, shown by a dashed line, has four inlets / outlets 122, 124, 126, and 128 in this case. The fluid path 110 is primarily constructed of medical-grade tubing, such as PVC tubing. Except for those inlets / outlets 122-128, the fluid path 110 is a functionally closed loop, sealed except at vents containing filters of submicron pore size to allow gas escape but prevent contamination ingress. Specifically, the mechanical components contained within the housing 12 do not come into contact with any fluid in the path, thereby maintaining the sterility of the path in use. The frame 105 also includes through-holes 120 and 130 extending from one side of the frame 105 to the other, providing areas where tubes crossing the fluid path through the orifices can be manipulated by the mechanical components from both sides of the frame. Where the fluid path crosses the orifices, these tubes need to be flexible, and therefore these tubes are preferably formed of silicon tubing.

[0014] The kit 100 also includes a tangential flow filter 140 and a removable processing reservoir 150, in this case, in the form of a molded plastic container. The processing kit 100 is inserted into and removed from the housing 12 in the direction of arrow y.

[0015] Figure 2To illustrate the side view of kit 100, the layout of fluid path 110 within support frame 105 and its external connections are shown, which are actually made outside housing 12 when kit 100 is inserted into housing 12 during use. Once kit 100 is inserted, it is connected to buffer / wash solution supply unit 123, suspended cell or similar biological material source 125, waste collector 129, and harvest collection chamber 127 respectively by means of corresponding sterile connectors 122, 124, 128, and 126. Alternatively, any one of buffer supply unit 123, source 125, waste collector 129, and harvest collection chamber 127 may be pre-connected to fluid path 110. In practice, the extended corresponding fluid connection tube initially coils near frame 105, terminating at the buffer supply unit 123, source 125, waste collector 129, and / or harvest collection chamber 127, and unfolds the extended tube once kit 100 is inserted into housing 12 for external supply. Through-hole 120 allows a pumping action to be applied to the fluid within the flexible tubular paths 120a, 120b, 120c, and 120d that cross the orifice. Similarly, through-hole 130 allows the tubular paths 130a and 130b that cross the orifice to be clamped to provide a valve action. Processing reservoir 150 serves as a fluid holding chamber and is part of a recirculation loop through which the cell-containing fluid is actively recirculated during most of the concentration and washing processes performed by device 10. It is important to determine the total fluid volume / mass throughout the entire processing loop, which includes fluid path 110, filter 140, and processing reservoir 150. This total will vary during use because, for example, the amount of waste fluid removed and the dosage of added buffers will change the total volume. However, since all components except processing reservoir 150 have a fixed working volume, the variable mass in processing reservoir 150 is the entirety that needs to be measured to determine the total processing loop volume / mass. Therefore, the reservoir 150 includes a hanger 152, which allows the weight of the reservoir 150 to be measured, and thereby the total fluid volume / mass can be determined.

[0016] Figure 3 A portion of the frame 105 inserted into the housing 12 is shown. In this case, the frame includes guide forming elements (e.g., I-shaped studs) or ribs 112 at the top and bottom, which are slidably positioned in open end recesses 23 formed in the top guide rail 22 (supported within the housing 12 by the rigid device frame 20) to slidably support and position the kit 100.

[0017] exist Figure 4 The bottom guide rail 24 is shown, which also includes a groove 25 for receiving a nail or 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 the top rail (in Figure 5Both the lower and lower guide rails (22) have grooves that mate with corresponding studs or ribs on the processing kit. The lower studs or ribs and grooves are wider than those on the top for two reasons: a) to inform the user which end is the top and to prevent incorrect insertion of the frame 105, and b) to make the lower guide rail easier to clean in case of processing kit leaks. To aid in cleaning, the bottom guide rail 24 has a large radius and is shaped to trap any leaks. An adjustable roller pawl feature (not shown) provides tactile feedback to the user to alert them to stop pushing the processing kit into the housing.

[0018] Figure 5 The device frame 20 is shown in more detail, with the housing 12 removed for clarity. The insertion direction of the kit 100 is indicated by arrows, thus the device frame 20 is shown in this figure from... Figure 1 Viewed from the rear of housing 12. The device frame 20 includes two plates 26 held at a distance by spacer fittings 28. A top guide rail 22 and a bottom guide rail 24 extend parallel to each other and are each mounted to both spaced plates 26. Also mounted to the plates are a bearing 30 (described in detail below) for reacting to forces acting on the peristaltic pump rotor, and an anvil (described in more detail below) for reacting to forces applied by the clamping valve. The bearing 30 and anvil 32 are aligned with through holes 120 and 130, respectively, during use.

[0019] Figure 6 A device frame 20 is shown, along with a pump assembly 40 pivotally mounted on the frame via a pivot 42. The pump assembly in use (with the treatment kit inserted into the housing 12 between guide rails 22 and 24) pivots relative to the stationary frame 20 about the pump pivot 42 in the direction of arrow R to interact with flexible tubes 120a, b, c, and d and flexible tubes 130a and b using the bearings 30 and anvil 32 as reaction surfaces. Additional alignment is achieved by guide pins 48 rigidly mounted to the assembly 40. The pump assembly 40 abuts against the treatment kit 100 to selectively pump fluid through a fluid path 110, in this case, with a peristaltic action. The assembly 40 includes a three-state clamping valve to properly guide flow by using cams of the compressed and closed mating flexible tubes. The pump and valve, each described in more detail below, are supported on the frame 20 such that operating forces are isolated from the surrounding housing. Disengagement of the pump and valve is achieved by pivoting in the direction opposite to arrow R before removing the used treatment kit 100.

[0020] Figure 7 The pump assembly, removed from frame 20, is shown in more detail and along... Figure 6See the direction of arrow A in the diagram. In this view, four pump heads 44a, 44b, 44c, and 44d are visible, interacting with flexible tubes 120a, b, c, and d, respectively. Each head is formed by a group of rollers mounted to rotate about roller pins, and each pin is mounted to rotate about the pump axis P, thus forming the head of the peristaltic pump. The four heads share the same pump axis P but can be independently rotated by four different servo motors 46 acting on a drive belt to provide a controlled and reversible fluid pressure differential in the fluid paths 120a to d. Pivoting of the entire pump assembly 40 to the pumping position is achieved by an electric actuator 44, which is mounted to the assembly 40 and reacts relative to the frame 20. During the movement of the pump assembly to the operating position, guide pins 48 engage with complementary forming elements on the processing kit support frame 105 to align the kit and pump heads more precisely than if relying solely on guide rails 22 and 24. The pump head has six generally evenly spaced rotors that always provide at least one roller to contact the tile when engaged with the tile at an approximately 70° arc, thereby preventing reverse fluid flow and fluid flow when the pump is not rotating.

[0021] Pump assembly in Figure 8 The diagram further shows in more detail that each of the four pump drive motors 46, along with one of the serrated drive belts 47 and tension screws 49, is visible, with the tension screws 49 used to apply tension in the drive belts 47. The pulleys of the drive belts are sized to provide an approximately 2:1 motor speed reduction at the pump head.

[0022] Figure 9 Another view of the pump assembly is shown. 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 axial direction P. This wider pump head 44d allows two or more flexible tubes to be engaged simultaneously, thereby providing increased fluid flow as needed. This wider head arrangement allows for a processing pump flow rate of up to 3000 mL / min at a motor speed of around 280 rpm.

[0023] Figure 10 Pump heads 44a, b, c, and d are shown. As can be seen from the label, four heads function to circulate fluid from the treatment reservoir 150 to the filter 140 and back to the reservoir or collection point 127 (head 44d acts on pipe 120d), introduce suspended cells from source 125 (head 44c acts on pipe 120c), introduce buffer / washing solution 123 (head 44b acts on pipe 120b), and remove waste permeate 129 from filter 130 (head 44a acts on pipe 120a). As mentioned above, for faster treatment, more than one pipe 120 can be provided for each pump head, so a wider head 44d can act on more than one pipe 120 in other arrangements.

[0024] Figure 11 A clamping valve assembly 50 is shown, mounted below the pump motor 464 and pump head 44 and pivoted to a position ready to operate in conjunction with the pump assembly 40. The clamping valve assembly 50 closes and opens the processing and collecting fluid paths by clamping tubes 130a and 130b relative to the anvil surface 32. The assembly includes a single linear actuator 55 comprising an electric stepper motor 56 for rotatably driving a lead screw 58 in both clockwise and counterclockwise directions, which in turn causes a carriage 57 to move linearly back and forth on a track 53 in the direction of arrow C. The carriage 57 includes two rollers 54a and 54b that act on cam profiles 51a and 51b formed on the back surfaces of two spring-loaded valve arms 53a and 53b. Arms 53a and 53b are forced against the corresponding rollers 54a and 54b. The arm has fingers 52a and 52b, the tips of which press against tubes 130a and 130b, which are aligned with anvil 32 in the valve's operating position. Cam profiles 51a and 51b have "open" portions (58a and 58b) that allow fluid flow and "closed" portions (59a and 59b) that prevent excessive 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. Figure 11 (as shown in the diagram); 130a closed, 130b closed (at the middle position of carriage 57); and 130a open, 130b closed (at the position shown in the diagram); Figure 11 When viewed from the same viewpoint shown, at the rightmost position of carriage 57. It will be noted that no power is required to hold the arm in the open or closed position, as such positions may need to be maintained for extended periods during possession. It should also be noted that the open / closed position is intentionally made impossible to prevent unwanted fluid flow.

[0025] Figure 12 A horizontal cross-section is shown passing through the anvil 32, through valve arms 53a and 53b, and through carriage rollers 54a and 54b, in which they are positioned in the middle, such that fingers 52a and 52b are used to compress and thereby close flexible tubes 130a and 130b (shown schematically in this figure). Note that the initial position of the tubes is also shown. To accommodate the thickness of the processing kit frame 105, fingers 52a and 52b are initially retracted (along with the pump head) and brought into an operational-ready position only by pivoting in front of the pump assembly 40 (once the processing kit 100 is in place). The fingers are then operated according to the operating protocol by opening or closing the tubes. Valve assembly 50 is initially adjustable independently of the position of pump assembly 40 to allow for the correct clamping load.

[0026] Figure 13 A transfer mechanism 60, housed within the housing 12, is shown for transferring the treatment reservoir 150 of the treatment kit 100 to a weighing hook 62 so that the volume of liquid in the reservoir can be estimated during use. Specifically, mechanism 60 removes the reservoir 150 from the treatment kit support frame 105, transfers it to the hook 62, supported by a load unit 61, where it remains for the duration of treatment operation, and then returns the reservoir 150 to the support frame 105. The treatment reservoir 150 is mounted on the support frame 105 when supplied to the user and inserted into the housing in that state. The treatment reservoir 150 is reattached to the support frame before the user removes the treatment kit from the housing. During operation, the treatment reservoir and connected tubing are freely suspended on the load unit hook for mass measurement.

[0027] The movement of mechanism 60 is controlled by a stepper motor 64 and a lead screw 66. As the lead screw 66 is rotated by the motor 64, it directly controls the X-direction movement of the rear carriage 65 (traveling on linear track 68). The rear carriage 66 supports an extension shaft 73 that moves together with the carriage 66. The shaft 73 has a distal end 71, which includes a contoured head 72 (…). Figure 14 The front carriage 70 can also move on the track 68, but not driven by the lead screw. Instead, its movement is controlled by the movement of the profiled head 74, as explained in more detail below.

[0028] Institution 60 Figure 14 Starting from the indicated position, Figure 14 For along Figure 5 The image shows a side view in the direction of arrow y. This position allows the processing kit 100 to be inserted into the housing 12 and aligns the processing reservoir's hanger 152 with the mechanism 60. The hanger 152 includes two resilient arms 154 located in support openings in the processing kit frame 105. In this initial position, the hanger arms support the processing reservoir and resiliently hold it in place on the frame 105. On the hanger 152, above the arms, is another opening 156 that receives a hook 62.

[0029] The rear carriage 65 then drives in the positive X direction, such as Figure 15 As shown in the diagram. This movement ultimately pushes the contoured head 72 into the latch arrangement with paired spring-loaded expansion arms 75. The spring force required to open the expansion arms 75 keeps the expansion arms closed, and the front carriage 70 is also driven forward in the positive X direction, as shown. Figure 15 As shown in the diagram, the front slide 70 is driven forward in this manner until it reaches the rigid stop formed by the reservoir clip on the support frame, as... Figure 16As shown in the diagram, frame 105 is immovable because it is held in place by the upper guide 22 and lower guide 24 of the guide rail. Therefore, the rear carriage continues to move forward while the front carriage stops, causing the contoured head 72 to force the expansion arm 75 apart and engage with the resilient arm 154 of the hanger 152 in a latching engagement. In this position, the expansion arm deforms the resilient arm to release its grip on the hanger 152, and the hook 62 enters the orifice 156.

[0030] Next, as Figure 17 As shown, the rear carriage is driven in the negative X direction by a motor 64 and a lead screw 68, thereby disengaging the hanger 152 from the frame 105 and moving the hanger 152, wherein the reservoir 150 is moved away from the frame 105.

[0031] The front slide 70 is dragged backward until it stops. In this position, the hanger 152 falls onto the load unit hook 62. The rear slide 65 continues to move, and the profiled head 72 is pulled out between the expansion arms 75, thus returning them to the neutral position shown. At this point, the hanger 152 is no longer held in place by the expansion arms and therefore slides down along the load unit hook 62, finally placing the load on the load unit 61. The rear slide 65 now retracts to its initial original position, and no part of the mechanism other than the hook 61 touches the reservoir 150 or its hanger 152.

[0032] Returning the reservoir 150 to the frame 105 is performed by reversing the steps described above. The front carriage 70 reaches the stop when the hanger 152 is flush with the support frame 105, which is held in place by the upper guide 22 and the lower guide 24. The rear carriage 65 continues to drive forward and pushes the expansion arms apart. This step ensures that the hanger 152 is properly positioned in the Z dimension and encounters the resilient arms 154 without resistance through their orifices in the frame 105. This action differs from the reservoir retrieval described above; the contoured head 72 drives past the end of the expansion arm 75, as... Figure 18 As shown in the image.

[0033] In this position, the hanger 152 is securely reattached to the support frame 105, and the expansion arm 75, profiled head 72, and load unit hook 62 are removable. The rear carriage 65 is driven rearward, using it to pull the front carriage 70. The front carriage 70 reaches the stop, while the rear carriage 65 continues to move rearward. This allows the profiled head to be pulled again through the expansion arm 75 and reset for use with new processing kits and new processing reservoirs, such as... Figure 19 As shown in the image.

[0034] In operation, the device 10 includes mechanical components such as a reusable pump, clamping valve, and weighing mechanism as described above, along with a removable and disposable low-cost processing kit 100 that includes all the fluid components required for cell harvesting (e.g., path 110, filter 140, and fluid processing reservoir 150). This combination of features results in a cell harvesting device that is easy to use and quickly ready for the next harvest batch. No mechanical parts come into contact with the fluid, meaning that cleaning of the mechanical parts between harvests is unnecessary. The device 10 is particularly well-suited for concentrating and / or washing human cells, for example, for subsequent use in cell therapy applications, where the device's easily achievable aseptic operating conditions provide a significantly improved chance of therapeutic success, as well as reduced costs and turnaround time.

[0035] While only certain features of the invention are shown and described herein, many modifications and variations will occur to those skilled in the art. Therefore, it will be understood that the appended claims are intended to cover all such modifications and variations falling within the scope of the invention. Furthermore, it is intended that combinations of features included in the dependent claims are combined for convenience, and that any one or more of these combined features may be removed, replaced, or moved to other claims without introducing something new.

Claims

1. A cell-harvesting process kit for separating cells from a fluid and / or for washing cells, the kit comprising a sealed fluid path, at least a portion of the fluid path being formed by a flexible tube adapted to be manipulated externally by mechanical elements, the kit further comprising a fluid-handling reservoir and a filter adapted to hold cells while passing fluid in the path, the kit being characterized in that the fluid path is supported by a generally planar support frame so that the path, reservoir and filter are disposed in a generally planar surface; wherein the support frame comprises through-holes and the flexible tube is disposed to extend across the holes so that the tube is accessible from both sides of the planar support frame to allow manipulation from the outside of both sides of the frame to open and close the tube; wherein the fluid-handling reservoir is in the form of a container that is generally laid flat in the frame and is detachable from and re-attachable to the frame when the cell-harvesting process kit is in use.

2. The cell harves ting process kit of claim 1, wherein, the fluid path comprises at least one inlet at an end of the extended flexible tube for further fluidic connection away from the frame.

3. The cell harves ting process kit of claim 2, wherein, a plurality of fluid paths and extended fluid paths form a closed fluid-handling system arranged so that fluid in the paths does not contact mechanical elements that effect fluid control, such as one or more fluid pumps and / or one or more fluid valves.

4. The cell harves ting process kit of claim 2, wherein, the fluid path further comprises at least one outlet.

5. A method for separating and / or washing cells comprising using a cell-harvesting process kit according to claim 1 to separate and / or wash cells.

6. The method of claim 5, wherein, the cells are human cells.

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