Patent Description:
Blood transfusions are used to treat many disorders and injuries, such as in the treatment of accident victims and during surgical procedures. According to current American Red Cross statistics, about <NUM> million people receive blood transfusions each year, in the United States alone. Thus, health care systems rely on the collection and distribution of blood. Typically, blood is obtained from a donor and then processed and stored; units of stored blood or blood products are then taken from storage as needed and transfused into a patient in need. In some cases, the blood may be an autologous donation, where an individual donates blood in expectation of receiving his or her own blood by transfusion during a medical procedure.

Donated blood is typically processed into components and then placed in storage until needed. When a subject is in need of a blood transfusion, a unit of blood is commonly removed from storage, rejuvenated, washed, and resuspended in an appropriate solution. A system for separating cells is described in <CIT>. In some instances, the red blood cells were lyophilized prior to storage, in which case they need to be resuspended, washed, and then resuspended again in an appropriate solution. The resuspended red blood cells are then transfused into the subject. In either scenario, washing the red blood cells is traditionally a tedious, time consuming and multistep process that requires a great deal of tubing, and the use of expensive centrifuges with rotating seals to separate the cells from the wash solution. Therefore, there remains a need to streamline and simplify the process for washing red blood cells prior to transfusion.

Preferred features are set out in the sub-claims. Various embodiments are included in the description, not all of which are encompassed by the claims. This Summary section provides a general summary of the disclosure, and is not a comprehensive disclosure of its full scope or all of its features.

The present technology provides a method for washing a suspension of cells, such as a plurality of cells in a suspension fluid. Methods are not encompassed by the claims. In one embodiment, the plurality of cells includes red blood cells and the suspension fluid includes an enhancement composition. The method includes introducing a wash solution into a compartment of a device having a cylindrical inner wall and rotating the device to a first centripetal force that causes the wash solution to form a layer of wash solution against the inner wall. The method also includes introducing a suspension of cells into the compartment. Rotating the device to a second centripetal force causes the suspension of cells to form a layer of suspended cells adjacent to the layer of washing solution. The method additionally includes rotating the device to a third centripetal force that causes the cells to pass through and displace the layer of wash solution to generate a layer of clean cells adjacent to the inner wall. The method also includes collecting the clean suspension of cells from the device.

A device for washing a suspension of cells is also provided, as defined in claim <NUM>. Methods for using the device are also provided, but are not encompassed by the claims.

Also provided is another device for washing a suspension of cells, which is disclosed for illustration purposes but is not encompassed by the claims. The device includes a housing that defines a cylindrical wall that extends about and along a central longitudinal axis from a first surface of a first end to a second surface of a second end. The device also includes a first planar separator and a second planar separator. The first and second planar separators are orthogonal to the axis and the wall. Additionally, the first and second planar separators define consecutive first, second, and third compartments from the first end to the second end. A first inlet is positioned on the first end and is in fluid communication with the first compartment. A second inlet is positioned on the first end and is in fluid communication with the second compartment by way of a conduit. Methods for using the device are also provided, but are not encompassed by the claims.

The present disclosure generally provides devices and methods (unclaimed) for washing a suspension of cells. The devices and methods rely on centripetal force to pass a suspension of cells through a wash solution. The cells are then isolated from the wash solution. Accordingly, the devices according to the present technology do not require much, if any, tubing. Rotating seals are not required in the device and the only movements necessary are spinning of a rotor and depression of a plunger or opening of at least one valve as described in detail below. The devices provide quick and easy methods for washing a suspension of cells, which, for example, can be administered to a human or other animal subject in need thereof.

The devices can be used to separate a component from a multicomponent mixture. For example, cells can be separated from a multicomponent mixture in order to separate the cells from an unwanted component, which results in washed cells. Different types of cells are often treated with various compositions, which results in a need to separate the cells from treatment solutions and particulate matter, such as cell fragments and cellular debris. According to the present technology, cells can be washed in any wash solution commonly known in the art. However, in various embodiments the wash solution is not less dense than the fluid in which the cells are suspended. In other embodiments, the wash solution is denser and/or has a higher specific gravity than the fluid in which the cells are suspended. Non-limiting examples of wash solutions include water, saline, dextrose, saline with <NUM>% dextrose, dextran <NUM> (such as, for example, <NUM>% dextran <NUM> in <NUM>% sodium chloride or in <NUM>% dextrose), hetastarch solutions (such as, for example, <NUM>% hetastarch in <NUM>% sodium chloride), phosphate buffered saline, and other solutions that are used to remove unwanted components from cells. The cells may then be administered to a subject, such as a human or non-human mammal, or otherwise manipulated or stored. Non-limiting examples of cells that can be treated and washed include cells in whole blood, red blood cells, platelets, adipocytes, chondrocytes, and mixtures thereof. For example, because stored red blood cells (RBCs) have a diminished capacity to oxygenate tissues, a suspension of RBCs removed from storage can be rejuvenated by adding an enhancement composition, such as Rejuvesol® Red Blood Cell Processing Solution (Citra Labs, LLC, Braintree, MA), to the RBCs to form a multicomponent mixture or a suspension of cells. The suspension of cells including rejuvenated RBCs and a wash solution are then introduced into a device provided herein, wherein the RBCs are washed. During the wash, the RBCs are separated and isolated from the enhancement composition and optionally from unwanted cellular debris. The RBCs can then be used as concentrated RBCs or they can be resuspended in a reconstitution solution to achieve a desired RBC concentration.

The present disclosure provides a method for washing a suspension of cells. This method is included for illustration purposes and is not encompassed by the claimed invention. The suspension of cells includes a suspension fluid and a plurality of cells, such as red blood cells. The method comprises introducing a wash solution into a cylindrical compartment of a device having a cylindrical inner wall that extends about and along a longitudinal central axis from a first surface at a first end to a second surface at a second end. In some embodiments, the wash solution is selected from the group consisting of water, saline, dextrose, saline with <NUM>% dextrose, dextran <NUM> (such as, for example, <NUM>% dextran <NUM> in <NUM>% sodium chloride or in <NUM>% dextrose), hetastarch solutions (such as, for example, <NUM>% hetastarch in <NUM>% sodium chloride), and phosphate buffered saline. The method then comprises rotating the device about the center axis at a first speed to generate a first centripetal force. The first centripetal force causes the wash solution to push against the inner wall to form a layer of wash solution at the inner wall. Then, the method includes introducing the suspension of cells into the cylindrical compartment and rotating the device at a second speed to generate a second centripetal force. The second centripetal force is greater than the first centripetal force. The second centripetal force causes the suspension of red blood cells to push against the layer of wash solution to form a layer of suspended cells adjacent to the layer of wash solution. In various embodiments, the density and/or specific gravity of the wash solution is equal to or greater than the density and/or specific gravity of the suspension fluid, which facilitates formation of two distinct layers, i.e., the layer of wash solution and the layer of suspended cells, which are separated by an interface. The denser the wash solution is relative to the suspension fluid, the more defined the interface between the wash solution and suspension of cells will be. The method then comprises rotating the device at a third speed to generate a third centripetal force. The third centripetal force is greater than the second centripetal force and causes the cells in the layer of suspended cells to migrate through the interface, through the layer of wash solution, and to the inner wall. This migration results in separating the red blood cells from the suspension of red blood cells and the wash solution and displacing the layer of wash solution to form a layer of clean cells. The method also includes isolating the clean cells by removing the wash solution and suspension fluid from the compartment. Finally, the method includes removing the isolated clean cells from the device.

A device <NUM> according to the present technology, which can be used to perform the unclaimed method for washing a suspension of cells, is shown in <FIG>. 1B and 1C show cross-sectional perspectives of the device <NUM> of <FIG> taken along lines 1B and 1C, respectively. The device <NUM> comprises a housing <NUM> that defines a cylindrical outer wall <NUM> that extends about and along a central longitudinal axis <NUM> from a first inner surface <NUM> at a first end <NUM> to a second inner surface <NUM> at a second end <NUM>. The first end <NUM> includes a first outer surface <NUM> and the second end <NUM> includes a second outer surface <NUM>. In various embodiments, the first and second inner and outer surfaces <NUM>, <NUM>, <NUM>, <NUM> are planar. The device <NUM> also includes an inner plate <NUM> that has a third inner surface <NUM>. The inner plate <NUM> is positioned between the first and second ends <NUM>, <NUM>. The inner plate <NUM> is planar and orthogonal to the axis <NUM>, and bifurcates the device <NUM> into an inner washing region <NUM> and an inner loading region <NUM>. Also, the device <NUM> includes a cylindrical inner wall <NUM> that extends about and along the axis <NUM> from the first inner surface <NUM> to the third inner surface <NUM> in the washing region <NUM> of the device <NUM>. The first inner wall <NUM> defines a first center compartment <NUM> and a second compartment <NUM> in the washing region <NUM>, wherein the second compartment <NUM> is adjacent to and concentric with, i.e., they share the same central axis <NUM>, the center compartment <NUM>.

The device <NUM> also comprises a first inlet port <NUM> and an outlet port <NUM>. The first inlet port <NUM> is positioned at the first end <NUM> and is in fluid communication with the first compartment <NUM>. The outlet port <NUM> is also positioned at the first end <NUM>, but is in fluid communication with the first compartment <NUM>. However, in various embodiments, the outlet port <NUM> is in fluid communication with the first compartment <NUM> by way of a conduit <NUM> that extends to or near the third inner surface <NUM>, such that when gravity pulls a composition, solution, or collection of cells to the third inner surface <NUM>, all or most of the composition, solution, or collection of cells can be removed from the device <NUM> through the outlet port <NUM>.

Additionally, the device <NUM> comprises a second inlet port <NUM> for introducing a suspension of cells into the device <NUM> and a plunger assembly <NUM> that includes a barrel <NUM> and a plunger <NUM>. In <FIG>, the second inlet port <NUM> is positioned at the outer wall <NUM> of the device <NUM>. However, in other embodiments, the second inlet port <NUM> may be positioned on another surface, such as the second outer surface <NUM>. The plunger assembly <NUM> is positioned in the loading region <NUM> of the device <NUM>. Therefore, in any configuration, the inlet port <NUM> is in fluid communication with the barrel <NUM> of the plunger assembly, such that a suspension of cells can be loaded into the barrel <NUM> of the plunger assembly <NUM> by way of the second inlet port <NUM>. However, once loaded, the contents of the barrel <NUM> cannot flow back and out of the device <NUM> through the second inlet port <NUM>. For example, the second inlet port can be capped or plugged to prevent leaking after the barrel <NUM> is loaded. Although the first and second inlet ports <NUM>, <NUM> and the outlet port <NUM> are shown protruding from the device <NUM>, it is understood that all the ports <NUM>, <NUM>, <NUM> can be in line, i.e., flat, with their respective surfaces to preclude protrusions when the device <NUM> is in use.

In various embodiments, a first valve assembly <NUM> comprising a tubular body <NUM> that extends from a first end <NUM> to a second end <NUM> and a first internal valve (not shown) is positioned between the barrel <NUM> of the plunger assembly <NUM> and the first compartment <NUM>, such that the barrel <NUM> is in fluid communication with the first compartment <NUM> when the valve is open. The valve of the first valve assembly <NUM> is normally in a closed position prior to use. During use, the valve is actuated to an open position mechanically, electrically, or magnetically, at a predetermined centripetal force or pressure. Non-limiting examples of valves that are actuated by centripetal force and that are suitable for the first valve assembly <NUM> are described in <CIT> on August <NUM>, <NUM>, and <CIT>. In some embodiments the valve of the valve assembly <NUM> is opened when the device <NUM> is rotated or spun about axis <NUM> to a speed in which a first centripetal force is reached. At or about the same speed and force, the plunger <NUM> of the plunger assembly <NUM> is actuated to introduce, for example, a suspension of cells into the first compartment <NUM> at a slow and steady rate. The speed at which the first valve assembly <NUM> opens can be from about <NUM> rpm to about <NUM>, or from about <NUM> rpm to about <NUM> rpm. In one embodiment, the valve <NUM> opens at a speed of about <NUM> rpm. In some embodiments, a baffle or dampener (not shown) is coupled to the second end <NUM> of the first valve assembly <NUM> to facilitate slow and gentle entry of the suspension of cells into the first compartment <NUM> such that the suspension of cells can be layered adjacent to a layer of wash solution, as described further below.

The device <NUM> further comprises a second valve assembly <NUM> having a tubular body <NUM> that extends from a first end <NUM> to a second end <NUM> and a second internal valve (not shown). In various embodiments, the device <NUM> comprises at least one of second valve assemblies <NUM>. The second valve assembly <NUM> is positioned through the first inner wall <NUM>, such that the first compartment <NUM> is in fluid communication with the second compartment <NUM> when the second valve is open. In some embodiments, the valve assembly <NUM> extends into the first compartment <NUM> and toward the central axis <NUM>. As described further below, when the device is in use, the first end <NUM> of the valve assembly <NUM> is positioned at or near an interface between a suspension of cells and a wash solution. In this regard, the length of the body <NUM> is tuned based on the volume of cells loaded into the device <NUM>. Therefore, device <NUM> can be made with various body <NUM> lengths. A user can select an appropriate device <NUM> based on the volume of cells to be washed. The second end <NUM> of the valve assembly <NUM> can either be in line with the inner wall <NUM> or it can extend radially into the second compartment <NUM>. The second valve of the valve assembly <NUM> is normally in a closed position when the device <NUM> is not in use. During use, the second valve is actuated to an open position mechanically, electrically, or magnetically, at a predetermined centripetal force or pressure. Non-limiting examples of valves that are actuated by centripetal force and that are suitable for the second valve assembly <NUM> are described in <CIT>. on August <NUM>, <NUM>, and <CIT>. The second valve of the second valve assembly <NUM> is opened when the device <NUM> is rotated or spun to a speed in which a second centripetal force is reached. The speed at which the second valve assembly <NUM> opens can be from about <NUM> rpm to about <NUM>, or from about <NUM> rpm to about <NUM> rpm. In various embodiments, the valve <NUM> opens at a speed of about <NUM> rpm or at a speed of about <NUM> rpm. The second centripetal force is greater than the first critical centripetal force as described further below.

The device <NUM> is configured to be received by a base unit <NUM> that comprises a rotor. When engaged with the base unit <NUM>, the base unit <NUM> is capable of spinning the device <NUM> about the axis <NUM> at various speeds to generate various centripetal forces.

With reference to <FIG>, a device <NUM>' is similar to the device <NUM> shown in <FIG>. However, in regard to the device <NUM>', the first inner wall <NUM> is positioned at a distance D1 from the axis and the device <NUM>' has a housing <NUM>' that further defines a porous screen <NUM> that extends about and along the longitudinal axis <NUM> from the first surface <NUM> to the third surface <NUM> in the washing region <NUM>. The porous screen <NUM> is positioned at a distance D2 from the axis <NUM>, wherein D2 is shorter than D1. Therefore, the first inner wall <NUM> is concentric with the porous screen <NUM>. In various embodiments, the position of the porous screen <NUM> is turned based on the volume of wash solution to be loaded into the device <NUM>. For example, as the device <NUM>' rotates about the axis <NUM>, wash solution is forced against the first inner wall <NUM> to generate a surface that opposes the inner wall <NUM>. The porous screen <NUM> is positioned at the surface of the wash solution opposing the first inner wall <NUM>. Therefore various devices <NUM>' can be made with the porous screen <NUM> located at various distances D2 from the axis <NUM>. A user can select an appropriate device <NUM>' based on the volume of wash solution to be used.

The porous screen <NUM> comprises pores that are sufficiently large to allow cells to pass through and sufficiently closely spaced such that cells will not jam or pile up significantly between the pores. In various embodiments, the porous screen <NUM> has pores with a mass cut off of from about <NUM> kDa to about <NUM> kDa, or from about <NUM> kDa to about <NUM> kDa. In one embodiment, the screen <NUM> has pores with a mass cutoff of about <NUM> kDa. However, as described in more detail below, the pores should be sufficiently restrictive so that the screen <NUM> has a sufficient surface area to aid a suspension of cells to layer against a wash solution when the device <NUM>' is in use. Therefore, the screen <NUM> can be a screen, frit, or open cell foam or mat. Accordingly, the screen <NUM> is positioned such that it will be immediately adjacent to, and in contact with, a surface of a wash solution when the device is rotating about the axis <NUM>. In such embodiments, the second valve assembly <NUM> is positioned such that the first end <NUM> protrudes into the first compartment <NUM> at least to the screen <NUM>.

With reference to <FIG>, the current disclosure provides a method (unclaimed) for washing a suspension of cells with the device <NUM>. As shown in <FIG>, the method comprises loading or introducing a suspension of cells <NUM> into the barrel <NUM> of the plunger assembly <NUM> by way of the second inlet <NUM>. The suspension of cells <NUM> comprises a suspension fluid <NUM> (which may include an enhancement composition) and a plurality of cells <NUM>. As described above, the suspension of cells <NUM> can be a suspension of any cells that requires washing. In one embodiment, the suspension of cells comprises red blood cells and an enhancement composition. In another embodiment, the barrel <NUM> is pre-loaded with an enhancement composition and red blood cells are loaded into the barrel <NUM> via the second inlet <NUM> where the red blood cells are incubated and enhanced to generate the suspension of cells <NUM>. The method also comprises loading or introducing a wash solution <NUM> into the first compartment <NUM> by way of the first inlet port <NUM>. Any wash solution described herein can be used to wash the suspension of cells. However, in some embodiments the wash solution <NUM> has a density or specific gravity equal to or greater than the density or specific gravity of the suspension fluid <NUM>. By having a wash solution <NUM> with higher density or specific gravity than the suspension fluid <NUM>, a sharp and distinct interface forms between the wash solution <NUM> and the suspension of cells <NUM>. After loading, the inlets <NUM>, <NUM> are capped or plugged to prevent the respective solutions <NUM>, <NUM> from leaking. Alternatively, in some embodiments the inlets <NUM>, <NUM> are configured to prevent flow-back, such as with one-way inlet valves.

With reference to <FIG>, the method comprises rotating the device <NUM> about the central axis <NUM> at a first speed to generate a first centripetal force by using the base unit <NUM>. As the device <NUM> rotates, the first centripetal force causes the wash solution <NUM> to form a layer of wash solution <NUM> against the inner wall <NUM>. Then, the device <NUM> is rotated at a second speed that is faster than the first speed until a second centripetal force is reached. As shown in <FIG>, at a predefined centripetal force, the first internal valve of the first valve assembly <NUM> is opened, such as at a centripetal force in which the wash solution <NUM> has formed a layer <NUM> adjacent to the inner wall <NUM>. At or near the same centripetal force, the plunger <NUM> of the plunger assembly <NUM> is actuated and depressed to slowly introduce the suspension of cells <NUM> into the first compartment <NUM>. In various embodiments, the suspension of cells <NUM> is introduced into the first compartment <NUM> at or near the central axis <NUM>. The centripetal force then causes the suspension of cells <NUM> to form a cell suspension layer <NUM> adjacent to the layer of wash solution <NUM>, wherein the cell suspension layer <NUM> is closer to the central axis <NUM> than the layer of wash solution <NUM>.

In embodiments including device <NUM>' shown in <FIG>, a volume of wash solution <NUM> is added such that it occupies a space between the inner wall <NUM> and the screen <NUM>, such that the screen <NUM> is at a surface of the layer of wash solution <NUM> that opposes the first inner wall <NUM>. The screen <NUM> encourages the suspension of cells <NUM> to form the cell suspension layer <NUM> against the layer of wash solution <NUM>, such that the screen <NUM> is located at an interface between the layers <NUM>, <NUM>.

With reference to <FIG>, the speed of rotation is increased to a third speed that is faster than the second speed to generate a third centripetal force. In various embodiments, the third speed is from about <NUM> rpm to about <NUM> rpm. In one embodiment, the third speed is about <NUM> rpm. Here, the cells <NUM> in the cell suspension layer <NUM> pass through the layer of wash solution <NUM>, thus separating the cells <NUM> from the suspension fluid <NUM> and wash solution <NUM> and forming a layer of clean cells <NUM> against the inner wall <NUM>. As the cells <NUM> pass through the layer of wash solution <NUM>, the cells <NUM> displace the layer of wash solution <NUM> and the wash solution <NUM> may blend with the suspension fluid <NUM>. When the device <NUM>' of <FIG> is used, the cells <NUM> pass through the pores of the screen <NUM> and then are forced to the inner wall <NUM>.

As shown in <FIG>, the method includes rotating the device <NUM> to a fourth speed that is faster than the third speed, until a fourth centripetal force is reached. At this centripetal force, or when all the cells <NUM> have passed through the layer of wash solution <NUM>, the second interval valve in the second valve assembly <NUM> opens. Because the second valve assembly <NUM> extends into the first compartment <NUM> to, or about to, a surface <NUM> of the layer of cells <NUM>, as described above, wash solution <NUM> and suspension fluid <NUM> flow through the second valve and into the second compartment <NUM>, thus isolating the cells <NUM> from the wash solution <NUM> and suspension fluid <NUM>. Loss of cells <NUM> can be minimized by ensuring that not too many cells are introduced into the device <NUM> such that the layer of cells <NUM> extends beyond the second valve assembly <NUM>. When all of the wash solution <NUM> and suspension fluid <NUM> has entered the second compartment <NUM>, rotation of the device <NUM> is stopped. As shown in <FIG>, the wash solution <NUM> and suspension fluid <NUM> is isolated in the second compartment <NUM> and the clean cells <NUM> are isolated in the first center compartment <NUM>. The method then comprises removing the clean cells <NUM> from the device <NUM> through the conduit <NUM> and outlet port <NUM>. Optionally, the clean cells <NUM> can be stored or administered to a subject in need thereof with or without reconstitution. By performing the current method, a suspension of cells can be cleaned in from about <NUM> minutes to about <NUM> minutes, or in from about <NUM> minutes to about <NUM> minutes.

Another device <NUM> that can be used in the unclaimed method for washing a suspension of cells is shown in <FIG>. This embodiment is included for illustration purposes and is not encompassed by the claimed invention. <FIG> shows a cross-sectional view of the device <NUM> of <FIG> taken along line 9B. The device <NUM> comprises a housing <NUM> that defines a cylindrical outer wall <NUM> that extends about and along a central longitudinal axis <NUM> from a first inner surface <NUM> of a first end <NUM> to a second inner surface <NUM> of a second end <NUM>. The first end <NUM> includes a first outer surface <NUM> and the second end <NUM> includes a second outer surface <NUM>. In various embodiments, the first and second inner and outer surfaces <NUM>, <NUM>, <NUM>, <NUM> are planar. The device <NUM> also includes a first cylindrical inner wall <NUM> and a second cylindrical inner wall <NUM> that extend about and along the axis <NUM> from the first inner surface <NUM> to the second inner surface <NUM>. The first inner wall <NUM> defines a first central compartment <NUM>. The second cylindrical wall <NUM> defines a second annular compartment <NUM> and a third annular compartment <NUM>. The second compartment <NUM> is between the first compartment <NUM> and the third compartment <NUM>. Additionally, the second and third compartments <NUM>, <NUM> are concentric with, i.e., they share the same central axis <NUM>, the first central compartment <NUM>. Also, the device includes a relief compartment <NUM> that is adjacent to the second outer surface <NUM> and that extends about the central axis <NUM>. The relief compartment <NUM> can have any cross-sectional geometry. However, in one embodiment, the relief compartment <NUM> has a three-dimensional shape of a cone and has a cross-sectional geometry of a triangle, such that it defines a collection point or sump <NUM>. The relief compartment <NUM> is in fluid communication with the second compartment <NUM> by means of at least one aperture, or an annular opening <NUM>.

The device <NUM> also comprises a first inlet port <NUM> and a second inlet port <NUM> positioned at the first end <NUM>. The first and second inlet ports <NUM>, <NUM> are in fluid communication with the first compartment <NUM> and the second compartment <NUM>, respectively. Also, the device <NUM> includes an outlet port <NUM>. The outlet port <NUM> is in fluid communication with the relief compartment <NUM> by way of a conduit <NUM> that traverses the first compartment <NUM>. In one embodiment, the conduit <NUM> extends to or near the collection point or sump <NUM> of the relief compartment. Due to the aperture or opening <NUM>, the outlet port <NUM> can optionally be used as an inlet port, equivalent to the second inlet port <NUM>, for introducing a substance to the second compartment <NUM>. Although the first and second inlet ports <NUM>, <NUM> and the outlet port <NUM> are shown protruding from the device <NUM>, it is understood that all the ports <NUM>, <NUM>, <NUM> can be in line, i.e., flat, with the first surface <NUM> to preclude protrusions when the device <NUM> is in use.

The device <NUM> also comprises a first valve assembly <NUM> comprising a tubular body <NUM> that extends from a first end <NUM> to a second end <NUM> and a first internal valve (not shown) positioned through the first inner wall <NUM>, such that the first compartment <NUM> is in fluid communication with the second compartment <NUM> when the first valve is open. The valve of the first valve assembly <NUM> is closed prior to use of the device. During use, the first valve is actuated to an open position mechanically, electrically, or magnetically, at a predetermined centripetal force or pressure. Non-limiting examples of valves that are actuated by centripetal force and that are suitable for the first valve assembly <NUM> are described in <CIT>. on August <NUM>, <NUM>, and <CIT>. In some embodiments the first valve of the first valve assembly <NUM> is opened when the device <NUM> is rotated or spun to a speed in which a first centripetal force is reached. The speed at which the first valve assembly <NUM> opens can be from about <NUM> rpm to about <NUM>, or from about <NUM> rpm to about <NUM> rpm. In one embodiment, the valve <NUM> opens at a speed of about <NUM> rpm. In one embodiment, the tubular body <NUM> of the first valve assembly <NUM> extends into the second compartment <NUM> to facilitate layering of cells against a layer of wash solution. In various embodiments, a baffle or dampener (not shown) is coupled to the second end <NUM> of the first valve assembly to facilitate gentle and efficient layering of a suspension of cells against a layer of a wash solution. The first valve assembly <NUM> is positioned anywhere along the first inner wall <NUM>. However, in some embodiments, the first valve assembly <NUM> is positioned through the first inner wall <NUM> at or near the second surface <NUM>.

The device <NUM> also comprises a second valve assembly <NUM> comprising a tubular body <NUM> that extends from a first end <NUM> to a second end <NUM> and a second internal valve (not shown) positioned through the second inner wall <NUM>, such that the second compartment <NUM> is in fluid communication with the third compartment <NUM> when the second valve is open. The valve of the second valve assembly <NUM> is normally closed prior to use of the device. During use, the second valve is actuated to an open position mechanically, electrically, or magnetically, at a predetermined centripetal force or pressure. Non-limiting examples of valves that are actuated by centripetal force and that are suitable for the second valve assembly <NUM> are described in <CIT>. on August <NUM>, <NUM>, and <CIT>. In some embodiments the second valve of the second valve assembly <NUM> is opened when the device <NUM> is rotated or spun to a speed in which a second centripetal force is reached. The speed at which the second valve assembly <NUM> opens can be from about <NUM> rpm to about <NUM>, or from about <NUM> rpm to about <NUM> rpm. In various embodiments, the valve of the second valve assembly <NUM> opens at a speed of about <NUM> rpm or at a speed of about <NUM> rpm. In one embodiment, the tubular body <NUM> of the second valve assembly <NUM> extends into the second compartment <NUM> such that the second end <NUM> is positioned at or near an interface between a layer of cells and a layer of wash solution when the device <NUM> is in use. In this regard, the length of the body <NUM> is tuned based on the volume of cells loaded into the device <NUM>. Therefore, devices <NUM> can be made with various body <NUM> lengths. A user can select an appropriate device <NUM> based on the volume of cells to be washed. The second valve assembly <NUM> is positioned anywhere along the second inner wall <NUM>. However, in some embodiments, the second valve assembly <NUM> is positioned through the second inner wall <NUM> at or near the first surface <NUM>. In various embodiments, the device <NUM> comprises at least one second valve assembly <NUM>.

In some embodiments, the device <NUM> further includes an optional porous screen <NUM> that extends about and along the longitudinal axis <NUM> from the first inner surface <NUM> to the second inner surface <NUM>. The optional porous screen <NUM> is positioned between the first and second inner walls <NUM>, <NUM>. Accordingly, the porous screen <NUM> is concentric with the first and second inner walls <NUM>, <NUM>. In various embodiments, the position of the porous screen <NUM> is turned based on the volume of wash solution to be loaded into the device <NUM>. For example, as this device <NUM> rotates about the axis <NUM>, wash solution is forced against the first inner wall <NUM> to generate a surface that opposes the inner wall <NUM>. The porous screen <NUM> is positioned at the surface of the wash solution opposing the first inner wall <NUM>. Therefore various devices <NUM> can be made with the porous screen <NUM> located at various distances D2 from the axis <NUM>. A user can select an appropriate device <NUM> based on the volume of wash solution to be used. The porous screen <NUM> comprises pores that allow cells and fluid to pass through and sufficiently closely spaced such that cells will not jam or pile up significantly between the pores. In various embodiments, the porous screen <NUM> has pores with a mass cutoff of from about <NUM> kDa to about <NUM> kDa, or from about <NUM> kDa to about <NUM> kDa. In one embodiment, the screen <NUM> has pores with a mass cutoff of about <NUM> kDa. However, as described in more detail below, the pores should be sufficiently restrictive so that the screen <NUM> has a sufficient surface area to aid a suspension of cells to layer against a wash solution when the device <NUM> is in use. Therefore, the screen <NUM> can be a screen, frit, or open cell foam or mat. Accordingly, the screen <NUM> is positioned such that it will be immediately adjacent to, and/or contact with, a surface of a wash solution when the device <NUM> is rotating about the axis <NUM>. In such embodiments, the second valve assembly <NUM> is positioned such that the second end <NUM> protrudes into the second compartment <NUM> at least to the screen <NUM>.

The device <NUM> is configured to be received by a base unit <NUM> that comprises a rotor (not shown). When engaged with the base unit <NUM>, the base unit <NUM> is capable of spinning the device <NUM> about the axis <NUM> at various speeds to generate various centripetal forces.

With reference to <FIG>, the current disclosure provides a method for washing a suspension of cells with the device <NUM>. This embodiment is included for illustration purposes and is not encompassed by the claimed invention. As shown in <FIG>, the method comprises loading or introducing a wash solution <NUM> into the second compartment <NUM> of the device <NUM> by way of the second inlet port <NUM>. In various embodiments, the wash solution <NUM> is selected from the group consisting of water, saline, dextrose, saline with <NUM>% dextrose, dextran <NUM> (such as, for example, <NUM>% dextran <NUM> in <NUM>% sodium chloride or in <NUM>% dextrose), hetastarch solutions (such as, for example, <NUM>% hetastarch in <NUM>% sodium chloride), and phosphate buffered saline. The wash solution <NUM> flows through the opening or aperture <NUM> and into the relief compartment <NUM>. Nonetheless, as shown in <FIG>, the volume of the wash solution <NUM> is sufficient to fill the relief compartment <NUM> and a portion of the second compartment <NUM>. In an alternative embodiment, the wash solution <NUM> is introduced to the device <NUM> by way of the outlet <NUM> and its corresponding conduit <NUM>.

As shown in <FIG>, the method also includes loading or introducing a suspension of cells <NUM> into the first compartment <NUM> of the device <NUM> by way of the first inlet port <NUM>. The suspension of cells <NUM> includes a suspension fluid <NUM> and a plurality of cells <NUM>. In various embodiments, the plurality of cells <NUM> is selected from the group consisting of red blood cells, white blood cells, platelets, and combinations thereof. The suspension of cells <NUM> may also include cell fragments and cellular debris. The suspension fluid <NUM> comprises a fluid for suspending the plurality of cells <NUM> and, in some embodiments, an enhancement composition. In some embodiments, an enhancement composition is preloaded into the device <NUM> through inlet <NUM> and a solution of cells is then added into the device through the first inlet <NUM> to generate the suspension of cells <NUM>. The suspension of cells <NUM> is then incubated for a predetermined period of time. Because the first valve in the first valve assembly <NUM> is closed, the suspension of cells will not flow into the second compartment <NUM> and mix with the wash solution <NUM>. It is understood that the wash solution <NUM> and suspension of cells <NUM> can be loaded in any order.

With reference to <FIG>, the method comprises rotating or spinning the device <NUM> about the axis <NUM>. Rotating or spinning can be performed in clockwise or counter-clockwise directions. As the device <NUM> is spinning, centripetal force pushes the suspension of cells <NUM> against the first inner wall <NUM>. Likewise, the centripetal force pushes the wash solution <NUM> against the second inner wall <NUM> to generate a layer of wash solution <NUM>. When the screen <NUM> is present, the pores receive a portion of the layer of the wash solution <NUM>. For example, the device <NUM> can be spun at a speed of from about <NUM> to about <NUM> rpm in order to push the suspension of cells <NUM> against the first inner wall <NUM>. In one embodiment, the suspension of cells <NUM> are pushed against the first inner wall <NUM> when the device <NUM> reaches a speed of about <NUM> rpm.

As shown in <FIG>, the method includes opening the first valve of the first valve assembly <NUM> when a first centripetal force is reached, such as the centripetal force in which the suspension of cells <NUM> is pressed against the first inner wall <NUM> and the layer of wash solution <NUM> has formed at the at the second inner wall <NUM>. The suspension of cells <NUM> flows through the first valve assembly <NUM> and into the second chamber <NUM>. When the porous screen <NUM> is included in the device <NUM>, it has a sufficient surface area to facilitate layering of the suspension of cells <NUM> against the layer of wash solution <NUM> to generate a layer of suspended cells <NUM>. Nonetheless, the layer of suspended cells <NUM> also forms when the screen <NUM> is not included. In various embodiments, not shown a baffle or dampener is coupled to the first valve assembly <NUM> to facilitate slow and gentle flow of the suspension of cells into the second compartment <NUM>.

As shown in <FIG>, as the speed of rotation increases, centripetal force forces the plurality of cells <NUM> through the layer of wash solution <NUM> and against the second inner wall <NUM>. As the plurality of cells <NUM> passes through the layer of wash solution <NUM>, unwanted components, such as cell fragments, cellular debris, or and suspension fluid <NUM> stays behind and may blend into the layer of wash solution <NUM>. Thus a layer of clean cells <NUM> forms against the second inner wall <NUM>. Simultaneously, the layer of clean cells <NUM> displaces the layer of wash solution <NUM> so that an interface <NUM> is formed between the layer of clean cells <NUM> and the layer of wash solution <NUM>. The interface <NUM> is positioned such that the second end <NUM> of the second valve assembly <NUM> is at or near the interface <NUM>.

With reference to <FIG>, when the device <NUM> is rotated or spun at a speed that generates a second centripetal force or the centripetal force in which the layer of clean cells <NUM> has been completely formed, the method includes opening the second internal valve of the second valve assembly <NUM>. Because the second valve assembly <NUM> extends into the second compartment <NUM> to, or near, a surface <NUM> of the layer of cells <NUM>, as described above, the centripetal force then forces the wash solution <NUM> and the suspension fluid <NUM> through the second valve assembly and into the third compartment <NUM>, thus isolating the layer of clean cells <NUM>. As shown in <FIG>, when the device stops rotating, gravity pulls the clean cells <NUM> through the opening or apertures <NUM> and into the relief compartment <NUM>. The clean cells are then removed from the device <NUM> through the conduit <NUM> and outlet <NUM>. By performing the current method, a suspension of cells can be cleaned in from about <NUM> minutes to about <NUM> minutes or in from about <NUM> minutes to about <NUM> minutes.

The present disclosure provides another device <NUM> for washing a suspension of cells, as shown in <FIG>, which is not encompassed by the present claims. The device comprises a housing <NUM> that defines a cylindrical wall <NUM> that extends about and along a central longitudinal axis <NUM> from a first inner surface <NUM> of a first end <NUM> to a second inner surface <NUM> of a second end <NUM>. The device <NUM> includes a first planar separator <NUM> positioned orthogonal to the axis <NUM> and to the wall <NUM> and a second planar separator <NUM> positioned orthogonal to the axis <NUM> and to the wall <NUM>. The first and second separators <NUM>, <NUM> define a first compartment <NUM>, a second compartment <NUM>, and a third compartment <NUM> consecutively from the first end to the second end. Additionally, the device <NUM> comprises a first inlet <NUM> positioned at the first end <NUM> that is in fluid communication with the first compartment <NUM> and a bi-functional port <NUM> positioned at the first end <NUM> that is in fluid communication with the second compartment <NUM> through a conduit <NUM>. The conduit <NUM> extends to or near the first separator <NUM>. In an alternative embodiment (not shown), the bi-functional port <NUM> is a second inlet and the device further comprises an outlet port that is in fluid communication with the second compartment <NUM> by way of a second conduit.

The device <NUM> also include a first valve assembly <NUM> that includes a tubular body <NUM> that extends from a first end <NUM> to a second end <NUM>, and a first internal valve (not shown). Non-limiting examples of valves that are actuated by centripetal force and that are suitable for the first valve assembly <NUM> are described in <CIT>. on August <NUM>, <NUM>, and <CIT>. The first valve assembly <NUM> is positioned in the first separator <NUM> such that the first and second compartments <NUM>, <NUM> are in fluid communication when the first valve is open. Additionally, the device <NUM> includes a second valve assembly <NUM> that includes a tubular body <NUM> that extends from a first end <NUM> to a second end <NUM>, and a second internal valve (not shown). Non-limiting examples of valves that are actuated by centripetal force and that are suitable for the second valve assembly <NUM> are described in <CIT>. on August <NUM>, <NUM>, and <CIT>. The second valve assembly <NUM> is positioned in the second separator <NUM> such that the second and third compartments <NUM>, <NUM> are in fluid communication when the second valve is open. In various embodiments, the length of the body <NUM> is tuned based on the volume of cells to be loaded into the device <NUM>. After rotating the device <NUM>, the first end <NUM> of the body <NUM> is positioned, at or near an interface between cells and a wash solution. Devices <NUM> can be made with various tube body <NUM> lengths. A user can select an appropriate device <NUM> based on the volume of cells to be washed. The first and second valves of the first and second valve assemblies can be actuated mechanically, electrically, or magnetically, at a predetermined centripetal force or pressure. In one embodiment, the first valve of the first valve assembly <NUM> is configured to open at a first centripetal force and the second valve of the second valve assembly <NUM> is configured to open at a second centripetal force. Typically, the second centripetal force is greater than the first centripetal force. For example, the first valve can be opened when the device <NUM> reaches a speed of from about <NUM> rpm to about <NUM> rpm. And the second valve can be opened when the device <NUM> reaches a speed of from about <NUM> rpm to about <NUM> rpm. The device <NUM> is configured to be rotated by a centrifuge rotor.

A method for using the device <NUM> for washing a suspension of cells is also disclosed, but not encompassed by the claims. With reference to <FIG>, the method comprises introducing a wash solution <NUM> into the second compartment <NUM> of the device <NUM> through the bi-functional port <NUM> and its corresponding conduit <NUM>. Here, the bi-functional port <NUM> is used as a second inlet. In various embodiments, the wash solution <NUM> is selected from the group consisting of water, saline, dextrose, saline with <NUM>% dextrose, dextran <NUM> (such as, for example, <NUM>% dextran <NUM> in <NUM>% sodium chloride or in <NUM>% dextrose), hetastarch solutions (such as, for example, <NUM>% hetastarch in <NUM>% sodium chloride), and phosphate buffered saline. The method also includes introducing a suspension of cells <NUM> into the first compartment <NUM> through the first inlet <NUM>. The suspension of cells <NUM> comprises a suspension fluid <NUM> and a plurality of cells <NUM>. The suspension of cells <NUM> can also comprise cell fragments, cellular debris, or a combination thereof. The plurality of cells <NUM> can include, for example, red blood cells, white blood cells, platelets, or combinations thereof. In various embodiments, the density or specific gravity of the wash solution <NUM> is equal to or greater than the density or specific gravity of the suspension fluid <NUM>. By having a wash solution <NUM> with higher density or specific gravity than the suspension fluid <NUM>, a sharp and distinct interface forms between the wash solution <NUM> and the suspension of cells <NUM>. It is understood that introducing the wash solution <NUM> and suspension of cells <NUM> can be performed in any order. In one embodiment, the first compartment <NUM> is preloaded with an enhancement composition and cells <NUM> are introduced into the enhancement composition through the first inlet <NUM> to generate the suspension of cells <NUM> within the device <NUM>. The suspension of cells is incubated for a predetermined period of time and the method is thereafter resumed.

After loading the device <NUM>, the device <NUM> is placed into a centrifuge rotor that is placed within a centrifuge. The centrifuge rotor is balanced by a blank device, a second loaded device <NUM>, or a centrifuge tube of the same weight. The centrifuge is turned on and the rotor begins to spin. With reference to <FIG>, when a first centripetal force is reached, the first valve of the first valve assembly is opened. The suspension of cells <NUM> is pulled through the first valve and into the wash solution <NUM>. Because they are denser than the wash solution <NUM>, the cells <NUM> are pulled through the wash solution <NUM> and settle on the second separator <NUM>, thus displacing the wash solution <NUM> and separating the cells <NUM> from the suspension fluid <NUM> and wash solution <NUM>. The suspension fluid <NUM> may blend with the wash solution <NUM> depending on their relative densities and an interface <NUM> is formed between the cells <NUM> and the wash solution <NUM> blended with suspension fluid <NUM>. Accordingly, the cells <NUM> that settle on the second separator <NUM> are clean. As shown in <FIG>, the first end <NUM> of the second valve assembly <NUM> extends into the second compartment <NUM> to or near the interface <NUM>.

As shown in <FIG>, when a second centripetal force is reached, or at the centripetal force in which all the cells <NUM> have passed through the wash solution <NUM>, the second valve of the second valve assembly <NUM> is opened. The wash solution <NUM>, suspension fluid <NUM>, and other unwanted components, such as cell fragments and cellular debris, are pulled through the second valve assembly <NUM> and into the third compartment <NUM>, thus isolating the clean cells <NUM>. When the centrifuge is stopped, the method includes removing the clean cells <NUM> through the conduit <NUM> and the bi-functional port <NUM>, which is now used as an outlet. By performing the current method, a suspension of cells can be cleaned in from about <NUM> minutes to about <NUM> minutes or in from about <NUM> minutes to about <NUM> minutes.

Throughout this disclosure, the numerical values represent approximate measures or limits to ranges to encompass minor deviations from the given values and embodiments having about the value mentioned as well as those having exactly the value mentioned. Other than in the working examples provided at the end of the detailed description, all numerical values of parameters (e.g., of quantities or conditions) in this specification, including the appended claims, are to be understood as being modified in all instances by the term "about" whether or not "about" actually appears before the numerical value.

Claim 1:
A device (<NUM>, <NUM>') for washing a suspension of cells (<NUM>), the device comprising:
a housing (<NUM>) defining a cylindrical outer wall (<NUM>) that extends about and along a central longitudinal axis (<NUM>) from a first inner surface (<NUM>) of a first end (<NUM>) to a second inner surface (<NUM>),
a first outer surface (<NUM>) at the first end (<NUM>);
a first cylindrical inner wall (<NUM>) that extends about and along the longitudinal axis (<NUM>) from the first inner surface (<NUM>) to the second inner surface (<NUM>), the first cylindrical inner wall (<NUM>) defining a first compartment (<NUM>) and a second compartment (<NUM>), wherein the second compartment (<NUM>) is adjacent to and concentric with the first compartment (<NUM>);
a second valve assembly (<NUM>) including a second internal valve, the second valve assembly (<NUM>) positioned through the first cylindrical inner wall (<NUM>) such that the first compartment (<NUM>) is in fluid communication with the second compartment (<NUM>) when the second internal valve is open at a second centripetal force;
a first inlet port (<NUM>);
a plunger assembly (<NUM>) comprising a barrel (<NUM>), a plunger (<NUM>), a second inlet port (<NUM>) for introducing the suspension of cells (<NUM>) into the barrel (<NUM>), and a first valve assembly (<NUM>) including a first internal valve, the first valve assembly positioned between the barrel and the first compartment (<NUM>) and in fluid communication with the first compartment (<NUM>) when the first internal valve is open at a first centripetal force, the second centripetal force being greater than the first centripetal force; and
an outlet port (<NUM>).