RIGID CHAMBER FOR CELL SEPARATION FROM A FLEXIBLE DISPOSABLE BAG

Abstract

Method consists of placing a flexible container within a rigid frame and expanding the container by pneumatic or hydraulic pressure such that the walls of the container conform to the inside walls of the rigid frame thus forming a well-defined chamber. The system has the capability of reducing the volume of the chamber by adjusting the distance between the walls of the rigid container. The methods and systems so described are applicable to closed sterile systems that employ immunomagnetic isolation or purging of components from blood products. By providing a fixed volume and at least one surface upon which targeted entities can be magnetically deposited, target cells in the case of positive isolations can be magnetically held, flushed with wash buffers over them to remove entrapped cells and finally the recovery of product of very high purifies and at high yields.

Claims

1. A method of converting a flexible container into a substantially rigid container, said flexible container having a first end and a second end and a pair of expansible panels joined along the periphery thereof such that facing interior surfaces of said panels form a collection space with an opening at each of said ends, said method comprising: a.) placing said flexible container into a rigid frame having oppositely facing wall members that define a confinement gap; and b.) expanding said flexible container by application of positive pressure such that exterior surfaces of said panels are urged into engagement with said oppositely facing wall members within said confinement gap, thereby rendering said container substantially inflexible.

2. The method of claim 1, wherein said flexible container is a sterile, disposable bag adapted for aseptically processing biological entities.

3. The method of claim 2, wherein said biological entities are viable cells.

4. The method of claim 1, wherein said container is expanded by applying pneumatic pressure.

5. The method of claim 1, wherein said container is expanded by applying hydraulic pressure.

6. The method of claim 1, wherein said container has a valved inlet port at said first end and a valved outlet port at said second end, thereby allowing controlled fluid flow through said container.

7. The method of claim 1, wherein said oppositely facing wall members of said rigid frame are generally planar and substantially parallel to one another.

8. The method of claim 1, wherein at least one of said oppositely facing wall members of said rigid frame has at least one surface projection that directs fluid flow through said container along a predetermined flow path.

9. The method of claim 1, wherein each of said oppositely facing wall members of said rigid frame has at least one surface projection that directs fluid flow through said container along a predetermined flow path.

10. The method of claim 1, wherein one of said oppositely facing wall members is generally planar, and one of said oppositely facing wall members has at least one surface projection that directs fluid flow through said container along a predetermined flow path.

11. The method of claim 1, wherein the distance within said confinement gap between said oppositely facing wall members is adjustable.

12. A method for separating a plurality of target cells from a mixed cell population, said method comprising: a.) providing a substantially rigid container formed according to the conversion method of claim 1; b.) introducing into said collection space of said container said mixed cell population and at least one magnetic labeling agent effective to selectively bind to and magnetically label said target cells; c.) separating said magnetically labeled target cells from said mixed cell population under the influence of a magnetic field gradient; and d.) recovering said magnetically labeled target cells.

13. The method of claim 12, wherein at least one of said oppositely facing wall members of said rigid frame comprises a magnet component effective to generate said magnetic field gradient in said collection space.

14. The method of claim 13, wherein said magnet component is reversibly disengageable from said at least one oppositely facing wall member such that said magnetic field gradient in said collection space is reversibly attenuated.

15. The method of claim 12, wherein each of said oppositely facing wall members of said rigid frame comprises a magnet component effective to generate said magnetic field gradient in said collection space.

16. The method of claim 15, wherein said magnet component is reversibly disengageable from each of said oppositely facing wall members such that said magnetic field gradient in said collection space is reversibly attenuated.

17. The method of claim 12, wherein a wash fluid is passed through said container at least once before said recovering step.

18. The method of claim 12, wherein said mixed cell population and said at least one magnetic labeling agent are simultaneously introduced into said collection space.

19. The method of claim 12, wherein said mixed cell population and said at least one magnetic labeling agent are sequentially introduced into said collection space.

20. A system for converting a flexible container into a substantially rigid container, said system comprising, in combination: a rigid frame having oppositely facing wall members defining a confinement gap; and said flexible container disposed in said confinement gap, said flexible container having a first end and a second end and a pair of expansible panels joined along the periphery thereof such that facing interior surfaces of said panels form a collection space with an opening at each of said ends, said flexible panels being expansible under the influence of positive pressure applied within said collection space, thereby urging said panels into engagement with said oppositely facing wall members within said confinement gap and rendering said container substantially inflexible.

21. The system of claim 20 further including a pressure source operatively connected to said container and effective to apply positive pressure within said collection space.

22. The system of claim 20, wherein one of said oppositely facing wall members of said rigid frame is movable in relation to the other, whereby said confinement gap is adjustable.

23. The system of claim 20, wherein said oppositely facing wall members of said rigid frame are movable in relation to each other, whereby said confinement gap is adjustable.

24. The system of claim 20, wherein at least one of said oppositely facing wall members of said rigid frame comprises a magnet component effective to generate a magnetic field gradient in said collection space.

25. The system of claim 24, wherein said magnet component is reversibly disengageable from said at least one oppositely facing wall member such that said magnetic field gradient in said collection space is reversibly attenuated.

26. The system of claim 20, wherein each of said oppositely facing wall members of said rigid frame comprises a magnet component effective to generate a magnetic field gradient in said collection space.

27. The system of claim 26, wherein said magnet component is reversibly disengageable from each of said oppositely facing wall members such that said magnetic field gradient in said collection space is reversibly attenuated.

Description

DESCRIPTION OF THE DRAWINGS

[0020] FIG. 1 shows an example of a disposable set of bags with inter-connecting tubing and valves that might be used to perform an immunomagnetic cell separation of some subset of cells derived from leukapheresis product, peripheral blood, bone marrow, or the like. The set consists of a source bag 1 containing the starting material (e.g., blood, leukapheresis product, washed cells, or the like), a buffer reservoir bag 2 that could be used for pushing solutions through the system, for washing away non-target cells, or re-suspension of product, a waste bag 3, and a disposable, rectilinear bag 4 wherein the magnetic separation takes place. Valves 5 and 6 are positioned at the inlet and outlet of the separation bag 4, respectively. A valve 7, which is positioned near the waste bag 3, is employed during the pneumatic pressurization of said waste bag and the subsequent pressurization of the separation bag 4.

[0021] FIG. 2 depicts the bag 4 where magnetic separation takes place. In the embodiment shown, the bag has inlet and outlet ports 8, 9 on its ends for filling and/or emptying, respectively. The ends of the bag are partially tapered 10, 11 and in the partially tapered regions are flow directors 12, 13 that, in combination with the partially tapered regions, promote laminar flow.

[0022] FIG. 3 shows the waste bag 3 connected to the separation bag 4 with the latter placed within a rigid frame 14 that can be rotated where one or both sides 15, 16 of the frame 14 could be, or serve as a support for, an array of magnets that could exert magnetic forces on one or both sides 17, 18 of the separation bag 4, respectively. Attached to the waste bag 3 is a port 19 that can be used to pneumatically pressurize the waste bag 3 and the separation bag 4 and a port 20 that is connected to a pressure relief valve 21.

[0023] FIG. 4 shows the separation bag 4 placed within the frame 14 where one or both sides 15, 16 of the frame 14 could be, or serve as a support for, an array of magnets that could exert magnetic forces on one or both sides 17, 18 of the separation bag 4, respectively. Inlet and outlet ports 8, 9 are connected to an inlet pump 22 and an outlet pump 23 that can independently or synchronously pump fluids (solutions or gases) into or out of the separation bag 4, respectively. A pressure gauge 24 is attached to the separation bag 4 via a port 25 and provides feedback control to pump 23.

[0024] FIG. 5 shows an adjustable rigid frame 26 where the spacing 27 between the sides 28, 29 of the frame 26 can be varied using a mechanism 30 that allows one side 29 of the frame 26 to be moved in relation to the other side 28 of the frame 26.

DETAILED DESCRIPTION OF THE INVENTION

[0025] To produce a rigid, sterile chamber to meet the foregoing needs, we disclose herein methods and devices whereby a flexible disposable bag can be converted into a rigid container having a predetermined, reproducible shape and collection surface(s). Further, these methods allow the flexible bag to maintain that predetermined shape throughout a series of manipulations that include filling and emptying of the bag, tilting the bag (useful for a variety of processing steps, particularly the introduction of bubbles with subsequent agitation for meniscus scrubbing), as well as during recovery of the product. We also disclose means for efficiently moving different solutions through the system and particularly within the separation chamber that promote plug flow and minimize mixing at boundaries between such solutions. This is accomplished by employing differential densities of sequential solutions in conjunction with tilting of the chamber to leverage gravitational effects or by decreasing the depth of the chamber and using air gaps to keep solutions separated from one another. These are key considerations for achieving the removal of non-target components and thus purification of the desired product.

[0026] FIG. 1 shows a disposable bag set that might be used for positive or negative immunomagnetic cell separations. The set comprises a source bag 1 that contains the sample to be separated, a buffer reservoir bag 2, a waste bag 3, and a bag 4 wherein magnetic separation takes place. Valves 5 and 6 are positioned at the inlet and outlet of the separation bag 4, respectively, and can be operated to control the flow of fluids into and out of the separation bag 4. Another valve 7 is employed to permit pneumatic pressurization of the waste bag 3 and the separation bag 4. Such an arrangement was marketed by Baxter Health Care for their Isolex system.

[0027] FIG. 2 shows the separation bag 4 with inlet and outlet ports 8, 9 and partially tapered ends 10, 11 to facilitate filling and emptying the separation bag 4, respectively. In concert with the partially tapered ends 10, 11, flow directors 12, 13 promote laminar flow.

[0028] FIG. 3 depicts an arrangement wherein the separation bag 4 is confined within a rigid frame 14 that can be rotated. In a preferred embodiment, the sides 15, 16 of the frame 14 are arrays of magnets that exert magnetic forces on one or both sides 17, 18 of the separation bag 4, respectively. In another embodiment, the sides 15, 16 of the frame 14 are passive containment walls that do not exert magnetic forces on one or both sides 17, 18 of the separation bag 4, respectively. In a more preferred embodiment, the sides 15, 16 of the frame 14 are passive containment walls which can be engaged and disengaged with arrays of magnets that exert magnetic forces on one or both sides 17, 18 of the separation bag 4, respectively. It should be noted that the frame 14 and the separation bag 4 are not horizontal so that fluids pumped into the separation bag 4 will form a meniscus that will traverse the sides 17, 18 of the separation bag 4 upon filling. Prior to filling the separation bag 4, sterile-filtered air is used to pressurize both the waste bag 3 and the separation bag 4 through a port 19. The pressure applied should be sufficient to cause the separation bag 4 to inflate so that the sides 17, 18 of the separation bag 4 engage with the sides 15, 16 of frame 14, but not excessive so as to rupture the separation bag 4 or hinder the introduction of fluids. Pressures of about 1.5 psi have been found to be appropriate to meet these requirements. A port 20 on the waste bag 3 is connected to a pressure relief valve 21 to maintain the pressure within the separation bag 4 and the waste bag 3.

[0029] The pressure relief valve 21 plays an important role in this invention. For example, in one embodiment, the separation bag 4 contains 270-315 mL and is paired with the waste bag 3 having a capacity of 2 L, where the latter is of sufficient size to accept waste from the initial magnetic separation as well as subsequent washes. The total volume of waste could be as much as 1.5 L, resulting in a four-fold pressure increase in the waste bag, potentially threatening the integrity of the system or hindering the pumping of fluids. Furthermore, without pressure relief, the increasing pressure could affect the viability of target cells. By incorporating a pressure relief valve 21 set to maintain constant pressure within the system as fluid is introduced, these problems are eliminated.

[0030] There are many advantages of using the pressurized system described above for maintaining the separation bag 4 in the rigid form as compared with a non-pressurized system. While this invention can be practiced with the separation bag 4 placed in a horizontal position, it is advantageous to rotate the frame 14 on an angle minimally sufficient to create a meniscus as the separation bag 4 is filled. When sample to be separated is pumped into the pressurized system, a clear and well-defined meniscus is visible and rises within the separation bag 4 upon filling. The visibility of the meniscus can be very helpful in the case where it is desirable to position sample to be separated within some specific region of the separation bag 4. For example, if it is advantageous to have a plug of sample positioned above the bottom of 4, a denser cell-compatible buffer (e.g., containing sucrose or some other substance that increases density) can be introduced after the appropriate quantity of sample is pumped into the separation bag 4. By that method and the ability to visualize a meniscus, sample for separation can be accurately positioned. We have found that an isotonic buffered saline solution containing 5% sucrose is adequate for positioning leukapheresis products containing as much as 10% hematocrit into the region where the sample is exposed to the magnetic gradient.

[0031] By incorporating constant pressurization, differential densities, and a rotatable frame 14, it is possible to create a process that results in recovery of highly pure target cells by 1) pumping a sample containing magnetically labeled target cells from an inlet end of a rigidly contained and pressurized separation bag 4 that is positioned on an angle sufficient to create a defined meniscus as sample is introduced and position that sample precisely within the magnetic gradient by following it with a denser buffer; 2) allowing separation to occur; 3) pumping the solution containing cells which did not magnetically separate through the outlet end of the separation bag 4 with the denser buffer; 4) introducing an air bubble into the separation bag 4 containing the denser buffer; 5) tilting the frame 14 back and forth to cause the meniscus created by the bubble therein to “scrub” the collection surface(s) to remove non-target bystander cells; and 6) repeating Steps 3-5 as required to yield the desired product. It is advantageous to alternate buffer densities in sequential steps to prevent mixing of solutions, which results in more efficient removal of non-target bystander cells. For example, after the first wash with the denser buffer is completed, the frame 14 can be tilted downwards so that the outlet end is lower than the inlet end, and less dense buffer can be pumped into the separation bag 4.

[0032] A second solution for creating a rigid container from a flexible disposable bag that would have similar benefits as the foregoing is depicted in FIG. 4. As in FIG. 3, the separation bag 4 is held within the frame 14, and the sides 15, 16 of the frame 14 engage with the sides 17, 18 of the separation bag 4, respectively. However, in this arrangement, the separation bag 4 is attached to an inlet pump 22 through the inlet port 8 and an outlet pump 23 through the outlet port 9, where both pumps can also serve as valves (e.g., in the case of peristaltic pumps). Also depicted in FIG. 4 is a pressure gauge 24 that is attached to the separation bag 4 via a port 25. In this embodiment, the separation bag 4 essentially devoid of any air is positioned within frame 14. With the outlet pump 23 deactivated (i.e., valve closed), sample to be separated is pumped into the separation bag 4 by the inlet pump 22, thus filling the separation bag 4 and rendering it sufficiently rigid. As before, a sufficiently denser buffer (e.g., 5% sucrose) can be used to position the sample for optimal separation. Following separation, the inlet pump 22 is activated to pump wash buffer into the separation bag 4 while synchronously pumping solution out of the separation bag 4 with the outlet pump 23. In this way, the separation bag 4 maintains its rigid shape throughout the entirety of the process. The pressure gauge 24 monitors the pressure inside the separation bag 4 during the process and provides feedback to both pumps 22, 23 to prevent over- or under-pressurization. With this embodiment, the benefits of the preceding positive-pressure embodiment are realized.

[0033] An alternative approach that can also be used for magnetic separation of targeted entities to achieve similar ends as the foregoing is to employ an adjustable rigid frame 26 where the spacing 27 between the sides 28, 29 of the frame 26 can be varied using a mechanism 30. In one embodiment, the mechanism 30 would allow one side 29 of the frame 26 to be moved in relation to the other side 28 of the frame 26, thereby varying the spacing 27 between the sides 28, 29 of the frame 26. In another embodiment, both sides 28, 29 of the frame 26 would be movable by the mechanism 30, thereby permitting variation of the spacing 27 between the sides 28, 29 of the frame 26.

[0034] Such an adjustable rigid frame 26 can be deployed in the preceding positive-pressure embodiment or in the preceding two-pump embodiment. In some embodiments, the spacing 27 between the sides 28, 29 of the frame 26 can be set prior to separation to define the chamber volume and maintained throughout the entirety of the process. In other embodiments, the spacing 27 between the sides 28, 29 of the frame 26 can be set prior to separation to define the chamber volume and subsequently changed during the process to increase or decrease the chamber volume, for example during the washing steps or product recovery. In still other embodiments, the spacing 27 between the sides 28, 29 of the frame 26 can be decreased to force solution out of the separation bag 4 and into the waste bag 3, and increased thereafter to permit refilling of the separation bag 4.

[0035] To test the concept of varying the spacing 27 between the sides 28, 29 of the frame 26 while maintaining the integrity of the collection surface(s), an arrangement similar to that depicted in FIG. 3 was fabricated where the spacing 27 between the sides 28, 29 of the frame 26 could be varied from 4-14 mm. The dimensions of the sides 28, 29 of the frame 26 were 19.5×26.5 cm. One of the sides was constructed from ⅜″-thick rigid Plexiglas to allow for visualization, while the other side comprised an array of magnets capable of inducing a magnetic field gradient. A flexible bag, approximately 12.5×25.5 cm, was obtained from a LOVO Cell Washing Disposable Kit (Fresenius Kabi, Lake Zurich, Ill.). The bag in that kit is referred to as the “in-process bag” and has ports on both ends of the longer dimension. This bag was utilized as the separation bag 4 and was positioned as in FIG. 3 with the top port of the bag connected to a second flexible bag serving as the waste bag 3. The bag used as the waste bag 3 was also obtained from the LOVO Cell Washing Disposable Kit and is referred to as the “filtrate bag” by the supplier. That bag was also connected to a pressure relief valve 21, as in FIG. 3. A peristaltic pump was connected to the inlet port 8 of the separation bag 4 identical in concept to the inlet pump 22 shown in FIG. 4. This created a system whereby, with the inlet pump 22 acting as a shutoff valve, both bags could be pressurized to 1.5 psi with a suitable fluid (e.g., air) such that the sides 17, 18 of the separation bag 4 engage with and are held tightly against the sides 28, 29 of the frame 26. As the waste bag 3 is not confined, it acts like an air bladder and inflates. For this arrangement, as liquid is introduced into the system via the peristaltic pump, it displaces an equivalent volume of air, which is released via the pressure relief valve 21 to maintain constant pressure, and thus, the system integrity.

[0036] Evaluations of the system were made at a spacing 27 of 4-12 mm. At a spacing 27 of 5 mm, the volume of the chamber was 180 mL, and at a spacing 27 of 10 mm, the volume approximately doubled, as expected. In all cases, the sides 17, 18 of the separation bag 4 engaged with and were held tightly against the sides 28, 29 of the frame 26. The functionality of this system in terms of separation performance was further tested with experiments performed using peripheral blood mononuclear cells. CD3+ cells (i.e., T cells) were magnetically labeled, and the mixture of cells (i.e., labeled target cells and unlabeled non-target cells) was pumped into chambers wherein the spacing 27 between the sides 28, 29 of the frame 26 was 5 or 10 mm. Following magnetic separation, the magnetically labeled target cells were subjected to several cycles of washes with buffer and meniscus scrubbing (i.e., rocking the entire chamber through 90°, allowing a meniscus to passage over collected cells) to remove non-target cells. At a spacing 27 of both 5 and 10 mm, the system performed satisfactorily as regards yield and purity of target cells.

[0037] The experiments described above demonstrate the utility of this invention wherein a flexible bag that can be converted into a rigid chamber of variable volume. The present system will be useful to accommodate variations in the number and concentration of total cells, the number and concentration of target cells, and the volume of sample to be processed. For example, processing 10.sup.8 TNC might be sufficient for many applications; however, 10.sup.9 TNC is typical for a leukapheresis product, while 10.sup.10 TNC is often required for stem-cell isolations. In cases where the volume to be processed becomes large enough that the magnetic field gradient applied to one side of the chamber is insufficient, it is a simple matter to employ an arrangement wherein magnetic field gradients are applied to both sides.

[0038] A variable-volume separation chamber obtained from a flexible bag in accordance with this invention is useful in other respects. The manipulations required to remove non-target cells (see co-pending application PCT/US2016/031528) involve the passage of wash buffers over the collected cells as well as the passage of menisci created by controlled introduction of air. Fora separation requiring a large spacing (e.g., 10-12 mm), the meniscus that is formed might not be sufficient to rid the collected cells of entrapped non-target cells. Accordingly, it would be advantageous to employ an adjustable frame wherein the spacing can be decreased, thus reducing the chamber volume. This could be advantageous in at least three ways. Firstly, there would be a reduction in the quantity of buffer that would be required to fill the chamber for the wash cycles. Secondly, the time required for emptying the chamber would also be reduced. Finally, a bubble passaging through two plates has a greater meniscus scrubbing effect as the spacing between those plates decreases. There is likely an optimal spacing between the plates to achieve the desired goals.

[0039] Another significant advantage to employing a variable-volume separation chamber constructed from a flexible bag is that by decreasing the volume of the chamber, there will be a point where an air gap will be capable of preventing two solutions from contacting each other and mixing. To illustrate this point, consider that a blood product to be separated is introduced into a separation chamber where the spacing is 10 mm, accommodating a sample volume of approximately 360 mL. After the separation has taken place, the volume of the separation chamber can be reduced, with appropriate valving, to force solution to go to waste. By selecting an appropriate spacing, it is possible to subsequently introduce an air gap such that fresh buffer can be introduced into the separation chamber to force the remaining solution to go to waste with little or no mixing of the fresh buffer and the remaining solution.

[0040] The following examples explain the invention in greater detail.

Example 1

[0041] The following steps have been used for separating magnetically labeled CD3+ cells from leukapheresis products employing air pressure, differential densities, and gravitational effects. [0042] 1. Begin by pressurizing the waste bag to about 1.5 psi to make it rigid and connect the waste bag to the separation bag that is placed within the rigid frame (the walls of the rigid frame being on one side a planar magnetic array and the other a containment wall). [0043] 2. With the separator tilted up (outlet at the top) and the inlet and outlet valves open, pump sample to be separated into the bag at high speed (e.g., 150 mL/min). [0044] 3. Once the sample has completely entered the separation bag, switch to 5% sucrose containing 75 mM NaCl (“sucrose solution”) until sucrose begins to enter the bag which positions the sample fully within the magnetic field. [0045] 4. Separate for 10 min. [0046] 5. Pump sucrose solution into the bag to push the negative fraction or supernatant up and out of the bag. [0047] 6. Once the negative fraction has been removed, tilt the separator down and begin to pump in air to form a bubble; after the appropriate amount of air (about 10% of the bag volume) has been pumped in, switch back to sucrose solution and pump just until it begins to enter the bag. [0048] 7. With both the inlet and outlet valves closed, rock the separator back and forth until the non-target cells have been re-distributed back into suspension. [0049] 8. With the separator in the upward-facing position (bubble near the outlet) and both valves open, pump in sucrose solution to remove the bubble. [0050] 9. Wait 5-10 min for any dislodged target cells to be re-captured, [0051] 10. Tilt the separator down and begin to pump non-sucrose-containing wash solution into the bag; once the sucrose wash solution has been removed, begin to pump in air to form a bubble. [0052] 11. With both the inlet and outlet valves closed, rock the separator back and forth until the non-target cells have been re-distributed back into suspension. [0053] 12. With both valves open, tilt the separator up (bubble near the outlet) and pump in non-sucrose-containing solution to remove the bubble, [0054] 13. Wait 5-10 min for any dislodged target cells to be re-captured. [0055] 14. Pump sucrose solution to push the non-sucrose-containing wash solution up and out of the bag. [0056] 15. If necessary, Steps 6-10 can be repeated to put the cells back into non-sucrose-containing solution for recovery; alternatively, the cell product can be directly recovered after Step 14 and removed from the magnetic field.

Example 2

[0057] The following steps could be used for separating magnetically labeled CD3+ cells from leukapheresis products employing air pressure, differential densities, gravitational effects, and a variable-volume chamber. [0058] 1. Begin by pressurizing the waste bag to about 1.5 psi to make it rigid and connect the waste bag to the separation bag that is placed within the rigid frame (the walls of the rigid frame being on one side a planar magnetic array and the other a translatable containment wall). [0059] 2. With the separator tilted up (outlet at the top) and the inlet and outlet valves open, pump sample to be separated into the bag at high speed (e.g., 150 mL/min). [0060] 3. Once the sample has completely entered the separation bag, switch to 5% sucrose containing 75 mM NaCl (“sucrose solution”) until sucrose begins to enter the bag which positions the sample fully within the magnetic field. [0061] 4. Separate for 10 min. [0062] 5. With the inlet valve closed and the outlet valve open, decrease the chamber spacing by translating the containment wall, thereby forcing the negative fraction out of the separation bag and into the waste bag. [0063] 6. Close the outlet valve, open the inlet valve, and pump sucrose solution into the bag while increasing the chamber spacing. [0064] 7. Once the bag is full of sucrose solution, tilt the separator down and begin to pump in air to form a bubble; after the appropriate amount of air (about 10% of the bag volume) has been pumped in, switch back to sucrose solution and pump just until it begins to enter the bag. [0065] 8. With both the inlet and outlet valves closed, rock the separator back and forth until the non-target cells have been re-distributed back into suspension. [0066] 9. With the separator in the upward-facing position (bubble near the outlet) and both valves open, pump in sucrose solution to remove the bubble. [0067] 10. Wait 5-10 min for any dislodged target cells to be re-captured. [0068] 11. With the inlet valve closed and the outlet valve open, decrease the chamber spacing by translating the containment wall, thereby forcing the sucrose solution out of the separation bag and into the waste bag. [0069] 12. Tilt the separator down, close the outlet valve, open the inlet valve, and pump non-sucrose-containing wash solution into the bag while increasing the chamber spacing. [0070] 13. Once the bag is full of non-sucrose-containing wash solution, begin to pump in air to form a bubble; after the appropriate amount of air has been pumped in, switch back to non-sucrose-containing wash solution and pump just until it begins to enter the bag. [0071] 14. With both the inlet and outlet valves closed, rock the separator back and forth until the non-target cells have been re-distributed back into suspension. [0072] 15. With both valves open, tilt the separator up (bubble near the outlet) and pump in non-sucrose-containing wash solution to remove the bubble. [0073] 16. Wait 5-10 min for any dislodged target cells to be re-captured. [0074] 17. With the inlet valve closed and the outlet valve open, decrease the chamber spacing by translating the containment wall, thereby forcing the non-sucrose-containing wash solution out of the separation bag and into the waste bag, [0075] 18. Close the outlet valve, open the inlet valve, and pump sucrose solution into the bag while increasing the chamber spacing. [0076] 19. If necessary, Steps 7-12 can be repeated to put the cells back into non-sucrose-containing solution for recovery; alternatively, the cell product can be directly recovered after Step 18 and removed from the magnetic field.

[0077] Disclosed herein are means for creating a rigid chamber from a flexible and desirably disposable bag that can be used to perform multiple operations with the same facility as a chamber constructed from rigid material without the high cost of such a chamber and the difficulties of sterilizing such a chamber. The present invention also obviates the need to employ a device that maintains the engagement of the bag with either the surface of the magnet or the surface of a containment wall that mates with the magnet, which could be accomplished by placing appropriately designed vacuum plates on either side of the bag.

[0078] The present invention has broader applications than for cell separations, which may include separating a wide array of “biological entities”, a term used herein to refer to various substances of biological origin, for example, cells, both eukaryotic (e.g., leukocytes, erythrocytes, or fungi) and prokaryotic (e.g., bacteria, protozoa, or mycoplasma), viruses, cell components, such as organelles, vesicles, endosomes, lysosomal packages, or nuclei, as well as molecules and macromolecules (e.g., proteins or nucleic acids, such as RNA or DNA).

[0079] The present invention has applications beyond creating a chamber for separation. It could be employed where a chamber needs to have a precise volume or some precise shape, which need not include parallel walls. It also could be employed to create a chamber within which is one or more barriers or channels. More specifically, if a bag were created while one or more surface projections were pressed into the sides of the bag, then by placing such a bag in a frame wherein the frame structure mirrors the bag structure, complex chambers can be created upon inflation of the bag. For example, if one wanted to construct a chamber that resembled a waffle, that could be accomplished as follows: 1) press the desired waffle structure into two deformable sheets, such as those used for blood bags or some other suitable material; 2) position two opposing pressed sheets and weld the edges to form a bag, with the appropriate numbers of ports in the appropriate locations; and 3) place that bag into a frame which the corresponding desired waffle structure and pressurize the bag to form the chamber. Alternatively, if sufficiently deformable materials are used for producing a bag, there would be no need to press in any structural elements as pressure exerted within the bag would be sufficient to cause the walls of the bag to conform to the desired frame structure.

[0080] A number of patent and non-patent publications are cited herein in order to describe the state of the art to which this invention pertains. The entire disclosure of each of these publications is incorporated by reference herein.

[0081] While certain embodiments of the present invention have been described and/or exemplified above, various other embodiments will be apparent to those skilled in the art from the foregoing disclosure. The present invention is, therefore, not limited to the particular embodiments described and/or exemplified, but is capable of considerable variation and modification without departure from the scope and spirit of the appended claims.

[0082] Furthermore, the transitional terms “comprising,” “consisting essentially of,” and “consisting of,” when used in the appended claims, in original and amended form, define the claim scope with respect to what unrecited additional claim elements or steps, if any, are excluded from the scope of the claim(s). The term “comprising” is intended to be inclusive or open-ended and does not exclude any additional, unrecited element, method, step, or material. The term “consisting of” excludes any element, step, or material other than those specified in the claim and, in the latter instance, impurities ordinarily associated with the specified material(s). The term “consisting essentially of” limits the scope of a claim to the specified elements, steps, or material(s) and those that do not materially affect the basic and novel characteristic(s) of the claimed invention. All devices, device components, and methods described herein that embody the present invention can, in alternate embodiments, be more specifically defined by any of the transitional terms “comprising,” “consisting essentially of,” and “consisting of.”