Abstract:
The invention relates to a multifunctional bioreactor for cell sorting and cell culture in vitro. Said bioreactor comprises five main elements, including an adjustable magnetic field, a multifunctional cell supporting system, a protective perfusion system and a computerized control system. Said methods are for the application of the said bioreactor. The said bioreactor has the functions of cell expansion, cell directed differentiation and cell separation (sorting). It allows its all functions carried out in one reaction chamber.

Description:
CROSS-REFERENCES TO RELATED APPLICATION 
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       FIELD OF THE INVENTION 
       [0003]    The invention relates to a multifunctional bioreactor for cell culture and cell sorting. 
       BACKGROUND OF THE INVENTION 
       [0004]    This application relates generally to bioreactors and more particularly bioreactors for growing and separating cells. 
         [0005]    Many kinds of cells, especially hematopoietic stem cells and immunocytes, need to be isolated from original samples before they can be efficiently expanded and directed differentiated in culture. This isolation procedure is also called cell sorting or cell separation. Previously, the cell sorting and cell culture have been respectively conducted in separated systems, in which the target cells are isolated first and then are transferred into culture containers. This traditional method is quite cumbersome and has a higher risk for cell contamination and cell loss from the cell sorting to cell culture, in which two completely different devices and systems were involved. Our current invention with a novel design allows these two different procedures completed in one container (chamber), and so minimizes the risk of contamination and target cell loss, and significantly increases the efficiency of the operation. 
         [0006]    Some cells are very sensitive to shear-stress in the culture. For example, shear-stress can cause the non-specific differentiation and the increased apoptosis in the stem cell culture, which significantly reduces the efficiency of the stem cell expansion and directed differentiation. The higher shear-stress also causes the more release of non-specific proteins in protein expression, in which the protein of interest takes less proportion in the culture and so result in the increase of protein purification. The static culture has the least shear stress but the cells in static culture normally sit at the bottom of the culture containers, some cells cannot get enough nutrition when cells are at higher density and so not suitable for large scale cell expansion. Some bioreactors, such as NASA&#39;s rotation wall vessel (RWV) bioreactor, were designed for reducing the shear-stress. However, these bioreactors have to keep cells in suspension by continuously moving, stirring or/and agitating cells. Once the bioreactor stop running, cells will accumulate somewhere of the bottom but are not evenly distributed, which is harmful for most cell growth. Therefore, though the shear-stress has been reduced in these bioreactors, the reduced shear-stress has to continuously exert on the cultured cells when these bioreactors running. In our current invention, when the bioreactor is at static status, cells are allowed to evenly distribute at the bottom of the culture chamber or on the surface of the magnetic beads. Thus, our invention provides cells the best growth condition in both suspension status and static status. 
         [0007]    Some bioreactors use magnet element (specifically blades or vans) controlled by magnet impeller to agitate culture media to keep cells in suspension status. This kind of bioreactor purposely enhances the shear-stress for the culture requirements of a certain cells. In addition to the differences in the application, the bioreactor in our invention does not use blades or canes to be the magnet element, and the magnet beads in our invention actually has no magnetism if they are not in magnet field and they can only gain magnetism when they are placed in magnet field. The magnet beads in our invention are not controlled by impeller but by the changes of magnetic field strength affecting the beads&#39; moving. The proper microenvironments, or so called niches, are very important for the growth of some cells, such as stem cells. Some devices use solid materials to form niches or use gel-like materials to form niches. With these devices, after cells culture, cells need to be rinsed out from niches with a special procedure or the niche-forming materials has to be melted or digested with enzyme to release the target cells. In our current invention, the interspaces among beads naturally from the niches for cell growth, and the cells can be easily released when the beads are lifted by changing the magnet field strength. Some other systems of cell culture attempt to build niches via welled culture plates (such as the common 96-welled plate), micro-chambers (Hung 2005) or micro-sieves (Zhang 2009). The current invented bioreactor is superior to the welled plate because the mobility of the beads creates dynamic microenvironments for cells needing increased flux of medium to grow. It is also superior to micro-chambers and micro-sieves because it poses less of a challenge to manufacture, is easier to sterilize after use, and the size of the niche can be easily modified by adjusting the size of the beads. 
         [0008]    Many cell culture containers (chambers) have been designed for bioreactor use, such as common cell culture flasks, gas-permeable bags, rotation wall vessels, and so on. These containers can be used with common perfusion system with which the cells can be diluted and media can be changed. However, during the media change with these containers, the cytokines, proteins and other expensive substances for cell growth and cells&#39; products are removed from the culture simultaneously. And, the efficiency of common dialysis process is not high enough. The cell culture chambers designed in our current invention take the advantage of colloid osmotic force differences between osmosis chambers at two side of culture chamber to allow the media exchange go through the dialysis membranes rapidly without any loss of cytokines, peptides, proteins and other materials in a certain size. This chamber design provides a novel perfusion strategy and the system with it is called as gradient osmosis perfusion system. 
         [0009]    Most bioreactors were designed for culturing either adherent or suspension cells. No bioreactors have been reported to support the growth of partial adherent cells yet. The bioreactor in our current invention can be used for the culture of almost any cell, including suspension cells, adherent cells and partial adherent cells. 
         [0010]    It is very common to use computer to control bioreactor running, and many bioreactors are programmable. It is emphasized in our current invention that (1) the strength and direction of magnetic fields, (2)the frequencies and speeds of cell culture chamber flip, and (3) frequencies and speeds of the magnet beads as results of above (1) and (2) are controlled by pre-selected programs or/and programs that response to the data it receives from detectors and send feedback to bioreactor. 
         [0011]    Similar to the application of computer in bioreactor control, several cell density detectors with some special light sources (such as laser projector) have been designed for monitoring the cell concentration during the cell culture. The data obtained from the detector or sensor is used to determine if the cells need to be diluted if the culture needs media change. In our current invented bioreactor, the cell density detector uses the common light source and the data is specifically used to adjust the strength of the magnetic field, the flipping speed and frequency of cell culture chamber, as well as indirectly adjust the moving speed and frequency of the magnet beads. 
       SUMMARY 
       [0012]    Bioreactors are typically employed to grow cells within a culture. However, many kinds of cells need to be isolated from original samples before they can be efficiently expanded and directed differentiated in culture. This isolation procedure is also called cell sorting or cell separation. Typically, the cell sorting and cell culture have been conducted in separated systems, in which the target cells are isolated first and then are transferred into culture containers. This traditional method is quite cumbersome and has a higher risk for cell contamination and cell loss from the cell sorting to cell culture, in which two completely different devices and systems are involved. 
         [0013]    Additionally, many bioreactors employ rotating impellers or the like for mixing the contents of the bioreactor. Unfortunately, this imparts a shear stress on the cells which may damage the cells, reduces the efficiency of the system, causes a release of waste products, such as non-specific proteins expression, or the like. Additionally, in some bioreactors, cells may aggregate on the bottom of the cell culture chamber or other locations within the cell culture chamber. The aggregation of these cells in the chamber is not conducive to efficient cell growth. Therefore, there exits a significant need for an efficient bioreactor capable of growing and separating cells therewithin while also minimizing shear-stress imparted to the cells. 
         [0014]    In one embodiment, a bioreactor system for growing and separating cells comprises a cell culture chamber comprising an interior having a first portion and a second portion; an agitator disposed within the chamber interior, the agitator capable of moving between the interior first portion and interior second portion; and a control system coupled to the cell culture chamber, the control system operable to cause the agitator to move between the interior first portion and interior second portion. 
         [0015]    In another embodiment, a method for growing and separating cells comprises providing a chamber comprising an interior having a first portion and a second portion; disposing an agitator within the interior, the agitator capable of moving between the interior first portion and interior second portion; delivering a cell culture media to the chamber interior; transferring target cells to the chamber interior; moving the agitator between the first portion and second portion so as to mix the cells and media; and removing waste from the chamber interior while maintaining a substantial number of the target cells within the chamber interior. 
         [0016]    In yet another embodiment, a cell culture chamber interior comprises: a first compartment, the first compartment adapted for fluid communication with a cell culture media reservoir such that the first compartment is capable of receiving cell culture media from the reservoir; a second compartment, wherein the agitator is disposed within the second compartment; a first membrane positioned between the first compartment and second compartment, the first membrane adapted to selectively permit cell culture media to flow from the first compartment to the second compartment; a third compartment, the third compartment adapted for fluid communication with a buffer reservoir such that the third compartment is capable of receiving buffer from the buffer reservoir and wherein the third compartment is further adapted for fluid communication with a waste reservoir such that waste is capable of flowing from the third reservoir to the waste reservoir; and a second membrane positioned between the second compartment and third compartment, the second membrane adapted to selectively permit flow of media and waste from the second compartment to the third compartment. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0017]    The drawings, when considered in connection with the following description, are presented for the purpose of facilitating an understanding of the subject matter sought to be protected. 
           [0018]      FIG. 1  is a schematic view showing an illustrative bioreactor; 
           [0019]      FIGS. 2   a - 2   f  show a series of schematic views of a bioreactor showing the movement of an agitator within a chamber in series; 
           [0020]      FIG. 3  is a schematic isometric view of a chamber; 
           [0021]      FIG. 4  is a schematic side-view of a chamber; and 
           [0022]    FIG. is a schematic view of an agitator. 
       
    
    
     DETAILED DESCRIPTION 
       [0023]    Referring now to  FIG. 1 , a bioreactor system  10  for growing and separating cells is shown. The system  10  includes a cell culture chamber  15 , an agitator  20  and a control system  30 . 
         [0024]    The cell culture chamber  15  includes an interior  35  for receiving and growing target cells in a cell culture media disposed therein, a first end  40  and a second end  45 . As used herein, “target cells” refers to cells disposed within the chamber  15  and which are grown within the chamber  15 . While the present disclosure is given the context of growing target cells, it will be appreciated that the system may be employed to mix chemicals or any other suitable solution or material. Also, while the first end  40  and second end  45  are shown as being at the top and bottom of the chamber  15  respectively, it will be appreciated that the ends  40 ,  45  may be in any suitable orientation relative to one another (e.g., in a horizontal plane) and remain within the scope of the present disclosure. As will be discussed below, the chamber  15  may include one or more interior compartments. In addition, as will be appreciated by those skilled in the art, the chamber  15  may be formed from any suitable material, including a rigid material, a flexible material, a combination of rigid and flexible materials, a gas permeable material or any other suitable material. The chamber  15  may further include one or more ports for providing fluid communication between the chamber interior  35  and one or more reservoirs. Illustrative reservoirs include, without limitation, a cell culture media reservoir, a waste reservoir, a buffer reservoir, a CO 2  reservoir, or any other suitable reservoir. 
         [0025]    Referring now to  FIG. 3 , an illustrative alternative chamber  15  is shown. In this embodiment, the chamber  15  includes a first compartment  75 , a second compartment  80  and a third compartment  85 . The first compartment  75  may include one or more ports  18   a  for providing fluid communication between the first compartment and one or more reservoirs such as: a cell culture media reservoir, such that media can be added to and/or removed from the first compartment; a buffer reservoir, such that buffer can be added to and/or removed from the first compartment; a waste reservoir, such that waste may be removed from the first compartment  75 ; a CO 2  reservoir such that CO 2  may be added to and/or removed from the first compartment; or any other suitable reservior. The first compartment  75  and second compartment  80  are separated by a first membrane  90 . The first membrane  90  is adapted to selectively permit media to flow from the first compartment  75  to the second compartment  80 . The membrane  90  may include perforations, apertures or any other suitable configuration and/or is otherwise semipermeable so as to permit the media to flow from the first compartment  75  to the second compartment  80 . In one embodiment, the membrane  90  is a dialysis membrane. The membrane  90  may be formed from any suitable material, including but not limited to cellulose and cellophane. 
         [0026]    The second compartment  80  is configured to retain the agitator therein such that the agitator may be moved within the compartment as previously discussed relative to  FIG. 1 . Target cells are typically grown and retained within the second compartment  80  as well. The second compartment  80  may include one or more ports  18   b  for providing fluid communication between one or more reservoirs such as the types of reservoirs discussed relative to the first compartment  75 . The second compartment  80  and third compartment  85  may be separated by a second membrane  95 . The second membrane  95  is adapted to selectively permit media and/or waste to flow from the second compartment  75  to the third compartment  85 . The membrane  95  may include perforations, apertures or any other suitable configuration and/or is otherwise semipermeable so as to permit the media and/or waste to flow from the second compartment  80  to the third compartment  85 . In one embodiment, the membrane  95  is a dialysis membrane. The membrane  95  may be formed from any suitable material, including but not limited to cellulose and cellophane. The third compartment  85  may include one or more ports  18   c  for providing fluid communication between the third compartment  85  and one or more reservoirs such as the types of reservoirs discussed relative to the first compartment  75 . Also, in at least one embodiment, at least a portion of the exterior of the chamber may be formed from a gas permeable material. When at least a portion of the chamber exterior is formed from a gas permeable material the system may, in some but not necessarily all, instances not include direct gas injection into the chamber and media pH maintaining components. The first compartment  75  and third compartment  85  may be located in any suitable position relative to the second compartment  80  and the present disclosure is not in any way limited to the second compartment  80  positioned between the first and third compartments  75 ,  80 . Additionally, it will be appreciated that any suitable number of compartments may be employed and the present disclosure is in not limited to only three compartments. 
         [0027]    In one embodiment, a media comprising a suitable solution having a relatively small number of large and/or large polar molecules is provided to the first compartment  75 , and the same or similar media with suitable colloid osmosis for cell growth is provided to the second compartment  80 . Suitable media may be any media that used for growing or maintaining the target cells. A buffer having a greater concentration of large and/or large polar molecules to generate and maintain higher colloid osmosis force, as compared to the media of the first and/or second compartments  75 ,  80 , is provided to the third compartment  85 . The materials used to generate and maintain higher colloid osmosis force for the third compartment  85  may include, but is not limited to, PEG 80000, albumin, and other proteins or any other suitable material or solution. The membranes  90 ,  95  may have the same or different permeability and are configured such that media and/or waste from the growth of new cells may flow from the first and/or second chambers  75 ,  80  to the third chamber  85  via osmosis. The control system may control the infusion and draining of media, buffer and/or waste from one or more compartments  75 ,  80 ,  85  so as to maintain a constant volume and osmosis force in each compartment  75 ,  80 ,  85  and maintain a supply of fresh media to the second compartment  80 . In addition, the control system, via any suitable detection device, mechanism or method, may monitor the any suitable parameter involved in the growth of the target cells, for example and without limitation, the change in the number of target cells, pH, CO 2 , glucose, calcium, potassium, sodium, temperature, humidity or any other suitable factor and adjust the interval, frequency and/or speed of the movement of the agitator within the second chamber and/or adjust the amount of media, the type of media, the amount of buffer, the type of buffer, the amount of CO 2 , or make any other suitable adjustment based on the control system measurement so as to enhance or promote the growth of the target cells within the chamber. 
         [0028]    Referring now to  FIG. 4 , another illustrative chamber  15  is shown. In this embodiment, the chamber may include one or more ports  100  for providing fluid communication between the chamber interior  35  and a waste reservoir. The chamber  15  may also include one or more ports  110  for providing fluid communication between the chamber interior  35  and a cell culture media reservoir. Also, the chamber  15  may include one or more ports  110  for providing fluid communication between the chamber interior  35  and a buffer reservoir. Further, the chamber may also include one or more ports  115  for receiving the agitator into the chamber interior  35 . In one embodiment, the chamber  15  is formed from Teflon FEP. This particular embodiment may be useful in instances where the target cells will adhere or otherwise be coupled to the agitator so that when waste is flushed from the chamber interior  35 , the target cells remain within the chamber interior  35 . However, it will be appreciated that the chamber  15  may be formed from any suitable material and remain within the scope of the present disclosure. 
         [0029]    The agitator  20  is disposed within the chamber interior  35  and is capable of moving between the chamber first end  40  and chamber second end  45 . Alternatively, the agitator  20  may be configured to be moved between any two or more points, or between any two or more portions, within the chamber interior  35 . In the illustrative embodiment, the agitator comprises a plurality of beads  21 . It will be appreciated that any illustrative embodiment showing beads  21  may use any alternative agitator configuration and remain with the scope of the present disclosure and that any particular illustrative embodiment is not limited to using beads exclusively as the agitator. In one embodiment, the beads  21  are be formed from a magnetizable material, such as silicon steel, Fe 3 O 4 , or any other suitable magnetizable material. As used herein, magnetizable means that the agitator, such as the beads, will hold a magnetic charge when subjected to a magnetic field but will not otherwise hold a magnetic charge once removed from the magnetic field, or the magnetic field removed from the vicinity of the agitator, for example, when a magnetic field generator is de-energized. The magnetizable material typically comprises the core of each bead  21 . The magnetizable core may then be coated with any suitable material. In one embodiment, the magnetizable core is coated with polystyrene; however, it will be appreciated that the magnetizable core may be coated with any suitable material and remain within the scope of the present disclosure. For example, and without limitation, the magnetizable core may be coated with any suitable thermoplastic or thermoset polymer. While the beads  21  are shown as being formed from a magnetizable material, it will be appreciated that the beads may be formed from any suitable material, magnetizable or non-magnetizable, and remain within the scope of the present disclosure. Additionally, it will be appreciated that the beads  21  may each be coated with any suitable material such that the target cells will adhere to the beads as the cells grow within the chamber  15 , yet it will be appreciated that beads not coated with a particular material to which target cells will adhere also remain within the scope of the present disclosure. In some embodiments, it may be desirable to have beads  21  that are buoyant within the cell culture media; therefore, the core of the beads may include air pockets or bubbles, a lightweight foam or plastic or any other suitable material for permitting the beads  21  to be buoyant within the media. 
         [0030]    The beads  21  may be formed such that one or more niches, or micoenvironments, may be formed or created in the voids between the beads  21  when the beads are stacked together. In some embodiments, these niches may promote growth of additional target cells therein. In one embodiment, where the beads are substantially spherical, the diameter of each bead  21  may be between 1 mm and 10 mm for the creation of suitable niches. However, it will be appreciated that the beads  21  may have any suitable size and/or shape such that one or more suitable niches may be formed when the beads  21  are stacked together. Also, it will be appreciated that at least some niches may be formed between some beads and one or more walls of the chamber interior. 
         [0031]    In an alternative embodiment, as shown in  FIG. 5 , the agitator  20   a  may be a plate member  21   a  having a plurality of apertures  22  therein. The cross-section of the plate member  21   a  may be complimentary to the cross-sectional shape of the chamber  15  such that the agitator  20   a  may move within the chamber interior  35 . The apertures  22  may permit the media to flow through the agitator  20   a  as the agitator  20   a  moves within the chamber interior  35 . The agitator  20   a  may be formed from magnetizable materials or non-magnetizable material, formed to be buoyant or non-buoyant, and/or coated as previously discussed with respect to the beads  20 . 
         [0032]    Referring again to  FIG. 1 , the control system  30  may include one or both of a controller  55  and computer  60  for controlling operation of the system  10 . Alternatively, the system  10  may be run manually. The control system  30  is configured to be releasably coupled to the chamber  15 . The control system  30  may include a cassette  50  for receiving the chamber  15  but it will be appreciated that the chamber  15  may be coupled to the control system  50  via any suitable means or configuration (e.g., clips, hooks, magnets, hook-and-loop assemblies, friction fit, etc.) and remain within the scope of the present disclosure. 
         [0033]    The control system  30  may also include a light source  2  and a cell detector  9  for detecting the number of cells within the chamber  15 , detecting the change in the number of cells within the chamber  15  or the like and reporting the results back to the control system  30 . However, it will be appreciated that any detector, mechanism or technique known in the art for monitoring the number of cells or the change in the number of cells may be employed and remain within the scope of the present disclosure. Additionally, the control system  30 , via any suitable detection device, mechanism or method, may monitor the any suitable parameter involved in the growth of the target cells, for example and without limitation, the change in the number of target cells, pH, CO 2 , glucose, calcium, potassium, sodium, temperature, humidity or any other suitable factor and adjust the frequency and/or speed of the movement of the agitator within the chamber and/or adjust the amount of media, the type of media, the amount of buffer, the type of buffer, the amount of CO 2 , or make any other suitable adjustment based on any control system measurement so as to enhance or promote the growth of the target cells within the chamber  15 . 
         [0034]    The control system  30  is operable to cause the agitator to move within the interior  35  of the chamber  15 . This may be accomplished a variety of ways. In the illustrative embodiment, the control system includes a motor  65  operable to rotate the chamber  15  between a first position and second position. As will be discussed below, the first position and second position are approximately 180° apart but it will be appreciated that first and second positions may have any suitable angular relationship relative to one another and remain within the scope of the present disclosure. The chamber  15  may be rotated in a horizontal plane, rotated in a vertical plane or rotated, shifted, slid or otherwise moved in any suitable manner to cause the agitator  20  to move within the chamber  15 . 
         [0035]    In addition, the control system  30  may include first and second magnetic field generators  70 ,  72  for exciting the beads  21 , or other agitator  20 , so as to move the beads  21  within the chamber  15  to mix the target cells and culture media. In the illustrative embodiment, each magnetic field generator is an electromagnet that generates a magnetic field when energized and ceases to create a magnetic field when de-energized. When energized, each magnetic field generator draws the agitator  20 , e.g. the beads  21 , toward the energized magnetic field generator. In an alternative embodiment, a permanent magnet may be used wherein the control system  30  is operable to remove the magnet from the vicinity of the chamber  15  or otherwise block the magnetic field from the magnet from penetrating into the chamber  15 . While the illustrative embodiment employs both chamber rotation and electromagnets for moving the agitator within the chamber, it will be appreciated that chamber rotation may be used alone or that electromagnets may be used alone. Moreover, it will be appreciated that any technique for moving the agitator within the chamber may be employed and remain within the scope of the present disclosure. 
         [0036]    Referring now to  FIG. 2   a - 2   f,  operation of the system  10  is illustrated by way of a non-limiting example. Target cells and cell culture media are delivered to the interior  35  of the chamber  15 . In this embodiment, the beads  21  are buoyant and float near the top of the cell culture media within the chamber  15 . In  FIG. 2   a,  the first magnetic field generator  70  is energized and the beads  21  are held near the first end  40  of the chamber  15 . The chamber  15  is then rotated approximately 180° to a position as shown in  FIG. 2   b  wherein the first magnetic field generator  70  maintains the beads  21  near the chamber first end  40 . The first magnetic field generator  70  may then be de-energized whereby the beads  21  begin to float towards the second end  45  of the chamber  15  as shown in  FIG. 2   c.  In embodiments where the beads  21  include a coating which target cells will adhere to, movement of the beads  21  from one end to the other will collect newly grown target cells. The target cells may adhere to the beads  21  while waste is flushed from the chamber  15  and/or when new media is introduced to the chamber  15  such that a substantial number of the target cells, original and newly grown, remain within the chamber. Alternatively, magnetizable antibodies specific to the target cells may be added to the interior of chamber  15  whereby the antibodies will bind themselves to the target cells, and when a magnetic field is introduced to the chamber, the antibody bound target cells will be releasably coupled to the magnetizable beads  21  and/or the chamber wall(s) adjacent to the magnetic field generator(s). In this embodiment, one or both of the magnetic field generators  70 ,  72  may remain energized while unbound cells and/or waste are flushed from the chamber and/or while new media is introduced to the chamber such that a substantial number of the target cells, original and newly grown, remain within the chamber  15 . Alternatively, magnetic reagents, such as Annexin V or other suitable reagent, may be employed to couple to damaged or dead cells to the beads and the healthy target cells flushed from the system  10 . Further, it will be appreciated that magnetizable antibodies and/or reagents may be employed in a chamber  15  without the use of an agitator whereby the target cells or damaged/dead cells may be held against the chamber when the chamber is flushed. 
         [0037]    Referring again to FIGS., once the beads  21  are near the second end  45  of the chamber, the second magnetic field generator  72  may be energized whereby the beads  21  are held near the chamber second end  45  ( FIG. 2   d ) and the chamber rotated to the position shown in  FIG. 2   e.  The second magnetic field generator  72  may then be de-energized whereby the beads  21  will float towards the chamber first end  40  as shown in  FIG. 2   f.  As will be appreciated by those skilled in the art, a variety of additives, media, buffers, CO 2  and the like may be selectively added to the chamber at any desired point during this process and/or waste selectively removed in order to promote or enhance new cell growth based on measurements taken by the control system as previously discussed. 
         [0038]    In an alternative embodiment, non-buoyant beads may be employed such that the beads are moved within the chamber by rotation of the chamber and without also being subjected to magnetic fields. Here, gravity and centrifugal force, by way of rotation of the chamber, are employed to move the beads between two or more points within the chamber  15 . In yet another alternative, the first and second magnetic field generators  70 ,  72  may be alternately energized so as to move the beads between two or more points within the chamber and without any rotation of the chamber  15 . While the forgoing example employs beads  21  as the agitator, it will be appreciated that suitable device may be employed as the agitator and remain within the scope of the present disclosure, including but not limited to that of  FIG. 5 . Moreover, it will be appreciated that any means or technique for moving the agitator within the chamber may be employed and remain within the scope of the present disclosure. 
         [0039]    Moreover, it will be appreciated that if the chamber  15  formed from gas permeable material or otherwise includes a gas permeable portion, the system may be disposed within a CO 2  incubator or CO 2  room. Without a CO 2  incubator or CO 2  room or without any gas permeable portion of the chamber, reagents, such as HEPES may be employed or, alternatively, CO 2  may be injected directly into the chamber from a CO 2  reservoir. 
         [0040]    Although the present invention and its advantages have been disclosed in the context of certain illustrative, non-limiting embodiments, it should be understood that various changes, substitutions, permutations, and alterations can be made without departing from the scope of the invention as defined by the appended claims. It will be appreciated that any feature that is described in a connection to any one embodiment may also be applicable to any other embodiment.