Patent Application
20150024922
This disclosure relates generally to the fluid handling arts and, more particularly, to systems for separating solids, such as cells, from a liquid, using centrifugal force.
The use of centrifugation to separate a solid fraction, such as cells, from a liquid fraction, of a suspension is well known. Typically, the centrifuges used for collecting cells from a bioreactor are not disposable components, and in any case require a halting of the centrifugation process in order to allow for cell recovery. Moreover, existing devices that attempt to achieve such semi-continuous centrifugation invariably require dynamic seals to introduce the cell suspension to and extract the supernatant from the centrifuge. This adds to the cost and complexity, risks breaching sterility, and also potentially results in the generation of heat and particles (which is deleterious in the case of autologous cell seperaration, and in many cases will necessitate a costly and time consuming added filtration step). These existing devices also typically rely on high flow rates and excessive g-forces, which may destroy fragile cells.
Thus, a need is identified for a manner of providing an improved centrifuge. The centrifuge may at least rotate, and possibly levitate, as well, while the process of solids recovery is completed. Also, the arrangement may be such that the capacity of the separation compartment would be minimized to allow for a high separation efficiency at a relatively low flow rate (e.g., <1 liter/minute, and possibly as low as 0.25-0.5 milliliters/minute), even with the use of dynamic seals.
According to one aspect of this disclosure, an apparatus for use in performing centrifugation with a liquid including solids is disclosed. In one embodiment, the apparatus comprises a container including an interior compartment for receiving the liquid and solids. The container is capable of rotating to urge the solids toward the periphery of the interior compartment. A fixed extraction conduit is provided for extracting at least a portion of the solids from adjacent the periphery of the interior compartment of the container. A motive device is also provided for forming a non-contact coupling with the container.
The apparatus may also include a vessel for receiving the container. The vessel may have an inlet for introducing the liquid and solids to the container and a drain for draining at least liquid from the vessel. Any one of the inlet, the extraction conduit, or the drain may comprise a tube connected to a wall of the vessel by a static seal.
The motive device may rotate the container via the non-contact coupling. The motive device may also be adapted to levitate the container via the non-contact coupling. The motive device may be adapted to levitate and rotate the container via the non-contact coupling. The motive device may comprise a magnet, a superconductor or an electromagnet.
The container may include a bottom wall and an upstanding sidewall forming an at least partially open top. A lip may be provided adjacent the sidewall for assisting in retaining solids, such as cells, in the interior compartment during rotation. The container may comprise a rigid material, and may carry at least one magnet.
A further aspect of this disclosure relates to an apparatus for use in performing centrifugation on a liquid including solids. The apparatus comprises a vessel and an open-ended container mounted for rotating within the vessel. The container includes an interior compartment for receiving the liquid and solids. A motive device is also provided for rotating the container by way of a non-contact coupling.
In one embodiment, the motive device comprises a superconductor connected to a motor, and the container is adapted for forming a magnetic coupling with the superconductor. Alternatively, the motive device may comprise a magnet, and the container is adapted for forming a magnetic coupling with the magnet of the motive device. In the case where the motive device comprises a superconductor for levitating the container, a permanent magnet may be adapted for rotating to rotate the container via a magnetic coupling with the superconductor. A mechanical bearing may support the container for rotation relative to the vessel.
The container may include a lip along an upper portion for assisting in retaining cells in the interior compartment during rotation. A fixed extraction conduit may also be provided for extracting at least a portion of the solid. The extraction conduit may be adjacent the periphery of the interior compartment of the container.
Another aspect of this invention is an apparatus for use in performing substantially continuous centrifugation to separate cells from a liquid. The vessel is adapted for receiving the liquid, and a container is mounted for rotating within the vessel. The container includes an interior compartment for receiving the liquid, and the vessel includes an inlet for introducing the liquid to the interior compartment of the container, an extraction conduit from extracting cells from the interior compartment of the container, and a drain for draining at least liquid from the vessel.
The arrangement may further include a motive device for rotating the container relative to the vessel. A motive device may also be provided for levitating the container relative to the vessel. The extraction conduit may comprise a partially non-linear tube in the container, which may have a substantially open top.
A further aspect of this disclosure is an apparatus for use in performing substantially continuous centrifugation to separate cells from a liquid. The apparatus comprises a collapsible vessel and a container mounted for rotating within the vessel. The container includes an interior compartment for receiving the liquid.
In one embodiment, the vessel comprises a flexible bag. A motive device may also be provided for forming a non-contact coupling with the container. In any of the foregoing situations, the liquid flow rate may be from about 250 ml/min to about 500 ml/min.
A further aspect of the invention is an apparatus for use in performing centrifugation to separate cells from a liquid. The apparatus comprises a container mounted for rotation, the container including a first conduit for conveying the liquid to an interior compartment of the container and a second conduit for conveying liquid from the interior compartment, the first and second conduits each being connected to the container by way of a dynamic seal. A flow rate of the liquid is from about 250 ml/min to about 500 ml/min, and may be through one or more of the first conduit, the second conduit, or the interior compartment of the vessel.
Yet another aspect of this disclosure relates to a system including a bioreactor and any of the above-described apparatuses.
A method of centrifugation using a liquid including solids comprises rotating a container including the liquid, and during the rotating step, removing a major portion of the solids from the container. A method of centrifugation also comprises rotating a container including a liquid and cells, and during the rotating step, removing a major portion of the cells from the container. The method may further include the step of levitating the container within a vessel, and the removing step may comprise extracting the solids from adjacent the sidewalls of the container. The method may further include the step of conveying liquid from the container during the rotating step, which may involve overflowing a liquid fraction substantially free of cells from the container.
Another method of centrifugation comprises rotating a container including a liquid and cells and, during the rotating step, transmitting liquid substantially free of cells from the container. The transmitting step may comprise overflowing the liquid from the container.
In any of the foregoing methods, the liquid flow rate may be from about 250 ml/min to about 500 ml/min. In any of the foregoing cases, the container may have has a capacity of about 100 ml to about 300 ml, and possibly about 135 ml.
A further method for performing centrifugation to separate cells from a liquid comprises providing a container mounted for rotation, the container including a first conduit for conveying the liquid to an interior compartment of the container and a second conduit for conveying liquid from the interior compartment, the first and second conduits each being connected to the container by way of a dynamic seal. The method further includes the step of flowing liquid through the container at a rate of about 250 ml/min to about 500 ml/min.
An apparatus for use in performing centrifugation on a liquid including solids is also disclosed. The apparatus comprises a container including an interior compartment for receiving the liquid and solids, said container being capable of rotating, and a motive device for levitating the container. One of the motive device or the container comprises a magnet. One of the motive device or the container comprises a superconductor. The container may comprise an open-top bowl, and a fixed extraction conduit may extend into the container.
A further aspect of the disclosure pertains to a centrifuge including a disposable bag for receiving the liquid and solids, and means for separating the liquid from the solids. The separating means may comprise a container for receiving the liquid and solids within the disposable bag, the container being coupled to a motive device (such as a motor for rotating a magnet).
a is a schematic diagram illustrating another specific embodiment of the disclosure;
a is a partially cross-sectional, partially schematic view of still another specific embodiment of the disclosure;
Reference is now made to
Turning to
The container 14 may also comprise a rigid or semi-rigid cup or bowl-shaped structure including a bottom wall 14a and an upstanding sidewall 14b forming an at least partially open top. The bottom wall 14a may support or carry one or more magnets 18, which are arranged to interface with the external motive device 16. The arrangement may be one that provides the container 14 with levitation and rotation in the absence of a physical bearing or the like. This may be achieved by using a field-cooled superconductor 20 as forming part of the motive device 16, which when rotated may provide both the levitational and rotational force for the container 14 via the magnetic coupling or pinning with the magnets 18. The details may be found in one or more of U.S. Pat. Nos. 6,416,215; 6,758,593; 6,837,613; 6,965,288 and 6,899,454, the disclosures of which are incorporated herein by reference. However, it is also possible to form other types of magnetic couplings, such as by using electromagnets or the like, that may achieve the levitation and rotation. Such systems are detailed in, for example, U.S. Pat. No. 5,141,327, the disclosure of which is incorporated herein by reference.
When the suspension is introduced into the rotating container 14, the interior compartment receives the liquid. The cells in this liquid are caused to move outwardly as the result of centrifugal force created by the rotation of the container 14. The extraction conduit formed by tube 12b is mounted adjacent to the periphery of the container 14, such as along the sidewall 14b. A pump (not shown) associated with the tube 12b may be used to apply a negative pressure and extract cell-rich liquid from the periphery of the container 14.
To provide continuous operation, it should be appreciated that the liquid will eventually line the vertical sides of the container 14 and may overflow from the open top. This liquid, which should be generally free of cells, flows into the interior compartment of the surrounding vessel 12. This liquid may be drained from the vessel 12 through tube 14c, and may be discarded or subjected to further processing (such as by recycling it to the inlet I). In one particular embodiment, shown in
Once processing is complete, the vessel 12 including the container 14 may be discarded. As should be appreciated, this single-use arrangement allows for these combined structures to be made of inexpensive disposable materials, which advantageously eliminates the risk of cross-contamination and cleaning costs. The vessel 12 including the container 14 along with the various connections for conveying fluid may also be provided as part of a cartridge for integrating with a system including other disposable components, such as perhaps a bioreactor or like cell culture device (see
While the vessel 12 is described as being rigid or semi-rigid, it could take the form of a flexible bag 112 or the like, as shown in
In an alternative embodiment, the container 114 may be arranged to be supported by a physical or mechanical bearing. For example, a roller bearing 120 may be provided between the magnet 118 and the rigid portion 112d (or, alternatively, between the matrix material M and the rigid portion 112d, or with the magnet 118 or the matrix material M and the retainer 112e). The bearing 120 may comprise a race 120a for retaining a rolling element, such as a ball 120b, roller, or the like. In such case, the motive device 116 need not supply a levitative force, but may instead serve to transmit rotational torque only (and thus may comprise a rotating magnet or like structure forming a non-contact coupling through the vessel 112). Examples of such bearing arrangements may be found in U.S. Patent Application Publication No. 20100157752, the disclosure of which is incorporated herein by reference. A removable retaining element 122 may also be provided for retaining the container 114.
Another possible embodiment of a centrifuge system 200 is shown in
To correspond to the ring-shaped levitation magnet, the motive device includes a superconducting element 210 that is generally annular. This element 210 can be fabricated of a single unitary piece of a high-temperature superconducting material (YBCO or the like), or may be comprised of a plurality of component parts or segments. Upon being cooled to the transition temperature in the presence of a magnetic field and aligning with the ring-shaped permanent magnet 206 producing the same magnetic field, the superconducting ring 210 thus provides the combined repulsive/attractive, spring-like pinning force that levitates the container 204 in the vessel 202 in an exceptionally stable and reliable fashion. In
As in other embodiments described, a motive device is used to impart rotary motion to the container 204, and may be positioned adjacent to and concentric with the annular superconducting element 210. One example of a motive device for use in the system 200 of this third embodiment includes driving magnets 212a, 212b that correspond to the driven magnets 208a, 208b on the container 204 and having opposite polarities to create a magnetic coupling. The driving magnets 212a, 212b may be coupled to a shaft 214 also forming part of the motive device. The driving magnets 212a, 212b may be attached directly to the shaft 214, or as illustrated in
The arrangement is this embodiment may be used in connection with specific process parameters to ensure optimum efficiency (e.g., maximum cell separation with minimum destruction). The volume of the container may be between about 100 ml and about 300 ml, and in particular about 135 ml. The corresponding flow may be less than one liter per minute, and may be in the range of about 250 milliliters per minute (0.25 L/min) to about 500 milliliters per minute (0.5 ml/min).
In the illustrated embodiment, no extraction conduit is located in the same position as the above-described arrangements. Accordingly, the segregated cells L may be collected at the end of the centrifugation process. This recovery may be aided by using a washing step (e.g., using a trypsinisation solution comprising 1.55 L tryposin (an enzyme) to release the cells and 7.45 L of a PBS buffer solution to keep the cells alive) that have accumulated on the container walls. The container 314 may be a single use component (e.g., a disposable bag or liner), and thus may be discarded after cell recovery.
The foregoing descriptions of several embodiments made according to the disclosure of certain inventive principles are presented for purposes of illustration and description. The embodiments described are not intended to be exhaustive or to limit the invention to the precise form disclosed and, in fact, any combination of the components of the disclosed embodiments is contemplated. The term “flexible” as used herein in the context of the vessel refers to a structure of the vessel that, in the absence of auxiliary support, may conform to the shape of the fluid contained in the vessel, as contrasted with a “rigid” structure, which retains a pre-determined shape when the fluid is in the vessel. Modifications or variations are possible in light of the above teachings. For example, various materials may be used to form the vessel in any combination, including polymers (such as, for example, polypropylene for any flexible portions, and high density polyethylene for any rigid portions). The embodiments described were chosen to provide the best illustration of the principles of the invention and its practical application to thereby enable one of ordinary skill in the art to utilize the invention in various embodiments and with various modifications as are suited to the particular use contemplated. All such modifications and variations are within the scope of the invention when interpreted in accordance with the breadth to which it is fairly, legally, and equitably entitled.
This application claims the benefit of and incorporates by reference U.S. Provisional Patent Application Ser. No. 61/594,077.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2013/024530 | 2/2/2013 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 61594077 | Feb 2012 | US |
