Abstract:
A vessel having a mixer that ensures a homogeneous cell distribution in dispensed quantities. The vessel has a mixer therein for stirring contents of the vessel and an orifice in a lower wall to which a cell dispenser is attached. The cell dispenser dispenses quantities of suspended cells having a homogeneous cell distribution.

Description:
RELATED APPLICATIONS 
       [0001]    The present application claims priority under 35 U.S.C. §119 to U.S. Provisional Application No. 62/286,294, filed Jan. 22, 2016. 
     
    
     TECHNICAL FIELD 
       [0002]    The invention pertains to vessels for dispensing cultured cells suspended in fluid and, more particularly, to a vessel having a mixer for dispensing quantities of cells suspended in fluid having a homogeneous cell distribution. 
       BACKGROUND OF THE INVENTION 
       [0003]    In the conventional therapeutic protein-based industry, recombinant cells are expanded and induced to produce target proteins, which are then isolated and purified before final formulation in chilled excipient designed to stabilize proteins. In such application, maintaining proteins in uniform suspension in the final fill/finish step is not a great concern, largely due to the fact that proteins do not settle very fast in the excipient relative to the time required for processing to create noticeable concentration gradient. 
         [0004]    On the other hand, in the growing field of cell therapy where animal cells in their native pluripotent, induced pluripotent, and/or differentiated form would be cultured and expanded, the cells themselves are the final product that must be isolated and dispensed into final vials. Maintaining cells in uniform suspension in the excipient during the dispensing step is more critical and challenging compared to maintaining proteins in suspension. Although there are a number of ways to dispense such cells in the art, such as withdrawing cells with a pipette from above, none as yet has been able to repeatedly and accurately dispense cultured cells from a vessel on demand. 
       SUMMARY OF THE INVENTION 
       [0005]    The present application discloses a vessel having a mixer that ensures a homogeneous cell distribution in dispensed quantities. 
         [0006]    An appreciation of the other aims and objectives of the present invention and an understanding of it may be achieved by referring to the accompanying drawings and the detailed description of the preferred embodiment. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0007]      FIG. 1  is a perspective view of an embodiment of the homogeneous cell-dispensing mixer; and 
           [0008]      FIG. 1  is a close-up sectional view of a dispensing portion of the mixer. 
       
    
    
     DESCRIPTION OF THE PREFERRED EMBODIMENTS 
       [0009]    The present application relates to vessels for dispensing cells suspended in fluid and, more particularly, to a vessel having a mixer that ensures a homogeneous cell distribution in dispensed quantities. 
         [0010]    In the growing field of cell therapy the final products are animal cells in their native pluripotent, induced pluripotent, and/or differentiated form. The cells themselves must be isolated and dispensed into final vials. Maintaining cells in uniform suspension in the excipient during the dispensing step is much more critical and challenging compared to maintaining proteins in suspension. This is due to the faster settling velocity of cells, the relatively large size of the cells (micrometer scale vs. nanometer scale) which limits the minimum size of the orifice required for accurate and low-shear dispensing, and the higher shear sensitivity level of cells which can impact the viability of cells dispensed. 
         [0011]    Further, the cell-dispensing step requires that a sterile vessel be used to mix the animal cells and excipient at a controlled refrigeration (2-8° C.) temperature and in an aseptic manner to ensure that the cell product is not contaminated with foreign particles or microorganisms. Typical lot release criteria for this cell-dispensing step are that the vials selected for QC inspection must meet a target cell concentration that fall within acceptable tolerance and that they must meet a minimum viability target. The process requires that cells be dispensed in accurate volume, at accurate cell concentration, within short processing time, and at controlled 2-8° C. temperature to ensure uniformity in cell concentration and high cell viability in the vials. 
         [0012]    This proposed solution for dispensing such animal cells includes a vessel for containing the cell suspension having a mixing device that allows the cells to be maintained in uniform suspension during dispensing at 2-8° C. condition into vials in a relatively low-shear manner to avoid damaging cells. The device would consist of a mixing vessel to hold the cells and excipient in a sterile manner, with an impeller that is rotated by any number of means—pneumatically, magnetically, or otherwise—to keep the cells suspended uniformly in the excipient. The rotational speed of the impeller should be controllable by the user in a repeatable manner and to the extent that would allow the cells to be suspended uniformly and dispensed within allowable tolerance. 
         [0013]    One embodiment of this invention, as depicted in Error! Reference source not found. and  FIG. 1 , consists of a vessel  20  to hold the chilled cells and excipient and an impeller  22  enclosed within the vessel for maintaining cells in suspension. The impeller  22  is oriented in a vertical plane and rotates about a horizontal axis  24  to allow maximum particle suspension at minimum power input and reduce shear effects on cells. Cells and excipient are introduced into the vessel by removing a threaded port cap  26  in a Class 100 clean room environment or equivalent, and then transferring the content into the vessel  20  via pipetting or pouring. The cap  26  may be threaded back onto the port to seal prior to cell dispensing to minimize potential for introducing foreign materials. A hydrophobic membrane  28  on the cap  26  allows improved thermal exchange with the air in the cold room to help maintain temperature. 
         [0014]    During cell dispensing, fluid is removed at a lower dispenser  30  via a vessel orifice  32  that extends through an outer wall near the bottom of the vessel  20 . The fluid travels down a bore  34  in a machined block  36  of the dispenser  30  which is affixed to the vessel  20  and sealed around the orifice  32 . A hose barb adaptor  38  open to the bore  34  that mates with the machined block  36  allows tubing to be secured to it to maintain a sterile fluid path. Prior to sterilization of this device, tubing would be attached and secured to the hose barb adaptor  38  and terminated with another adaptor depending on how the user wishes to connect it to a dosing pump (not shown). 
         [0015]    The impeller  22  consists of a plurality of paddles  40  along its outer periphery that generate strong sweeping motion of the liquid as it rotates to counteract cell settling in the excipient. The paddles  40 , which are hollow, can encapsulate permanent magnets, which are used to couple with magnets on the agitation controller (not shown) to drive the rotation of the impeller  22 . The impeller  22  also consists of two diametrically-opposed vanes  42  extending from the paddles to an inner hub that create bi-axial fluid flow as the impeller rotates to ensure homogeneity of cells suspended in the excipient. That is, the vanes  42  have curved surfaces that urge flow axially when the impeller is rotated in one direction. 
         [0016]    Desirably, there is a minimum of one port for adding cells and excipient into the vessel and a minimum of one port for dispensing the cells and excipient, both of which could be sealed as needed to prevent foreign contaminants, biological or not, from contacting the cell product. The dispensing port should allow for flexibility by the user to specify how to connect the device to a dosing pump—either by using an aseptic connector (GE ReadyMate Disposable Aseptic Connector, Pall Kleenpak™ Sterile Connector, or equivalent), a dead-ended thermoplastic tubing that may be heat welded onto another dead-ended thermoplastic tubing, or tubing that is terminated with fittings that may be connected to another tubing inside a Class 100 clean room environment. The dosing pump would be a calibrated instrument to allow accurate metering of liquid dispensed into vials. 
         [0017]    Since 2-8° C. temperature would be maintained in the vessel either by placing the mixing device in a cold room or a refrigerator or by applying cold packs, the vessel wall would therefore be composed of material and at thickness that allows relatively high thermal transfer. If the addition port is positioned at the top of the vessel, the cap on the port could further contain a hydrophobic, sterilizing-grade (0.22-micron or finer) membrane to allow gas exchange with chilled gas in the cold room or refrigerator for improved thermal transfer. Additionally, the material could be clear in appearance to allow visual confirmation of impeller rotation and cell suspension. 
         [0018]    All of the components of this mixing device that come in contact with the chilled excipient and cells should be manufactured from medical-grade materials that have been certified to USP Class VI, ISO 10993, or equivalent, to ensure they meet the regulatory requirements of the user. The mixing device would also need to be sterilizable to ensure Sterility Assurance Level (SAL) of 10 −6 —either by gamma radiation, steam sterilization, or other applicable means. 
         [0019]    It is understood that the foregoing examples are considered illustrative only of the principles of the invention. Further, since numerous modifications and changes will readily occur to those skilled in the art, it is not desired to limit the invention to the exact construction and operation shown and, accordingly, all suitable modifications and equivalents may be resorted to, falling within the scope of the invention.